DE102015011441A1 - Fluorescence light detection system and microscopy system - Google Patents

Abstract

For the detection of fluorescent light of a plurality of different fluorescent dyes, a fluorescent light detection system (1) is proposed, which comprises a beam splitter system (35), a spatially resolving first camera (21) and a second camera (23) different from the first camera (21), the beam splitter system (35) configured to transmit light (35) entering the beam splitting system according to a first transmission spectrum to the first camera (21) and to the second camera (23) according to a second transmission spectrum different from the first transmission spectrum; wherein the first transmission spectrum in a red spectral range has an average transmittance of at least 10%, in particular at least 20% or 50%, and wherein the second transmission spectrum in a blue and green spectral range in each case has an average transmittance of at least 90%.

Description

The present invention relates to a fluorescent light detection system for detecting observation and fluorescent light, and to a microscopy system comprising a fluorescent light detection system. In particular, the present invention relates to fluorescent light detection systems for detecting fluorescence of the fluorescent dyes protoporphyrin IX (PpIX), fluorescein and indocyanine green (ICG).

The fluorescent dyes PpIX, fluorescein and ICG are used in the medical field, in particular for coloring biological material, for example blood cells, tumors or other tissue. When the tissue stained with a fluorescent dye is illuminated with light of a wavelength which is in the absorption spectrum of the fluorescent dye, the fluorescent dye emits fluorescent light. This fluorescent light can in turn be detected, whereby the area of a particular tissue dyed with the fluorescent dye can be made visible and distinguishable from the surrounding biological material. Frequently, several different fluorescent dyes are used in an operation to color different biological materials. On the one hand, it may be advantageous for the surgeon to simultaneously observe different biological material, each colored with different fluorescent dyes. On the other hand, it may also be advantageous to selectively visualize certain biological materials by fluorescent light, so as not to be simultaneously distracted from another fluorescence.

In the field of surgical applications, fluorescence observation systems are usually integrated with or provided by conventional surgical microscopes. The fluorescence observation system is therefore usually located above the area to be operated and should therefore be as compact as possible, so as not to hinder the surgeon, i. H. take up little space. Conventional fluorescence observation systems are designed, for example, as a stereomicroscope, whereby the surgeon can take an ocular view of the surgical area. The observation beam path of the stereomicroscope can be superimposed by means of another optical system, a fluorescence image displayed on a display so that the surgeon can see the surgical area as well as the fluorescence image simultaneously.

Conventional fluorescent light detection systems use a variety of illumination and detection filters, each suitable for excitation and detection of only one fluorescence of a fluorescent dye. In order to excite and detect the fluorescence of several fluorescent dyes, such fluorescent light detection systems include filter changers comprising a plurality of filters. However, this requires a larger space, which hinders the surgeon.

Other conventional fluorescent light detection systems comprise a beam splitter, which splits light coming from an object, which may comprise fluorescent light but also illumination light, into two radiation beams. Usually, light of the visible spectral range, for example from 400 nm to 750 nm, is guided in the first beam, whereas the second beam is guided by infrared light. Furthermore, such conventional fluorescent light detection systems comprise two cameras, wherein the first beam is supplied to the first camera and the second beam is supplied to the second camera. While the first camera is configured to detect an image of visible light, the second camera is configured to detect an image of infrared light. This configuration is particularly useful in fluorescent light detection systems used to detect indocyanine green. In this configuration, detectors with RGB Bayer matrix or RGGB Bayer matrix are usually used for the first camera. In this case, the camera is divided into regularly repeating color filter cells, each color filter cell consisting of four pixels, one of which can transmit the red spectral range, two the green spectral range and one blue spectral range, so that these spectral ranges can each be selectively detected. In contrast, the second camera is configured to detect infrared light.

This configuration provides high sensitivity to infrared light, i. H. a high number of pixels for detecting infrared light, but relatively few pixels are provided for detection of other spectral regions (red, green, blue). Therefore, this configuration is suitable only for the fluorescent dye indocyanine green, but not for other fluorescent dyes whose fluorescence is outside the infrared spectral range.

It is therefore an object of the present invention to provide a fluorescent light detection system and a microscopy system, with which a plurality of different fluorescent dyes can be used and whose fluorescent light can be detected with a high sensitivity. In particular, it is an object of the present invention to provide a fluorescent light detection system to provide which manages without removable components, can detect different fluorescent dyes and thereby requires a small amount of space.

As schematically in 1A The fluorescent dye protoporphyrin IX (PpIX) has an absorption spectrum which has a normalized absorption intensity of more than 0.2 between 350 nm and 430 nm. The normalized absorption intensity is normalized to the maximum absorption intensity, ie the normalized absorption spectrum has only values between 0 and 1. In the range from 350 nm to 430 nm, therefore, the fluorescent dye PpIX can be efficiently excited. The maximum of the absorption, the fluorescent dye at about 405 nm. The fluorescent dye PpIX emits fluorescent light in a spectral range of about 600 nm to 750 nm, with a main maximum of the emission intensity at 635 nm and a secondary maximum at about 705 nm.

As schematically in 1B The fluorescent dye fluorescein has a normalized absorption intensity of greater than 0.2 between about 450 nm and 530 nm. In this area, the fluorescent dye fluorescein can therefore be well stimulated. The absorption spectrum of fluorescein peaks at about 495 nm. The fluorescent dye fluorescein emits emission light in the range of about 490 nm to 650 nm. The maximum of the emission spectrum is about 520 nm.

As schematically in 1C As shown, the fluorescent dye indocyanine green (ICG) has a normalized absorption intensity of greater than 0.2 between 700 nm and about 840 nm. Indocyanine green can therefore be well stimulated in this area. The maximum of the absorption spectrum of indocyanine green is about 800 nm. The fluorescent dye indocyanine green emits emission light in the range of about 750 nm to 1000 nm. The maximum of the emission spectrum is about 835 nm.

According to one embodiment, a fluorescent light detection system for detecting observation and fluorescent light comprises a beam splitting system, a spatially resolving first camera and a second resolving second camera different from the first camera, the beam splitter system being configured to transmit light entering the beam splitter system according to a first transmission spectrum to the first Camera and according to a different from the first transmission spectrum second transmission spectrum to the second camera to transmit; wherein the first transmission spectrum in a red spectral range has an average transmittance of at least 10%, in particular at least 20% or 50%, and wherein the second transmission spectrum in a blue and green spectral range in each case has an average transmittance of at least 90%.

The light entering the beam splitter system may be, for example, light emanating from an object or object region and may comprise light in a wavelength range of about 400 nm to 1000 nm. The light entering the beam splitter system may further include a portion which is illumination light reflected at an object. Further, the light entering the beam splitter system may include fluorescent light generated by one or more fluorescent dyes excited in an object. Usually, the intensity of the fluorescent light is much smaller than the intensity of the reflected illumination light, the latter being used to produce an overview image.

The beam splitter system is used to spectrally separate the light entering the beam splitter system, i. That is, the light entering the beam splitter system is transmitted to a first output of the beam splitter system according to the first transmission spectrum and to a second output of the beam splitter system according to the second transmission spectrum. The light emerging at the first output of the beam splitter system is supplied to the first camera and the light emerging at the second output of the beam splitter system is supplied to the second camera. The feeding of the light emerging from the beam splitter system can take place via one or more optical components and / or systems.

Herein, the transmission spectrum refers to the wavelength-dependent transmittance through the beam splitter system. The degree of transmission of the beam splitter system accordingly denotes the ratio of light of a wavelength λ entering the beam splitter system from the beam splitter system at one of the outputs of light of wavelength λ exiting. Since the beam splitting system inevitably absorbs light or may include specially provided absorption filters, the first transmission spectrum and the second transmission spectrum are not necessarily complementary to each other.

The first and second cameras are spatially resolved, d. H. these are configured to take pictures. In particular, the cameras can each be configured to detect images of a specific spectral range, for example a blue image which is generated exclusively from detected light of the blue spectral range.

The first and second camera preferably have the same sensor format, ie the spatial resolution or pixel number of the two cameras either the same or one of the two cameras has an integral multiple of the pixel number of the other camera in at least one spatial direction. The camera with the smaller number of pixels then has a correspondingly larger light-sensitive detection area. As a result, the same imaging optics between the beam splitter system and the cameras can be used. Furthermore, the camera on which less light intensity is expected to be specially adapted for low light conditions. Alternatively or additionally, by electrical interconnection / summing ("binning") of several pixels, the signal-to-noise ratio for the camera, to which less light hits, can be improved. The two cameras can be adapted in their design to the wavelength ranges to be detected by you, for example, in CMOS sensors by adjusting the thickness of the photosensitive sensor layer.

The first transmission spectrum has an average transmittance of at least 10%, 20% or 50% for a red spectral range, i. e., that at least 10%, 20% or 50% of the light entering the beam splitter system of the red spectral range is transmitted to the first output of the beam splitter system and supplied to the first camera. As previously explained, this does not necessarily mean that the second transmission spectrum has the complementary proportion of 90%, 80% or 50%. The second transmission spectrum has in each case a mean transmittance of at least 90% in a blue spectral range and in a green spectral range. This means that 90% of the light entering the beam splitter system of the blue or green spectral range is transmitted to the second output of the beam splitter system and the second camera can be supplied.

