Laser microscope for histological examination of a tissue sample, and histological examination method

Abstract

The invention relates to a laser microscope (20), in particular multiphoton microscope, for the histological examination of a tissue sample (14), comprising: a laser light source (27), laser optics (21), a measurement signal acquisition module (28) and a sample receiver (30) for receiving the tissue sample (14) and / or a sample holder for a tissue sample (14), wherein: the laser light source (27) is designed to produce laser light (L) having an excitation wavelength of between 950 nm and 1200 nm, in particular between 1000 nm and 1100 nm, more particularly between 1030 nm and 1070 nm, preferably 1050 nm; and the measurement signal acquisition module (28) is arranged and designed to detect fluorescent light (XF) reflected by the tissue sample (14) into the laser optics (21), in particular having a fluorescence wavelength of between 500 nm and 700 nm, preferably between 550 nm and 650 nm, and / or ultraviolet THG light (XT), in particular having a THG wavelength of between 300 nm and 400 nm, in particular between 330 nm and 370 nm, in particular between 340 nm and 360 nm, preferably 350 nm. The invention is characterized in that the sample holder (30) is arranged above the laser optics (21) in such a way that laser light (L) emitted by the laser optics (21) is incident on the tissue sample (14) in particular only through the sample receiver (30), and in particular the sample receiver (30) is freely accessible on a side remote from the laser optics (21).

Description

[0001] Laser microscope for histological examination of a tissue sample and histological examination procedure

[0002] The invention relates to a laser microscope, in particular a multiphoton microscope, for examining a tissue sample according to the preamble of patent claim 1. Furthermore, the invention relates to a method for the histological examination of a dye-enriched tissue sample, in particular using such a laser microscope. A laser microscope of the type mentioned above is described, for example, by Borah, BJ, Tseng, YC, Wang, KC, et al. in "Rapid digital pathology of H&E-stained fresh human brain specimens as an alternative to frozen biopsy," Communications Medicine 3, 77 (2023).

[0003] The surgical removal of cancerous tumors is a significant medical procedure, but it is often associated with challenges in accurately identifying tumor margins. A key dilemma is that excessive removal of tissue can cause unnecessary harm to the patient, while too conservative removal could necessitate subsequent surgery. To overcome this problem, a tissue sample is taken from the area of ​​the surgical margins during the tumor removal procedure. The tissue sample is transported to pathology, where it is processed into very thin frozen sections a few micrometers thick. Microscopic examination in pathology determines whether the tissue sample still contains tumor cells or is free of tumor cells. The latter is an indication that the tumor has been completely removed. During this time, the surgical team waits for feedback from pathology. In Fig.Figure 1 shows this well-known procedure, which takes an average of 40-60 minutes, during which the patient is anesthetized and the operating room and the surgical team are not available for other operations.

[0004] Borah et al. present an approach to reduce this time expenditure, specifically by replacing the frozen section step. Instead, a virtual or digital cross-sectional image of a tissue sample is created using laser microscopy and transmitted electronically to pathology.

[0005] Specifically, Borah et al. used a multiphoton microscope with a laser light source with a central wavelength of 1070 nm. The laser light is passed through a tissue sample stained with the dyes hematoxylin and eosin. The eosin dye is fluorescent, with the emitted fluorescence wavelength being between 550 and 650 nm. Although the hematoxylin dye does not fluoresce, it causes the generation of so-called third-order harmonics (THG). THG refers to a frequency tripling phenomenon. If a laser light with an excitation wavelength of 1000 nm is introduced into the tissue sample stained with hematoxylin, photons with three times the frequency, i.e., one-third of the excitation wavelength, namely 333 nm, are generated. Filters within the multiphoton microscope allow the excitation wavelength and the THG wavelength to be separated and detected separately.The measurement signals thus acquired are then processed to create a virtual cross-sectional image of the tissue sample.

[0006] In the multiphoton microscope according to Borah et al., the laser objective is arranged above a sample holder, so that laser light hits the tissue sample from above. This has several disadvantages. On the one hand, the design of the multiphoton microscope is bulky, and on the other hand, handling the tissue sample, which is usually arranged on a glass plate and covered with a thin coverslip, is difficult. Such a microscope is intended for use in an operating room or laboratory, where operating the device while standing is advantageous. Operation is made more difficult for the user if the sample cannot be placed on or in the device from above and an optical system is located above the sample insertion device, which is the case in the design described here. Such additional optical technology also increases the height of the device, assuming a comfortable working height for the user.In particular, there is a risk that focusing the laser lens could damage the coverslip, rendering the tissue sample unusable. Furthermore, covering the tissue sample with liquid is time-consuming and may be necessary to improve image quality and / or prevent the sample from drying out. Furthermore, to ensure good quality virtual cross-sectional images, it is advantageous if the surface of the tissue sample onto which the laser light strikes is as flat or even as possible. However, if the laser light strikes a tissue sample from above, this cannot be guaranteed, even when using a thin coverslip. When changing tissue samples to those of a different thickness, there is the additional problem that the laser lens must be refocused. This is time-consuming and increases the duration of the histological examination.