As a result, light of the red spectral range can be transmitted to the first camera and detected by it. Furthermore, as a result, light of the blue and green spectral range is transmitted to the second camera and can be detected by it.

Here, the blue, green, red and infrared spectral range can be defined as follows: The blue spectral range can be between 350 nm and 480 nm, the green spectral range can be between 480 nm and 580 nm, the red spectral range can be between 580 nm and 750 nm in particular between 580 nm and 680 nm or between 630 nm and 680 nm, and the infrared spectral range can be between 750 nm and 1000 nm. Furthermore, the blue, green, red and infrared spectral ranges can each have a spectral width of at least 50 nm, in particular 70 nm.

Herein denotes the average transmittance T an average of the transmittances T (λ) within a limited wavelength range. For example, the average transmittance T be defined as:

According to an exemplary embodiment, the first transmission spectrum in the blue and / or green spectral range has an average transmittance of at most 10%. Furthermore, the second transmission spectrum in the red spectral range has an average transmittance of at most 50%, in particular at most 80% or 90%. In this embodiment, only a small proportion of light of the blue and / or green spectral range is transmitted to the first camera, so that the light supplied to the first camera of other spectral ranges is only slightly superimposed with light of the blue and / or green spectral range. In this way it can be prevented that light of the blue and / or green spectral range is detected as light of another spectral range, whereby the detection of the other spectral range is improved.

According to another exemplary embodiment, the first camera is configured to detect light of the red spectral region and the second camera is configured to selectively detect light of the blue and green spectral regions, respectively.

In this embodiment, the first camera is configured to detect the light of the red spectral range transmitted to the first camera. In particular, the first camera can be configured to detect a spatially resolved intensity distribution of light of the red spectral range supplied by the first camera. For example, the first camera may have detection surfaces that are configured to exclusively or predominantly detect light of the red spectral range and to generate a value that corresponds to the intensity of the light of this spectral range. In an analogous manner, the second camera may be configured to detect light of the blue spectral range and light of the green spectral range. In particular, the second camera can be configured to detect a spatially resolved intensity distribution of light of the blue spectral range or of light of the green spectral range. For this purpose, the second camera may, for example, have detection surfaces which exclusively or predominantly detect light of the blue spectral range and have detection surfaces which exclusively or predominantly detect light of the green spectral range.

It means predominantly that less than 10%, in particular less than 1% or less than 0.1%, of an intensity value which represents the intensity of light of a specific spectral range is caused by light outside the specific spectral range.

According to an exemplary embodiment, a camera of the first and second cameras is configured to detect a spatially resolved first intensity distribution of light of a first spectral range; wherein a controller of the fluorescent light detection system is configured to determine, based on the first intensity distribution and a spatially resolved second intensity distribution, a spatially resolved third intensity distribution representing the intensity distribution of light of a third spectral region, the second intensity distribution comprising a spatially resolved intensity distribution of or detected by the controller Represents light of a second spectral range and wherein the first, second and third spectral range are different from each other.

In this embodiment, the fluorescent light detection system comprises a controller which determines a spatially resolved third intensity distribution based on a spatially resolved first and second intensity distribution. The first intensity distribution represents the spatially resolved intensity distribution of light of a first spectral range, the light being detected by the first or second camera. By way of example, the first camera can be configured to detect light of the red spectral range, such that the first intensity distribution corresponds to an intensity distribution of light of the red spectral range. The second intensity distribution may be an intensity distribution detected by one of the cameras or an intensity distribution determined by the controller. For example, the second intensity distribution corresponds to the intensity distribution of light of the red and infrared spectral ranges, which are detected jointly by the first camera. The controller may now be configured to determine the third intensity distribution, for example the intensity distribution of light of the infrared spectral range, based on the first intensity distribution (light of the red spectral range) and the second intensity distribution (light of the red and infrared spectral range). In the present example, the third intensity distribution can be obtained by subtracting the first intensity distribution from the second intensity distribution. Various mathematical operations may be used to determine the third intensity distribution, such as addition, subtraction, multiplication and division. Further, more complex formulas or algorithms for determining the third intensity distribution implemented by the controller may also be used.

In the present embodiment, two spectral ranges are different from each other when they include different spectral ranges, i. h., Two spectral ranges are different from each other when the two spectral ranges do not overlap or at most partially overlap or one spectral range completely covers the other spectral range, but also includes more spectral ranges.

According to an exemplary embodiment herein, the second intensity distribution is based on a spatially resolved intensity distribution detected by those of the first and second cameras other than the camera that detects the first intensity distribution. For example, when the first camera detects the first intensity distribution, the second intensity distribution is based on a spatially resolved intensity distribution detected by the second camera. Otherwise, if the first intensity distribution is detected by the second camera, the second intensity distribution is based on a spatially resolved intensity distribution detected by the first camera. The second intensity distribution may be equal to or determined based on the spatially resolved intensity distribution.

In this embodiment, the third intensity distribution is determined based on the first and second intensity distributions, wherein the first and second intensity distributions are based on intensity distributions detected by different cameras.

For determining the third intensity distribution, the controller may use characteristics of the first and / or second transmission spectrum.

According to an exemplary embodiment, the first transmission spectrum for an infrared spectral range has an average transmittance of at least 90% and the second transmission spectrum for the infrared spectral range has an average transmittance of at most 10%. Alternatively, the second transmission spectrum for the infrared spectral range may have an average transmittance of at least 90% and the first transmission spectrum for the infrared spectral range an average transmittance of at most 10%.

In this embodiment, light of the infrared spectral range, for example, fluorescent light of indocyanine green, becomes predominantly the first camera or predominantly to the second camera. In addition, the first or second camera may be configured to detect light of the infrared spectral range. In particular, the first or second camera may be configured to detect a spatially resolved intensity distribution of light of the infrared spectral range. This makes it possible to detect fluorescent light of indocyanine green either by the first camera or by the second camera.

According to an exemplary embodiment, a controller of the fluorescent light detection system is configured to provide a spatially resolved infrared intensity distribution representing a spatially resolved intensity distribution of light of the infrared spectral range detected or determined by the controller, a spatially resolved red intensity distribution that is detected or determined by the controller represents spatially resolved intensity distribution of red spectral region light, a spatially resolved green intensity distribution representing a detected or controlled spatial resolution intensity distribution of green spectrum light, and a spatially resolved blue intensity distribution representing detected or determined by the controller; spatially resolved intensity distribution of light of the blue spectral range represents.

The infrared, red, green and blue intensity distributions provided by the controller can then be used to display an overview image and a fluorescent light image. An intensity distribution of a specific spectral range may correspond to the intensity distribution detected by a camera configured to detect the particular spectral range. By way of example, the infrared intensity distribution may correspond to the spatially resolved intensity distribution of light of the infrared spectral range detected by a camera. Alternatively, the intensity distribution of a specific spectral range can also be determined by the controller from a plurality of spatially resolved intensity distributions of different spectral ranges.

According to an exemplary embodiment, the beam splitter system comprises a beam splitter and at least one optical filter. In this embodiment, the beam splitter and the at least one optical filter provide the first and / or second transmission spectrum. That at least one optical filter can be advantageous in particular if the beam splitter can not provide the spectral separation of the light striking the beam splitter system with sufficient accuracy.

According to an exemplary embodiment, the beam splitter is configured to spectrally and / or spatially and / or polarize the light entering the beam splitter system. In particular, the beam splitter may be configured to separate the light entering the beam splitter system into mutually orthogonal polarizations.

According to an exemplary embodiment herein, the first transmission spectrum furthermore has an average transmittance of at least 90% in the red and infrared spectral range and an average transmittance of at most 10% in the blue and green spectral range. In addition, the second transmission spectrum also has an average transmittance of at most 10% in the red and infrared spectral ranges. A controller of the fluorescent light detection system is configured to determine a spatially resolved intensity distribution of light of the red spectral region and a spatially resolved intensity distribution of light of the infrared spectral region based on intensity distributions detected by the first camera. Furthermore, the controller is configured to determine a spatially resolved intensity distribution of light of the blue spectral range and a spatially resolved intensity distribution of light of the green spectral range based on intensity distributions detected by the second camera.

In this embodiment, light of the blue spectral range and light of the green spectral range are transmitted substantially exclusively to the second camera. On the other hand, essentially no light of the red and infrared spectral range is transmitted to the second camera. Light of the red spectral range and light of the infrared spectral range is transmitted substantially exclusively to the first camera. On the other hand, essentially no light of the blue and green spectral range is transmitted to the first camera.

Furthermore, the first and second cameras are each configured to detect spatially resolved intensity distributions. The two cameras can be configured to detect multiple intensity distributions of light of different spectral ranges. From the intensity distributions detected by the first camera, the controller of the fluorescent light detection system determines a spatially resolved intensity distribution of light of the red spectral region and a spatially resolved intensity distribution of light of the infrared spectral region. This means that, based on the intensity distributions detected by the first camera, which need not necessarily correspond to the red spectral range and the infrared spectral range, a spatially resolved Intensity distribution is determined, which represents the incident on the first camera spatially resolved intensity distribution of light of the red spectral range. In the same way, based on the intensity distributions detected by the first camera, a spatially resolved intensity distribution is determined by the controller, which represents an intensity distribution of light of the infrared spectral range incident on the first camera.

From the intensity distributions detected by the second camera, the controller of the fluorescent light detection system determines a spatially resolved intensity distribution of light of the blue spectral region and a spatially resolved intensity distribution of light of the green spectral region. This means that, based on the intensity distributions detected by the second camera, which need not necessarily correspond to the blue spectral range and the green spectral range, a spatially resolved intensity distribution is determined which represents the spatially resolved intensity distribution of light of the blue spectral range impinging on the second camera. In the same way, based on the intensity distributions detected by the second camera, a spatially resolved intensity distribution is determined by the controller, which represents an intensity distribution of light of the green spectral range incident on the second camera.

According to an exemplary embodiment herein, the first camera comprises a spatially resolving detector having a first Bayer matrix, wherein the first Bayer matrix comprises a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the first Bayer matrix having a plurality of different non-overlapping ones A color filter, wherein at least a first color filter of the color filter of each color filter cell of the first Bayer matrix is a filter of a filter group, wherein at least a second color filter of the color filter of each color filter cell of the first Bayer matrix one of the at least one first color filter different filter of the filter group , wherein the filter group comprises a filter which predominantly transmits the red spectral range, a filter which predominantly transmits the infrared spectral range, and a filter which predominantly transmits the red and infrared spectral range.

In this embodiment, the first camera is equipped with a spatially resolving detector and a first Bayer matrix. The spatially resolving detector is configured to output a signal representing the spatially resolved intensity distribution of light striking the detector. Bayer matrices are optical filters which comprise a plurality of non-overlapping, regularly arranged color filter cells. Usually, the color filter cells are arranged in a square grid, i. H. Color filter cells are arranged side by side and one above the other and adjacent to each other. However, the color filter cells can also be arranged in other regular grating structures, for example a hexagonal grating. A detection surface of the detector superposed by a color filter cell is commonly referred to as a pixel. Each color filter cell includes a plurality of, typically different, spatially non-overlapping color filters that are configured to transmit different spectral regions. The individual color filters can be designed, for example, as absorption filters.

In the present embodiment, each of the color filter cells has at least a first and at least a second color filter of a filter group, wherein the at least one first color filter and the at least one second color filter are different from each other, i. H. are configured to transmit different spectral ranges. Spectral ranges are different if they are not identical. That is, an exemplary first color filter may be configured to transmit a first spectral range and the second color filter may be configured to transmit a second spectral range wherein the first and second spectral ranges may not overlap or partially overlap. Furthermore, for example, the first spectral range may comprise the second spectral range and comprises at least one further spectral range.

In the present embodiment, the filter group comprises a filter that predominantly transmits the red spectral region, a filter that predominantly transmits the infrared spectral region, and a filter that predominantly transmits the red and infrared spectral regions. The first Bayer matrix may therefore have at least the following combinations for the at least one first color filter and the at least one second color filter: (R, IR); (R, R + IR) and (IR, R + IR). The first combination means that the first or second color filter is a filter that predominantly transmits the red spectral range, and the other of the two color filters is a filter that predominantly transmits the infrared spectral range. The second combination means that the first or second color filter is a filter that predominantly transmits the red spectral region, and the other of the two color filters is a filter that predominantly transmits the red and infrared spectral regions. The third combination means that the first or second color filter is a filter that predominantly transmits the infrared spectral range, and the other of the two color filters is a filter that predominantly transmits the red and infrared spectral range.

The sensitivity for detecting a specific spectral range, here the red or infrared spectral range, can be adjusted by a suitable configuration of the first Bayer matrix. For example, if a high sensitivity is required to detect the infrared spectral range, a configuration can be chosen in which all or many color filters of each color filter cell can transmit the infrared spectral range. Such a configuration allows, for example, a high sensitivity for detecting the fluorescent light of indocyanine green. Alternatively, if the sensitivity to fluorescent light of the fluorescent dye PpIX is to be increased because the fluorescence of this fluorescent dye is usually weak, all or many color filters of each color filter cell may be configured to transmit the red spectral region.

According to an exemplary embodiment herein, half of the color filters of each color filter cell of the first Bayer matrix are configured to predominantly transmit the red spectral region or predominantly the infrared spectral region, and half of the color filters of each color filter cell of the first Bayer matrix are configured predominantly to transmit the red and infrared spectral range.

In this embodiment, each color filter cell of the first Bayer matrix comprises only color filters of the second and third above-mentioned combination. Accordingly, the first camera is exclusively configured to detect a spatially resolved intensity distribution of light of the red spectral region or a spatially resolved intensity distribution of light of the infrared spectral region next to a spatially resolved intensity distribution of light of the red and infrared spectral regions. In a PpIX preferred embodiment for detecting fluorescent light, half of the color filters of each color filter cell of the first Bayer matrix are configured to transmit predominantly the red spectral region and half of the color filters of each color filter cell of the first Bayer matrix are configured predominantly to transmit the red and infrared spectral range. Each color filter of the first Bayer matrix is therefore configured to transmit the fluorescent light from PpIX in the red spectral region to the detector.

According to another exemplary embodiment herein, the first camera is configured to detect a spatially resolved first intensity distribution of the light transmitted through the first color filters and incident on the detector and configured to have a spatially resolved second intensity distribution of the transmitted through the second color filters and incident on the detector To detect light. Further, the controller is configured to determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first and second detected intensity distribution and / or to determine the spatially resolved intensity distribution of light of the red spectral range based on the first and second detected intensity distribution.

For example, if half of the color filters of each color filter cell of the first Bayer matrix predominantly transmits the red spectral region and half of the color filters of each color filter cell of the first Bayer matrix predominantly transmits the red and infrared spectral regions, the first intensity distribution on the detector represents the incident light of Red spectral range and the second intensity distribution represents the light striking the detector of the red and infrared spectral range. The controller now determines the spatially resolved intensity distribution of light of the infrared spectral range from the first and second intensity distribution, for example by subtracting intensity values of an (R) subpixel from an (R + IR) subpixel. In this way, both a spatially resolved intensity distribution of light of the red spectral range and a spatially resolved intensity distribution of light of the infrared spectral range can be provided and at the same time a high sensitivity for light of the red spectral range can be made possible.

According to a further exemplary embodiment, the second camera comprises a spatially resolving detector having a second Bayer matrix, wherein the second Bayer matrix comprises a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer matrix being several different ones includes overlapping color filters.

The second Bayer matrix can be designed as RGGB matrix, i. H. One quarter of the color filters of each color filter cell transmits predominantly the blue spectral range, one quarter of the color filters of each color filter cell transmits predominantly the red spectral range and half of the color filters of each color filter cell transmits predominantly the green spectral range.

Alternatively, the second Bayer matrix may be formed as an RGGB matrix, ie half of the color filters of each color filter cell predominantly transmit the blue spectral range and half of the color filters of each color filter cell transmit predominantly the green spectral range. In the present embodiment, since substantially no light of the red spectral region is transmitted to the second camera, the (R) sub-pixel can be replaced by a (B) sub-pixel and thereby the Sensitivity for light of the blue spectral range can be improved.

Alternatively, the second Bayer matrix may be formed as a GGGB matrix, i. H. One fourth of the color filters of each color filter cell transmits predominantly the blue spectral range and three quarters of the color filters of each color filter cell predominantly transmit the green spectral range. Since none of the fluorescence dyes emits PpIX, fluorescein and ICG in the blue spectral range, the sensitivity to the green spectral range, in which, for example, fluorescein emits, can be improved at the expense of the sensitivity to the blue spectral range.

According to a further exemplary embodiment, the first transmission spectrum further has an average transmittance of at least 90% in the red spectral range and an average transmittance of at most 10% in the blue, green and infrared spectral ranges, and the second transmission spectrum also has an average transmittance in the infrared spectral range of at least 90% and in the red spectral region an average transmittance of at most 10%. Further, a controller of the fluorescent light detection system is configured to determine a spatially resolved intensity distribution of red spectral region light based on intensity distributions detected by the first camera, and a spatially resolved intensity distribution of blue spectral region light, a spatially resolved intensity distribution of green spectral region light, and a spatially resolved intensity distribution of To determine light of the infrared spectral range based on intensity distributions detected by the second camera.

In this embodiment, light of the red spectral range is transmitted substantially exclusively to the first camera and light of the blue, green and infrared spectral range is transmitted substantially exclusively to the second camera. Therefore, the first camera can be optimized for detecting light of the red spectral region, whereby, for example, fluorescent light of PpIXX can be detected with high sensitivity. As explained above in connection with the first camera, the second camera may be configured to detect spatially resolved intensity distributions of different spectral ranges. The controller can then determine the spatially resolved intensity distribution of light of the blue, green and infrared spectral range on the basis of the detected intensity distributions.