[0007] In their publication “Slide-free imaging of hematoxylin-eosin stained whole-mount tissues using combined third-harmonic generation and three-photon fluorescence microscopy,” J. Biophotonics, 2019, 12, Sun, CK, Kao, CT, Wei, ML, et al. propose using a chromium forsterite laser (Cr:F laser) that emits laser light with a central excitation wavelength of 1260 nm. The wavelength of the THG light is 420 nm. The laser light is introduced into a tissue sample from below via a sample holder. The laser objective is therefore positioned below the sample holder. This inverted multiphoton microscope setup improves tissue sample handling. However, the Cr:F laser used is not very robust against environmental influences and requires intensive maintenance, which increases the operating costs of the multiphoton microscope.

[0008] Against this background, the object of the invention is to provide a laser microscope, in particular a multiphoton microscope, for the histological examination of a tissue sample that is cost-effective and allows for easy handling of a tissue sample. In particular, the laser microscope should generate a virtual cross-sectional image that can be interpreted by experienced pathologists with minimal training. A further object of the invention is to provide a method for the histological examination of a tissue sample.

[0009] According to the invention, this object is achieved with regard to the laser microscope by the subject matter of patent claim 1 and with regard to the examination method by the subject matter of patent claim 15.

[0010] The invention is based on the idea of ​​providing a laser microscope, in particular a multiphoton microscope, for the histological examination of a tissue sample, comprising a laser light source, a laser objective, a measurement signal acquisition module, and a sample holder for receiving a tissue sample and / or for receiving a sample holder for a tissue sample. The laser light source is configured to generate laser light with an excitation wavelength between 950 nm and 1200 nm, in particular between 1000 nm and 1100 nm, in particular between 1030 nm and 1070 nm, preferably 1050 nm.The measurement signal acquisition module is arranged and configured to detect fluorescent light reflected back into the laser objective by the tissue sample, in particular with a fluorescence wavelength between 500 nm and 700 nm, preferably between 550 nm and 650 nm, and / or ultraviolet THG light, in particular with a THG wavelength between 300 nm and 400 nm, in particular between 330 nm and 370 nm, in particular between 340 nm and 360 nm, preferably 350 nm. According to the invention, the sample holder is arranged above the laser microscope such that laser light emitted by the laser objective strikes the tissue sample, in particular only through the sample holder. In particular, the sample holder can be freely accessible on a side opposite the laser objective.

[0011] A key aspect of the invention is the arrangement of the sample holder above the laser objective or the arrangement of the laser objective below the sample holder. In other words, the laser microscope according to the invention can be designed as an inverted laser microscope. The laser light, like the fluorescent light and / or the THG light, is thus guided through the sample holder. The advantage of this arrangement of the laser objective is that the handling of tissue samples is significantly easier. In particular, a tissue sample can be inserted into the sample holder directly or from above using a sample holder. Damage to the tissue sample by the laser objective is also virtually impossible, particularly because access to the sample holder is possible without being obstructed by the laser objective.

[0012] Arranging the sample holder above the laser objective has the further advantage that the tissue sample lies flat on the sample holder or a sample holder arranged in the sample holder due to gravity, thus creating the conditions for good cross-sectional imaging. In particular, the focus of the laser objective can be maintained even when changing the tissue sample, as the distance between a slice of the tissue sample to be scanned and the laser objective remains constant. The invention has further advantages. For example, the laser light emitted by the laser light source, with a central wavelength between 950 nm and 1200 nm, protects the tissue sample. In particular, the likelihood of photodamage to the tissue sample is reduced at such wavelengths, which are in the infrared range.The laser light with the excitation wavelength preferred according to the invention can also be generated using inexpensive and robust laser light sources, so that the manufacturing and operating costs of the laser microscope are low. Further cost savings can be achieved by using a commercially available light microscope objective as the laser objective, which is preferred in this respect.