According to an exemplary embodiment herein, the second camera comprises a spatially resolving detector having a second Bayer matrix, wherein the second Bayer matrix comprises a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer matrix being several different ones includes overlapping color filters.

The second Bayer matrix may be configured according to the pattern (RGGB) + IR, i. H. One quarter of the color filters of each color filter cell transmits predominantly the blue and infrared spectral range, one quarter of the color filter of each color filter cell transmits predominantly the red and infrared spectral range and half of the color filter of each color filter cell transmits predominantly the green and infrared spectral range.

According to an exemplary embodiment herein, the second camera is configured to detect a spatially resolved first intensity distribution of the light transmitted through the red and infrared spectral regions and incident on the detector, and the controller is configured to provide the spatially resolved intensity distribution of infrared spectral region light based on the first intensity distribution.

Since substantially no light of the red spectral range is transmitted to the second camera due to the configuration of the beam splitter system, the (R + IR) subpixel of the spatially resolving detector essentially strikes exclusively the light of the infrared spectral range. The intensity detected by these subpixels can therefore be used as a spatially resolved intensity distribution of light of the infrared spectral range. Alternatively, using the knowledge about the transmission spectra of the beam splitter system and the intensity distribution of light of the red spectral range detected by the first camera, the intensity of the light of the red spectral range incident on the second camera can be compensated.

Further, after the controller is configured to determine the spatially resolved intensity distribution of light of the infrared spectral range, the controller may be configured to have the spatially resolved intensity distribution of blue spectral region light and the spatially resolved green light spectral region intensity based on that of the second camera detected intensity distributions and the spatially resolved intensity distribution of light of the infrared spectral range to determine.

For this purpose, the second camera can be configured to have a spatially resolved second intensity distribution of the color filter transmitting through the blue and infrared spectral ranges to detect transmitted and incident on the detector light and to detect a spatially resolved third intensity distribution of the transmitted through the green and infrared spectral region color filter transmitted and incident on the detector light. Further, the controller may be configured to determine the spatially resolved intensity distribution of blue spectral region light based on the second intensity distribution and the intensity distribution of infrared spectral region light and / or the spatially resolved intensity distribution of green spectral region light based on the third intensity distribution and the intensity distribution of light of the infrared spectral range. In particular, the controller may determine the spatially resolved intensity distribution of light of the blue or green spectral range by subtracting the intensity distribution of light of the infrared spectral range from the second and third intensity distribution, respectively.

In this embodiment, four subpixels per color filter cell are available for detecting light of the infrared spectral range, one subpixel per color filter cell for detecting light of the blue spectral range and two subpixels per color filter cell for detecting light of the green spectral range.

Alternatively to the configuration of the second Bayer matrix discussed above, the second Bayer matrix may be formed as a G (G + IR) (G + IR) (B + IR) matrix, i. H. One quarter of the color filters of each color filter cell transmits predominantly the blue and infrared spectral range, one fourth of the color filters of each color filter cell transmits predominantly the green spectral range and half of the color filters of each color filter cell transmits predominantly the green and infrared spectral range.

According to an exemplary embodiment herein, the second camera is configured to detect a spatially resolved first intensity distribution of the light transmitted through the green and infrared spectral regions and incident on the detector, and the controller is configured to provide the spatially resolved intensity distribution of infrared spectral region light based on the first intensity distribution and the intensity distribution of light of the green spectral range to determine.

In this embodiment, since a sub-pixel is configured to detect only light of the green spectral range, the spatially resolved intensity distribution of light of the infrared spectral range can be determined by subtracting the intensity distribution of light of the green spectral range from the first intensity distribution.

Furthermore, the second camera may be configured to detect a spatially resolved second intensity distribution of the light transmitted through the blue and infrared spectral regions and incident on the detector, and the controller may be configured to base the spatially resolved intensity distribution of blue spectral region light on the second intensity distribution and the intensity distribution of light of the infrared spectral range. Herein, the intensity distribution of light of the infrared spectral range previously determined by the controller is used to determine the spatially resolved intensity distribution of light of the blue spectral range, for example by subtracting the intensity distribution of light of the infrared spectral range from the second intensity distribution.

In this embodiment, three subpixels per color filter cell are available for detecting light of the green spectral range, three subpixels per color filter cell for detecting light of the infrared spectral range and one subpixel per color filter cell for detecting light of the blue spectral range.

According to a further embodiment, the first transmission spectrum furthermore has an average transmittance of at least 90% in the infrared spectral range, an average transmittance between 10% and 90%, in particular between 25% and 75% or between 40% and 60% in the red spectral range in the blue and green spectral range in each case an average transmittance of at most 10%. In addition, the second transmission spectrum also has an average transmittance in the red spectral range of between 10% and 90%, in particular between 25% and 75% or between 40% and 60%, and in the infrared spectral range an average transmittance of at most 10%. Further, a controller of the fluorescent light detection system is configured to determine a spatially resolved intensity distribution of light of the infrared spectral region and a spatially resolved intensity distribution of light of the red spectral region based on intensity distributions detected by the first and second cameras and a spatially resolved intensity distribution of light of the blue spectral region and a spatially resolved one Determine intensity distribution of light of the green spectral range based on detected by the second camera intensity distributions.

In this embodiment, light of the blue spectral range and light of the green spectral range are transmitted substantially exclusively to the second camera and light of the infrared spectral range becomes substantially transmitted exclusively to the first camera. Light of the red spectral range is transmitted to both the first and the second camera. The first and second cameras may be configured to detect intensity distributions of different spectral regions which are used by the controller to determine the spatially resolved intensity distribution of infrared spectral region light and the spatially resolved red-light intensity intensity distribution of light. Furthermore, the controller may determine the spatially resolved intensity distribution of light of the blue spectral range and the spatially resolved intensity distribution of light of the green spectral range from intensity distributions of light of different spectral ranges detected by the second camera.

In particular, the first camera may be configured to detect a spatially resolved first intensity distribution of light of the red and infrared spectral ranges. This means that the pixels of the first camera together detect both light of the red spectral range and light of the infrared spectral range and output a corresponding intensity value.

According to an exemplary embodiment herein, the second camera comprises a spatially resolving detector having a second Bayer matrix, the second Bayer matrix comprising a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer matrix having a plurality of different non-overlapping ones Includes color filter. In particular, the second Bayer matrix may be formed as an RGGB matrix, i. H. One quarter of the color filters of each color filter cell transmits predominantly the blue spectral range, one quarter of the color filters of each color filter cell transmits predominantly the red spectral range and half of the color filters of each color filter cell transmits predominantly the green spectral range.

In this embodiment, the spatially resolved intensity distribution of light of the blue spectral range and the spatially resolved intensity distribution of light of the green spectral range can be detected directly by the second camera. Furthermore, the second camera detects a spatially resolved intensity distribution of light of the red spectral range, which can be used to determine a spatially resolved intensity distribution of light of the infrared spectral range, which is detected by the first camera. This can be implemented as follows.

The controller may be configured to calculate the spatially resolved intensity distribution of light of the infrared spectral range based on the first intensity distribution of light of the red and infrared spectral range detected by the first camera and an intensity distribution of the color filter transmitted through the red spectral range and detected by the second camera to determine the light striking the detector. Furthermore or alternatively, the controller may be configured to determine the spatially resolved intensity distribution of light of the red spectral region based on the first intensity distribution detected by the first camera and the intensity distribution of the color filter transmitted through the red spectral region and incident on the detector detected by the second camera Determine the light.

For example, the intensity distribution of light of the infrared spectral range can be determined by subtracting the intensity distribution of the light of the red spectral range detected by the second camera from the first intensity distribution of light of the red and infrared spectral range detected by the first camera.

Alternatively, the second camera may comprise a second Bayer matrix which is an (R + G) GGB matrix, i. H. One quarter of the color filters of each color filter cell transmits predominantly the blue spectral range, one quarter of the color filters of each color filter cell transmits predominantly the red and green spectral range and half of the color filters of each color filter cell transmits predominantly the green spectral range.

In an exemplary embodiment thereof, the second camera is configured to detect a spatially resolved second intensity distribution of the light transmitted through the green and red spectral region and incident to the detector, and the controller is configured to provide a spatially resolved third intensity distribution of red light Spectral range based on the second intensity distribution and an intensity distribution of light of the green spectral range to determine.

In this embodiment, the intensity distribution of light of the blue spectral range and the intensity distribution of light of the green spectral range can be detected directly by the second camera. Furthermore, the second camera detects the second intensity distribution which represents the intensity distribution of the light of the light transmitted through the color filters transmitting the green and red spectral ranges. The third intensity distribution, which represents an intensity distribution of light of the red spectral range incident on the second camera, may be obtained from the controller by, for example, subtracting the intensity distribution of light from the green Spectral range of the second intensity distribution can be determined.

The third intensity distribution can also be used to determine the intensity distribution of light of the infrared spectral range. For this purpose, the controller can be configured to determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first intensity distribution detected by the first camera, which represents light of the red and infrared spectral range, and the third intensity distribution, for example by subtracting these intensity distributions. Furthermore or alternatively, the controller may be configured to determine the spatially resolved intensity distribution of light of the red spectral region based on the first intensity distribution detected by the first camera and the third intensity distribution. In this case, the intensity distributions can be added, for example.

According to an exemplary embodiment, the wavelength-dependent transmittance T (λ) of the first and / or second transmission spectrum within the red spectral range applies: T ₀ -ΔT ≦ T (λ) ≦ T ₀ + ΔT; 15% ≤ T ₀ ≤ 85%; 0 ≤ ΔT ≤ 5%.

In this embodiment, the light of the red spectral region incident on the beam splitter system is transmitted to both the first and the second camera. Within the red spectral range, the transmittance T (λ) of the first and second transaction spectrum varies by a maximum of 2ΔT by a value of T ₀ . Within the red spectral range, the transmittance has an approximately constant value. For the value T ₀ , a different value can be selected for the first transmission spectrum than for the value T _{0 of} the second transmission spectrum. In particular, it may be approximated that the value T _{0 of} the first transmission spectrum and the value T _{0 of} the second transmission spectrum add up to approximately 1 in total. In particular, T ₀ can be equal to 50%.

According to a further embodiment, a first spectral range of the second transmission spectrum within the red spectral range exclusively comprises wavelengths that are smaller than a cut-off wavelength λ ₀ , and has an average transmittance T ₁ on. Furthermore, a second spectral range of the second transmission spectrum within the red spectral range only comprises wavelengths that are greater than the limit wavelength λ ₀ , and has an average transmittance T ₂ on. Furthermore: T ₁ ≥ 70%, preferably T ₁ ≥ 80% or T ₁ ≥ 90%; and T ₂ ≤ 30%, preferred T ₂ ≤ 20% or T ₂ ≤ 10%.

In this embodiment, the red spectral range of the second transmission spectrum (and, correspondingly, also the red spectral range of the first transmission spectrum) is divided into a first spectral range below the cutoff wavelength λ ₀ and a second spectral range above the cutoff wavelength λ ₀ . The first and second spectral range each have significantly different average transmittances T ₁ respectively. T ₂ on why in the transition region of the two spectral ranges around the cut-off wavelength λ _0, the transmittance of the second transmission spectrum T2 drops sharply or the degree of transmittance of the first transmission spectrum T1 increases sharply.

For example, the cut-off wavelength λ ₀ may be chosen to be below the maximum of the emission spectrum of PpIX, for example: 600 nm ≦ λ ₀ ≦ 635 nm. Alternatively, the cut-off wavelength λ _{0 may} be between the main maximum of the emission spectrum of PpIX at 635 nm and the minor maximum at 705 nm. For example: 640 nm ≦ λ ₀ ≦ 680 nm.

In the former case (600 nm ≦ λ ₀ ≦ 635 nm), the fluorescent light of PpIX is transmitted substantially exclusively to the first camera. This can then be optimized especially for the detection of the fluorescence light of PpIX. In the latter case (640 nm ≤ λ ₀ ≤ 680 nm) the main maximum of the emission spectrum of PpIXX is transmitted to the second camera and the secondary maximum is transmitted to the first camera.

Another embodiment provides a microscopy system that includes at least one light source, a lens system, and a fluorescent light detection system. The light source is configured to generate illumination light and excitation light to excite fluorescent dyes and direct it to an object area. The illumination light serves to illuminate the object area and to generate an overview image of the object area from the light reflected thereby. The excitation light serves to excite at least one fluorescent dye. The fluorescent light detection system may be, for example, one of the fluorescent light detection systems described herein.

The objective system and the fluorescence light detection system are arranged so that at least a section of the object area through the objective system and the Fluorescence light detection system is imaged on the first and second camera of the fluorescent light detection system.

The microscopy system may further comprise a display system for displaying an image, wherein the image may be generated based on intensity distributions provided by a controller of the fluorescent light detection system. In particular, the image may be a superposition of an overview image and a fluorescent light image. For improved representation, the spectral ranges used for the display do not have to correspond to the actually detected spectral ranges. In particular, the fluorescent light of indocyanine green, which lies in the infrared spectral range, can be represented in any desired color or by another marking. In particular, the image can be generated from the spatially resolved intensity distributions of the red, infrared, green and blue spectral regions or the infrared, red, green and blue intensity distributions provided by the controller.

Furthermore, a detection filter can be arranged in the beam path between the object area and the fluorescence light detection system, which is configured to suppress at least a part of the excitation light. The suppression can take place both in terms of intensity and in terms of the spectral range.

1A shows the normalized absorption and emission spectrum of the fluorescent dye PpIX.

1B shows the normalized absorption and emission spectrum of the fluorescent dye fluorescein.

1C shows the normalized absorption and emission spectrum of the fluorescent dye indocyanine green.

2 shows an exemplary embodiment of a microscopy system.

3 shows an exemplary embodiment of a fluorescent light detection system.

4 shows another exemplary embodiment of a fluorescent light detection system.

5 shows another exemplary embodiment of a fluorescent light detection system.

6 shows another exemplary embodiment of a fluorescent light detection system.

2 shows an exemplary embodiment of a microscopy system 1 , The microscopy system 1 includes at least one light source 3 , which lighting and excitation light 5 on an object area 7 directed. The excitation light is suitable for use in an object 9 contained fluorescent dyes, such as PpIX and / or fluorescein and / or ICG, excite. Furthermore, the microscope system has a lighting optical system 11 on which of the at least one light source 3 generated light on the object area 7 directed. In the beam path 13 from the at least one light source 3 to the object area 7 is a lighting filter 15 arranged, which the illumination and excitation light 5 in terms of its intensity spectrum can form.

The microscopy system 1 further includes a lens system 17 and a fluorescent light detection system 19 , The lens system 17 and the fluorescent light detection system 19 are configured and arranged so that at least a portion of the object area 7 on a first camera 21 and a second camera 23 of the fluorescent light detection system 19 what is reflected by the detection beam path 25 is indicated.

The lens system 17 may include multiple optical components, such as a lens 27 and a zoom lens system consisting of the optical elements 29 and 31 , Within the detection beam path 25 can be a detection filter 33 be arranged, which is configured to suppress excitation light for exciting fluorescent dyes and to transmit illumination light to produce an overview image.

The fluorescent light detection system 19 includes next to the first camera 21 and the second camera 23 a beam splitter system 35 which enters the beam splitter system at its entrance 37 entering light on two outputs 41 transfers.

The fluorescent light detection system 19 further includes a controller 43 , The control 43 is with the at least one light source 3 through a connection 45 connected. About this connection 45 can the controller 43 the at least one light source and, in general, a light generating system comprising the at least one light source 3 includes, controls. Further, the controller 43 about connections 47 with the first camera 21 and the second camera 23 connected. About these connections 47 For example, the controller may receive signals from the cameras, which may represent, for example, spatially resolved intensity distributions of light of different spectral ranges. The control 43 is also with a display device 49 of the microscopy system 1 connected. By means of the display device 49 the controller can display images of the object area.

3 shows an exemplary embodiment of a fluorescent light detection system 19A as it is for example in the in 2 shown microscope system can be used. The fluorescent light detection system 19A includes a first camera 21A , a second camera 23A and a beam splitter system 35A ,

Through the configuration of the fluorescent light detection system explained below 19A This is configured to detect fluorescent light of the fluorescent dyes PpIX, fluorescein and ICG, without having to make individual components of the fluorescent light detection system interchangeable or interchangeable. Instead, the fluorescent light of the three fluorescent dyes as well as an overview image can be detected by the configuration explained below.

The beam splitter system 35A is configured to light entering the beam splitter system 51 , which in the 3 to 6 is represented by four arrows, according to a first transmission spectrum T1 to the first camera 21A and according to a second transmission spectrum T2 different from the first transmission spectrum T1 to the second camera 23A transferred to. The representation 53A shows the first transmission spectrum T1 as a function of the wavelength λ. The representation 55A shows the transmission spectrum T2 as a function of the wavelength λ.

In the representations of the first transmission spectrum T1 and the second transmission spectrum T2 in the 3 to 6 For example, a "B" represented in a circle represents a blue spectral region, a "G" shown in a circle represents a green spectral region, an "R" shown in a circle represents a red spectral region and an "IR" represented in a circle represents an infrared spectral region.

The red spectral range covers the emission range of the fluorescent dye PpIX, d. H. the spectral range within which the fluorescent dye PpIX emits fluorescent light. The spectral range IR comprises the emission range of the fluorescent dye ICG. The emission region of the fluorescent dye fluorescein is partially covered by the green spectral region and partly by the red spectral region.

As in the presentation 53A shown, the first transmission spectrum T1 in the blue spectral range and in the green spectral range in each case a low average transmittance. In particular, the average transmittance of the blue and green spectral range is less than 10% in each case. Furthermore, the first transmission spectrum T1 in the red and infrared spectral range in each case has a high average transmittance, which is for example at least 90%. Therefore, light of the red spectral region represented by long-dashed line arrows and infrared spectral region light represented by solid line arrows become the first camera 21A transfer. On the other hand, essentially no light of the blue and green spectral range becomes the first camera 21A over amount.