[0013] In a preferred embodiment of the laser microscope according to the invention, the measurement signal acquisition module has at least one detection channel for detecting the THG wavelength and at least one further detection channel for detecting a wavelength in the range from 400 nm to 800 nm. The further detection channel can be configurable or configured in particular for detecting a counterstain, in particular eosin, and / or an immunostain and / or a molecular pathological stain. At least two, in particular at least three, further detection channels can also be provided, wherein a first further detection channel is configured for detecting a counterstain, in particular eosin, a second further detection channel is configured for detecting an immunostain and / or a third further detection channel is configured for detecting a molecular pathological stain.In this way, not only can the cell nuclei visible with hematoxylin and the emitted GHG light be identified in the same tissue sample, but also, for example, a counterstain can be applied to facilitate orientation within the tissue sample. Furthermore, the laser microscope can also be used to examine other stains of unsectioned tissue samples, particularly immunostains or molecular pathological stains. This significantly expands the potential uses of the laser microscope.

[0014] In this respect, it is preferred if the measurement signal acquisition module has an optical spectral filter for spectral separation, which is configured to effect spectral separation of the wavelength detected by the further detection channel in a range between 520 nm and 590 nm, in particular between 540 nm and 580 nm, in particular between 570 nm and 580 nm, preferably at 575 nm. Overall, the measurement signal acquisition module can have one or more measurement sensors and one or more optical spectral filters, each assigned to a detection channel. Each detection channel can comprise a corresponding optical spectral filter that separates corresponding wavelengths for each detection channel from the spectrum of the light reflected back into the laser lens. Furthermore, each detection channel can comprise an independent measurement sensor.

[0015] The optical spectral filter can significantly expand the functionality of the laser microscope. In particular, the separation of wavelengths in the range between 520 nm and 590 nm makes it possible to analyze tissue samples that have a different staining than hematoxylin and eosin (H&E staining). An alternative to H&E staining is, for example, staining the cell nuclei with acridine orange and counterstaining with sulforhodamine 101. The additional optical spectral filter also allows the laser microscope to be used for this type of staining. In other words, the spectral filter enables the laser microscope to be used for differently stained tissue samples. Consequently, a buyer of a laser microscope does not have to commit to a specific staining method.

[0016] At least one of the measurement sensors can be designed, for example, as a photomultiplier. In particular, the measurement signal acquisition module preferably has a photomultiplier that is designed and / or configured to detect a fluorescence wavelength of a dye contained in the tissue sample, in particular eosin. Furthermore, a further photomultiplier can be provided that is designed and / or configured to detect the THG wavelength. Furthermore, the measurement signal acquisition module can have at least one analog-to-digital converter channel, in particular a separate one for each detection channel, each of which is coupled to a measurement sensor, in particular to a photomultiplier. The analog signals generated in the measurement sensor are thus converted into digital data and can then be digitally post-processed or further processed by means of data processing.

[0017] The laser lens and / or optical components of the measurement signal acquisition module, in particular optical spectral filters, are preferably permeable to ultraviolet light. In this context, the ultraviolet light preferably has a wavelength between 300 nm and 400 nm, in particular between 350 nm and 360 nm, preferably 355 nm. Particularly preferably, the optical components of the measurement signal acquisition module are opaque to infrared light above 950 nm in order to protect the measurement sensors.

[0018] The preferred laser light source is a fiber laser comprising an ytterbium-doped core fiber. An ytterbium-doped fiber laser is efficient and reliably achieves the desired central excitation wavelength of approximately 1000 nm to 1100 nm, particularly 1050 nm. Compared to other laser light source designs, a fiber laser, especially one with an ytterbium-doped core fiber, has low manufacturing and operating costs and is characterized by high robustness and low maintenance requirements.

[0019] In the context of this application, specifications regarding wavelengths generally refer to the respective central wavelength or central wavelength. Laser light sources can generate comparatively broadband laser light, especially in pulsed operation. The central wavelength or central wavelength refers to the midpoint of the spectral bandwidth of the respective light, in particular laser light.

[0020] Furthermore, in the laser microscope, it can be provided that the laser light source, in particular the fiber laser, is configured for pulsed operation, wherein a laser pulse rate of at most 60 MHz, in particular between 10 MHz and 40 MHz, in particular between 15 MHz and 50 MHz, in particular between 15 MHz and 30 MHz, preferably 20 MHz, is adjustable or set.

[0021] The relatively low laser pulse rate has the advantage of keeping the average power on the tissue sample comparatively low. This offers a good compromise between scanning speed and protecting the tissue sample. Alternatively, a higher signal can be generated per laser pulse at a constant average power, which advantageously leads to improved signal strength. This eliminates the need for amplifying electronic components, such as a transimpedance amplifier, which disadvantageously contributes to increased image noise.