As in the presentation 55A illustrated, the second transmission spectrum T2 in the blue and green spectral range in each case a high average transmittance, which may be in particular at least 90%. On the other hand, the second transmission spectrum T2 in the red and infrared spectral regions respectively has only low mean transmittances, which are for example at most 10%. Accordingly, light of the blue spectral region, represented by short dashed line arrows, and green spectrum light, represented by dashed line arrows, become the second camera 23A transfer. For simplified understanding, the figure corresponds to the line of arrows, in the illustrations 53A and 55A , the shape of the circle of the respective spectral range. Accordingly, the blue spectral region "B" is represented by a short-dashed circle, the green spectral region "G" by a dot-dashed circle, the red spectral region "R" by a long-dashed circle, and the infrared spectral region "IR" by a solid-line circle.

The first camera 21A includes a spatially resolving detector 59A with a first Bayer matrix 61A , The first Bayer matrix 61A comprises a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the first Bayer matrix 61A includes several different non-overlapping color filters.

An exemplary color filter cell 63A the first Bayer matrix 61A consists of four non-overlapping color filters, represented by labeled squares. The regular non-overlapping arrangement of multiple color filter cells 63A is by the protruding edges 65 the color filter cell indicated. The color filter cell 63A comprises color filter "R", which predominantly transmit the red spectral range. Furthermore, the color filter cell comprises 63A Color filter "R + IR", which predominantly transmit the red and infrared spectral range.

A detection surface of the detector 59A which corresponds to the size of a color filter cell is called a pixel. A detection surface of the detector 59A the area of a color filter one Color filter cell corresponds, is called Subpixel. When using the color filter cell 63A Accordingly, four subpixels per color filter cell are available for detecting the red spectral range. Two subpixels per color filter cell are available for the detection of the infrared spectral range. This is the first camera 21A For fluorescence light of PpIX in the red spectral region particularly sensitive and can also detect the fluorescent light of ICG.

A spatially resolved intensity distribution of light of the infrared spectral range may be provided by the controller 43 be determined. For this purpose, for example, the intensity of light transmitted through the color filters "R" can be subtracted from the intensity of the light transmitted through the color filters "R + IR".

The second camera 23A includes a spatially resolving detector 67A with a second Bayer matrix 69A , The second Bayer matrix 69A Like the first Bayer matrix, it comprises a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer matrix comprising a plurality of different non-overlapping color filters. An exemplary color filter cell 71A The second Bayer matrix comprises four color filters, wherein a color filter denoted by "R" predominantly transmits the red spectral range, a color filter designated by "G" predominantly transmits the green spectral range and a color filter designated "B" predominantly transmits the blue spectral range. This is the second camera 23A configured to detect a spatially resolved intensity distribution of light of the red spectral region, a spatially resolved intensity distribution of light of the green spectral region and a spatially resolved intensity distribution of light of the blue spectral region.

In this configuration of the fluorescent light detection system 19A Fluorescence light of the fluorescent dye fluorescein is detected both by the first camera 21A as well as the second camera 23A detected. In particular, the proportion in the yellow spectral range of the fluorescent light is through the green subpixels of the second camera 23A , that is, detected by the subpixels having the color filters "G" in front of them, and the proportion in the red spectral range of the fluorescence light is detected by the red subpixels of the first camera 21A , ie the subpixels to which the color filters "R" are located upstream, detected.

Another exemplary color filter cell 73A the second Bayer matrix 69A has equal parts color filter "B" and color filter "G". Because of the configuration of the beam splitter system, no light of the red spectral range of the second camera 23A is supplied, can be dispensed with the red subpixel in favor of a blue subpixel.

Another exemplary color filter cell 75A the second Bayer matrix contains a color filter "B" and three color filters "G". Accordingly, the second camera 23A more subpixels are available for light of the green spectral range, which also improves the sensitivity for light of the yellow spectral range, and thus for fluorescent light of fluorescein.

4 shows a further embodiment of a fluorescent light detection system 19B , The beam splitter system 35B is configured to be in the beam splitter system 35B entering light 51 according to the in the presentation 53B shown first transmission spectrum T1 to the first camera 21B to transfer and according to the in the presentation 55B shown second transmission spectrum on the second camera 23B transferred to. The beam splitter system 35B is configured to emit light of the red spectral range substantially exclusively to the first camera 21B and transmit light of the blue, green and infrared spectral range each substantially exclusively to the second camera 23B transferred to. For this purpose, the first transmission spectrum T1 in the red spectral range have an average transmittance of at least 90% and in the blue, green and infrared spectral range each have an average transmittance of at most 10%. Furthermore, the second transmission spectrum in the blue, green and infrared spectral range can each have an average transmittance of at least 90% and in the red spectral range an average transmittance of at most 10%.

The first camera 21B is configured to detect light of the red spectral region, in particular it is configured to detect a spatially resolved intensity distribution of light of the red spectral region. The first camera 21B can be a symbolically indicated filter 63 which is configured to transmit exclusively or predominantly light of the red spectral range. Because the first camera 21B is provided exclusively for the detection of light of the red spectral range, provides the fluorescent light detection system 19B high sensitivity for the detection of fluorescent light from PpIX.

The second camera 23B includes a spatially resolving detector 67B with a second Bayer matrix 69B ,

An exemplary color filter cell 71B the second Bayer matrix 69B contains four color filters, of which one predominantly transmits the blue and infrared spectral range, one transmits the red and infrared spectral range and two transmit the green and infrared spectral range.

Since according to the second transmission spectrum T2 substantially no light of the red spectral range to the second camera 23B the subpixel "R + IR" detects only light of the infrared spectral range. With knowledge of the illumination light spectrum, the first transmission spectrum T1 and the second transmission spectrum T2, the spatially resolved intensity distribution of light of the red spectral range, which is emitted by the first camera, can also be determined 21B is detected, to compensate for the second camera 23B the incident light of the red spectral range. This compensation can be done by the controller 43 be performed.

According to this configuration, the second camera is 23B configured to transmit a spatially resolved intensity distribution of the color filter transmitted through the blue and infrared spectral regions and to the detector 67B to detect the incident light. The spatially resolved intensity distribution of light of the blue spectral range can be determined from this intensity distribution and the intensity distribution of light of the infrared spectral range, for example by subtracting the intensities of these intensity distributions. Analogously thereto, the intensity distribution of light of the green spectral range can also be determined from the intensity distribution of light of the infrared spectral range and the intensity distribution of light of the light transmitted through the color filter transmitting the green and infrared spectral range and detected by the detector.

Another exemplary color filter cell 73B The second Bayer matrix comprises four color filters, one of which predominantly transmits the green spectral range, one predominantly transmits the blue and infrared spectral range and two transmits predominantly the green and infrared spectral range.

In this configuration, the intensity distribution of light of the infrared spectral range can be determined, for example, by subtracting the intensity distribution detected by the "G" sub-pixel from the intensity distribution detected by the "G + IR" sub-pixels. Further, by using the intensity distribution of light of the infrared spectral range determined in this way, an intensity distribution of light of the blue spectral range can be determined.

In the in 4 illustrated embodiment, the entire detection surface of the first camera 21B provided for the detection of fluorescent light from PpIX. For detection of fluorescent light of ICG are each three subpixels per color filter cell of the second camera 23B provided. Fluorescence light of the fluorescent dye fluorescein is partly from the first camera 21B and partly from the second camera 23B detected. For the proportion in the red spectral range of the fluorescent light is the entire detection area of the first camera 21B are available and for detection of the proportion in the yellow spectral range are two or three the green spectral range detecting subpixels of the second camera 23B to disposal.

5 shows a further embodiment of a fluorescent light detection system 19C , In this embodiment, the beam splitter system 35C configured so that light of the red spectral range both to the first camera 21C as well as to the second camera 23C is transmitted. The first transmission spectrum T1 has an average transmittance of at most 10% for the blue and green spectral range and an average transmittance of at least 90% for the infrared spectral range. In the red spectral range, which may include, for example, the spectral range from 580 nm to a wavelength between 680 nm and 800 nm, the first transmission spectrum T1 has an average transmittance of about 40%. In particular, for the transmittance T (λ) of the first transmission spectrum T1 within the red spectral range, T ₀ -ΔT ≦ T (λ) ≦ T ₀ + ΔT, wherein T _{0 is} about 40% and ΔT is about 5%.

Accordingly, the transmittance T (λ) of the first transmission spectrum T1 within the red spectral range has a substantially constant value T ₀ of about 40%.

For this purpose, the second transmission spectrum T2 for the blue and green spectral range in each case has an average transmittance of at least 90% and for the infrared spectral range an average transmittance of at most 10%. Within the red spectral range, the transmittance of the second transmission spectrum T2 has a relatively constant value of about 60%.

Through this configuration, fluorescent light from PpIX and fluorescein is applied to both the first camera 21C as well as the second camera 23C transfer. The fluorescent light from ICG is essentially exclusively on the first camera 21C transfer.

The first camera 21C is configured to detect light of the red and infrared spectral regions and for this purpose has a corresponding filter which passes through the color filter cell 63C is shown. The second camera 23C includes a spatially resolving detector 67C and a second Bayer matrix 69C , An exemplary color filter cell 71C the second Bayer matrix 69C may have the previously explained RGGB pattern.

In this configuration, the spatially resolved intensity distribution of light of the blue spectral range and the spatially resolved intensity distribution of light of the green spectral range may be directly from the second camera 23C be detected. The spatially resolved intensity distribution of light of the red spectral range transmitted by the second camera 23C can be used to determine the spatially resolved intensity distribution of light of the infrared spectral range, for example, by subtracting that from the second camera 23C detected intensity distribution of light of the red spectral range from that of the first camera 21C detected intensity distribution. Furthermore, those from the first camera 21C detected intensity distribution with that of the second camera 23C detected intensity distribution of light of the red spectral range are added to improve the sensitivity for detecting light of the red spectral range.

To improve the sensitivity to light of the green spectral range, as in the exemplary color filter cell 73C represented, the color filter "R" of the color filter cell 71C be replaced by the color filter "R + G", which predominantly transmits the red and green spectral range. According to this configuration, the second camera is 23C configured to transmit a spatially resolved first intensity distribution of the color filter transmitting through the red and green spectral regions and to the detector 67C to detect the incident light. The spatially resolved intensity distribution of light of the green spectral range transmitted by the second camera 23C can be used, the spatially resolved intensity distribution from the second camera 23C determining the incident light of the red spectral range, for example by subtracting the detected intensity distribution of light of the green spectral range from the first intensity distribution.

6 shows a further embodiment of a fluorescent light detection system 19D , Analogous to the in 5 shown fluorescence light detection system 19C is also the fluorescent light detection system 19D configured to light the red spectral range on both the first camera 21D as well as the second camera 23D transferred to. Unlike the in 5 embodiment shown the red spectral region is not shared by intensities, but separated by spectral regions, that is, light of the red spectral region with a wavelength that is less than a cutoff wavelength λ _0, according to the first transmission spectrum T1 does not substantially to the first camera 21D transmitted and according to the second transmission spectrum T2 substantially exclusively to the second camera 23D transfer. On the other hand, light of the red spectral region having a wavelength larger than the cut-off wavelength λ ₀ becomes substantially exclusive to the first camera according to the first transmission spectrum T1 21D transmitted and according to the second transmission spectrum T2 substantially not to the second camera 23D transfer. The cut-off wavelength λ ₀ can be, for example, in the range between 600 nm and 630 nm. In this configuration, the fluorescent light from PpIX becomes substantially exclusive to the first camera 21D transfer. Alternatively, the cutoff wavelength λ _{0 may be} in a range of 640 nm to 680 nm. In this configuration, emission light from PpIX is partly onto the first camera 21D and partly on the second camera 23D transfer.

The first camera 21D can the in 5 shown first camera 21C be equal. Furthermore, the second camera 23D the in 5 shown second camera 23C be equal. In particular, the in 5 shown exemplary color filter cells of the second Bayer matrix 71C and 73C also as a color filter cell of the second Bayer matrix 69D the second camera 23D be used.

In this embodiment, the fluorescent light of indocyanine green is substantially exclusively from the first camera 21D detected and the fluorescent light from fluorescein is essentially exclusively from the second camera 23D detected.

The intensity distributions of the red and infrared spectral range can be determined by the controller according to the schemes already explained above.

Claims (37)

A fluorescent light detection system for detecting observation and fluorescent light comprising: a beam splitting system, a spatially resolving first camera, and a second resolving second camera other than the first camera, the beam splitter system being configured to transmit light entering the beam splitting system according to a first transmission spectrum to the first camera and to transmit to the second camera according to a second transmission spectrum different from the first transmission spectrum; wherein the first transmission spectrum in a red spectral range has an average transmittance of at least 10%, in particular at least 20% or 50%, and wherein the second transmission spectrum in a blue and green spectral range in each case one average transmittance of at least 90%. Fluorescent light detection system according to claim 1, wherein the first transmission spectrum in the blue and / or green spectral range has an average transmittance of at most 10%; and wherein the second transmission spectrum in the red spectral range has an average transmittance of at most 50%, in particular at most 80% or 90%. Fluorescent light detection system according to claim 1 or 2, wherein the first camera is configured to detect light of the red spectral region; and wherein the second camera is configured to selectively detect light of the blue and green spectral ranges, respectively. Fluorescent light detection system according to one of claims 1 to 3, wherein a predetermined camera of the first and second cameras is configured to detect a spatially resolved first intensity distribution of light of a first spectral range; wherein a controller of the fluorescent light detection system is configured to determine a spatially resolved third intensity distribution representing the intensity distribution of light of a third spectral range based on the first intensity distribution and a spatially resolved second intensity distribution, wherein the second intensity distribution represents a spatially resolved intensity distribution of light of a second spectral range detected or controlled by the controller, and wherein the first, second and third spectral range are different from each other. The fluorescent light detection system according to claim 4, wherein the second intensity distribution is based on a spatially resolved intensity distribution detected by the camera of the first and second cameras other than the predetermined camera. Fluorescent light detection system according to claim 4 or 5, wherein the controller uses the first and / or second transmission spectrum to determine the third intensity distribution. Fluorescent light detection system according to one of claims 1 to 6, wherein the first transmission spectrum for an infrared spectral range has an average transmittance of at least 90% and the second transmission spectrum for the infrared spectral range has an average transmittance of at most 10%; or wherein the second transmission spectrum for the infrared spectral range has an average transmittance of at least 90% and the first transmission spectrum for the infrared spectral range has an average transmittance of at most 10%. Fluorescent light detection system according to one of claims 1 to 7, wherein the first or second camera is configured to detect light of the infrared spectral range. A fluorescent light detection system according to any one of claims 1 to 8, wherein a controller of the fluorescent light detection system is configured to: A spatially resolved infrared intensity distribution which represents a spatially resolved intensity distribution of light of the infrared spectral range detected or determined by the control, A spatially resolved red intensity distribution which represents a spatially resolved intensity distribution of light of the red spectral range detected or determined by the control, A spatially resolved green intensity distribution, which represents a spatially resolved intensity distribution of light of the green spectral range detected or determined by the control, and A spatially resolved blue intensity distribution, which represents a detected or determined by the control, spatially resolved intensity distribution of light of the blue spectral range to provide. Fluorescent light detection system according to one of claims 1 to 9, wherein the beam splitter system comprises a beam splitter and at least one optical filter. Fluorescent light detection system according to claim 10, wherein the beam splitter is configured to separate the light entering the beam splitter system spectrally and / or spatially and / or polarizations, in particular mutually orthogonal polarizations. Fluorescent light detection system according to one of claims 1 to 11, wherein the first transmission spectrum in the red and infrared spectral range each having an average transmittance of at least 90% and in the blue and green spectral range in each case a mean transmittance of at most 10%, wherein the second transmission spectrum in the red and infrared Spectral range each having a mean transmittance of at most 10%, wherein a controller of the fluorescent light detection system is configured to To determine a spatially resolved intensity distribution of light of the red spectral region and a spatially resolved intensity distribution of light of the infrared spectral region based on intensity distributions detected by the first camera, and a spatially resolved intensity distribution of light of the blue spectral region and a spatially resolved intensity distribution of light of the green spectral region based on to determine intensity distributions detected by the second camera. Fluorescent light detection system according to claim 12, wherein the first camera comprises a position-resolving detector having a first Bayer matrix, the first Bayer matrix comprising a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the first Bayer matrix comprising a plurality of different, non-overlapping color filters, wherein at least a first color filter of the color filters of each color filter cell of the first Bayer matrix is a filter of a filter group, wherein at least a second color filter of the color filters of each color filter cell of the first Bayer matrix is a filter of the filter group different from the at least one first color filter, wherein the filter group comprises a filter that predominantly transmits the red spectral region, a filter that predominantly transmits the infrared spectral region, and a filter that predominantly transmits the red and infrared spectral regions. Fluorescent light detection system according to claim 13, wherein half of the color filters of each color filter cell of the first Bayer matrix predominantly transmits the red spectral range or predominantly the infrared spectral range and half of the color filters of each color filter cell of the first Bayer matrix predominantly transmits the red and infrared spectral range. Fluorescent light detection system according to claim 13 or 14, wherein the first camera is configured to detect a spatially resolved first intensity distribution of the light transmitted through the first color filters and incident on the detector, wherein the first camera is configured to detect a spatially resolved second intensity distribution of the light transmitted through the second color filters and incident on the detector; and wherein the controller is configured to determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first and second intensity distribution, and / or wherein the controller is configured to determine the spatially resolved intensity distribution of red spectral region light based on the first and second intensity distributions. Fluorescent light detection system according to one of claims 12 to 15, wherein the second camera comprises a position-resolving detector having a second Bayer matrix, the second Bayer matrix comprising a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer matrix comprising a plurality of different non-overlapping color filters, wherein a quarter of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the blue spectral range, one fourth of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the red spectral range and half of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the green spectral range; or wherein half of the color filters of each color filter cell of the second Bayer matrix predominantly transmit the blue spectral range and half of the color filters of each color filter cell of the second Bayer matrix predominantly transmit the green spectral range; or wherein a quarter of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the blue spectral range and three quarters of the color filters of each color filter cell of the second Bayer matrix predominantly transmit the green spectral range. Fluorescent light detection system according to one of claims 1 to 11, wherein the first transmission spectrum has an average transmittance of at least 90% in the red spectral range and an average transmittance of at most 10% in the blue, green and infrared spectral range, wherein the second transmission spectrum has an average transmittance of at least 90% in the infrared spectral range and an average transmittance of at most 10% in the red spectral range, wherein a controller of the fluorescent light detection system is configured to To determine a spatially resolved intensity distribution of light of the red spectral region based on intensity distributions detected by the first camera, and - To determine a spatially resolved intensity distribution of light of the blue spectral range, a spatially resolved intensity distribution of light of the green spectral range and a spatially resolved intensity distribution of light of the infrared spectral range based on detected by the second camera intensity distributions. The fluorescent light detection system of claim 17, wherein the second camera comprises a location-resolving detector having a second Bayer matrix, the second Bayer matrix comprising a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer array having a plurality of different non-overlapping color filter, wherein a quarter of the color filter of each color filter cell of the second Bayer matrix predominantly transmits the blue and infrared spectral range, a quarter of the color filter of each color filter cell of the second Bayer matrix predominantly transmits the red and infrared spectral range and half of the color filter each color filter cell of the second Bayer matrix predominantly transmits the green and infrared spectral range. Fluorescent light detection system according to claim 18, wherein the second camera is configured to detect a spatially resolved first intensity distribution of the light transmitted through the color filters transmitting the red and infrared spectral regions and incident on the detector, wherein the controller is configured to determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first intensity distribution. Fluorescent light detection system according to claim 19, wherein the second camera is configured to detect a spatially resolved second intensity distribution of the light transmitted through the color filters transmitting the blue and infrared spectral regions and incident on the detector, wherein the controller is configured to determine the spatially resolved intensity distribution of light of the blue spectral range based on the second intensity distribution and the intensity distribution of light of the infrared spectral range; and or wherein the second camera is configured to detect a spatially resolved third intensity distribution of the light transmitted through the color filters transmitting the green and infrared spectral regions and incident on the detector, wherein the controller is configured to determine the spatially resolved intensity distribution of light of the green spectral range based on the third intensity distribution and the intensity distribution of light of the infrared spectral range. Fluorescent light detection system according to claim 17, wherein the second camera comprises a position-resolving detector having a second Bayer matrix, the second Bayer matrix comprising a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer matrix comprising a plurality of different non-overlapping color filters, wherein one fourth of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the blue and infrared spectral range, one fourth of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the green spectral range and half of the color filters of each color filter cell of the second Bayer Matrix predominantly transmits the green and infrared spectral range. Fluorescent light detection system according to claim 21, wherein the second camera is configured to detect a spatially resolved first intensity distribution of the light transmitted through the color filters transmitting the green and infrared spectral regions and incident on the detector, wherein the controller is configured to determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first intensity distribution and the intensity distribution of light of the green spectral range. Fluorescent light detection system according to claim 22, wherein the second camera is configured to detect a spatially resolved second intensity distribution of the light transmitted through the color filters transmitting the blue and infrared spectral regions and incident on the detector, wherein the controller is configured to determine the spatially resolved intensity distribution of light of the blue spectral range based on the second intensity distribution and the intensity distribution of light of the infrared spectral range. Fluorescence light detection system according to one of claims 1 to 11, wherein the first transmission spectrum in the infrared spectral range has an average transmittance of at least 90%, in the red spectral range an average transmittance between 10% and 90%, in particular between 25% and 75% or between 40% and 60 %, and in the blue and green spectral range each have an average transmittance of at most 10%, the second transmission spectrum in the red spectral range having an average transmittance between 10% and 90%, in particular between 25% and 75% or between 40% and 60%, and in the infrared spectral range has an average transmittance of at most 10%, wherein a control of the fluorescence light detection system is configured to - a spatially resolved intensity distribution of light of the infrared spectral range and a determine spatially resolved intensity distribution of light of the red spectral range based on intensity distributions detected by the first and second camera, - determine a spatially resolved intensity distribution of light of the blue spectral range and a spatially resolved intensity distribution of light of the green spectral range based on intensity distributions detected by the second camera. The fluorescent light detection system of claim 24, wherein the first camera is configured to detect a spatially resolved first intensity distribution of red and infrared spectral region light. Fluorescent light detection system according to claim 25, wherein the second camera comprises a position-resolving detector having a second Bayer matrix, the second Bayer matrix comprising a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer matrix comprising a plurality of different non-overlapping color filters, wherein a quarter of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the blue spectral range, one fourth of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the red spectral range and half of the color filters of each color filter cell of the second Bayer matrix predominantly transmitted the green spectral range. Fluorescent light detection system according to claim 26, wherein the controller is configured to To determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first intensity distribution detected by the first camera and an intensity distribution of the light transmitted through the color filter transmitting the red spectral range and detected by the second camera; and or To determine the spatially resolved intensity distribution of light of the red spectral range based on the first intensity distribution detected by the first camera and the intensity distribution of the light transmitted through the color filter transmitting the red spectral range and detected by the second camera. Fluorescent light detection system according to claim 25, wherein the second camera comprises a position-resolving detector having a second Bayer matrix, the second Bayer matrix comprising a plurality of non-overlapping, regularly arranged color filter cells, each color filter cell of the second Bayer matrix comprising a plurality of different non-overlapping color filters, wherein one quarter of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the blue spectral range, one quarter of the color filters of each color filter cell of the second Bayer matrix predominantly transmits the red and green spectral range and half of the color filters of each color filter cell of the second Bayer Matrix predominantly transmits the green spectral range. Fluorescent light detection system according to claim 28, wherein the second camera is configured to detect a spatially resolved second intensity distribution of the light transmitted through the color filters transmitting the green and red spectral regions and incident on the detector, wherein the controller is configured to determine a spatially resolved third intensity distribution of light of the red spectral region based on the second intensity distribution and an intensity distribution of light of the green spectral region. Fluorescent light detection system according to claim 29, wherein the controller is configured to To determine the spatially resolved intensity distribution of light of the infrared spectral range based on the first intensity distribution detected by the first camera and the third intensity distribution; and or To determine the spatially resolved intensity distribution of light of the red spectral region based on the first intensity distribution detected by the first camera and the third intensity distribution. Fluorescent light detection system according to one of Claims 24 to 30, wherein the following applies to the wavelength-dependent transmittance T (λ) of the first and / or second transmission spectrum within the red spectral range: T ₀ -ΔT ≦ T (λ) ≦ T ₀ + ΔT; 15% ≤ T ₀ ≤ 85%; 0 ≤ ΔT ≤ 5%. Fluorescent light detection system according to one of claims 24 to 30, wherein a first spectral range of the second transmission spectrum within the red spectral range comprises only wavelengths which are smaller than a cut-off wavelength λ ₀ , and an average transmittance T ₁ and wherein a second spectral range of the second transmission spectrum within the red spectral range comprises exclusively wavelengths which greater than the cut-off wavelength λ ₀ , and an average transmittance T ₂ where: T ₁ ≥ 70%, preferably T ₁ ≥ 80% or T ₁ ≥ 90%; and T ₂ ≤ 30%, preferred T ₂ ≤ 20%, or T ₂ ≤ 10%. Fluorescent light detection system according to claim 32, wherein: 600 nm ≤ λ ₀ ≤ 635 nm or 640 nm ≦ λ ₀ ≦ 680 nm. Fluorescent light detection system according to one of claims 1 to 33, wherein the blue spectral range is between 350 nm and 480 nm and / or wherein the green spectral range is between 480 nm and 580 nm and / or wherein the red spectral range is between 580 nm and 750 nm, in particular between 580 nm and 680 nm or between 630 nm and 680 nm and / or wherein the infrared spectral range between 750 nm and 1000 nm. Microscopy system comprising: at least one light source configured to generate illumination light and excitation light to excite fluorescent dyes and direct it to an object area, an objective system and a fluorescent light detection system according to any one of claims 1 to 35, wherein the objective system and the fluorescent light detection system are arranged so that at least a section of the object area is imaged by the objective system and the fluorescent light detection system on the first and second camera. The microscopy system of claim 35, further comprising: a display system for displaying an image, wherein the image is generated based on intensity distributions provided by a controller of the fluorescent light detection system. The microscopy system of claim 35 or 36, further comprising: a detection filter disposed in the optical path between the object region and the fluorescent light detection system, which is configured to suppress at least a part of the excitation light.

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