[0022] In a preferred embodiment of the invention, the measurement signal acquisition module can be designed and / or configured to acquire at least 2, in particular at least 3, in particular at least 5, in particular at least 10, in particular at least 20, in particular at least 50, preferably at least 100, data points per laser pulse. The measurement signal acquisition module, which has one or more photomultipliers or photomultiplier tubes (PMTs), converts the light emitted by the dye into an analog voltage signal. This is then converted by at least one analog-to-digital converter channel of the measurement signal acquisition module into a digital value or data point, from which the displayed image is calculated over several steps. Preferably, 140 data points are acquired per laser pulse. This requires a comparatively powerful and correspondingly expensive electronic data acquisition card.The advantage, however, is that further data processing can result in improved quality of the cross-sectional image.

[0023] In general, it is advantageous if the laser microscope has an image processing module for processing the measurement data acquired by the measurement signal acquisition module, preferably digitized, and for generating a virtual or digital cross-sectional image of the tissue sample. With the image processing module, the data obtained via the measurement signal acquisition module can, for example, be processed so that they resemble a classic, i.e., manually generated tissue section with hematoxylin and eosin staining (so-called H&E tissue section) or an immunohistological staining. This makes it easier, especially for experienced pathologists, to evaluate the tissue sample quickly and reliably and contributes to rapid diagnosis and reporting during histological examination.

[0024] For the purposes of the application, a cross-sectional image is defined as a data compilation that can be graphically displayed on a screen or display as a virtual section through the tissue sample. The cross-sectional image can also be a file on a data storage medium, provided the file contains all the necessary data to enable the graphical representation of the virtual section through the tissue sample.

[0025] The image processing module can be adapted to generate a three-dimensional representation from multiple digital cross-sectional images. For example, a digital or virtual three-dimensional model of the tissue sample can be created and displayed on a screen. Such a three-dimensional model can accelerate diagnosis and lead to more accurate results.

[0026] The laser microscope preferably also includes a display for showing the image generated by the image processing module. Such a display can be, for example, a screen or monitor, preferably a TFT screen. Using the display, the surgical team can roughly evaluate whether a sufficiently good digital cross-sectional image has been generated to enable pathological evaluation or diagnosis. This can avoid time loss that could otherwise occur due to transmitting a digital cross-sectional image unsuitable for diagnosis to pathology, resulting in a repetition of the histological examination.

[0027] In a preferred embodiment of the invention, the laser objective is linearly movable to focus on different layers of the tissue sample such that a distance between the sample holder and the laser objective is variable. The laser objective is preferably coupled to a controller programmed to automatically focus the laser objective on different layers of the tissue sample during a single image acquisition process. The laser microscope can, in particular, have an acquisition mode in which multiple image planes, particularly in the direction of laser light emission from the laser objective (Z direction), can be acquired in one image without the user having to intervene in the meantime and / or without control commands having to be sent from outside the laser microscope.

[0028] The laser objective of the laser microscope preferably has a numerical aperture of 0.9 or less, in particular between 0.6 and 0.8, preferably 0.7. Such a low numerical aperture has the advantage that no immersion medium is required for the tissue sample to ensure good resolution of the acquired cross-sectional image. This avoids an additional step for preparing the tissue sample, resulting in overall time savings during histological examination.

[0029] For objectives with a higher numerical aperture, corrections are necessary to avoid a loss in resolution. Such corrections are usually only available for cover glasses for covering a tissue sample, which have a standard thickness of 170 μm. However, such a thin cover glass can bend easily when the tissue sample is flattened, leading to insufficient flattening of the tissue sample. In particular, there is a risk that the cover glass will break and injure the user. The lower numerical aperture of a maximum of 0.9 preferably provided in the laser microscope according to the invention, on the other hand, enables correction for sample holders or sample holders with a laser-transparent plate that has a thickness of more than 170 μm. This way, good resolution is achieved even when using stable sample holders or sample holders.

[0030] In this respect, it is preferably provided that the sample holder for directly receiving a tissue sample has a laser-transparent plate which has a thickness of at least 0.25 mm, in particular between 0.4 mm and 2 mm, in particular between 0.5 and 1.8 mm, in particular between 0.6 mm and 1.2 mm, preferably 1 mm. Such a sample holder is particularly stable and therefore requires less attention when handling the laser microscope. In particular, the risk of injury to the user is reduced. In conjunction with the preferred embodiment of an inverted laser microscope, in which the specimen carrier is arranged between the tissue sample and the laser objective, which also receives the light reflected back from the tissue sample, actively flattening the tissue sample is also not absolutely necessary.Rather, gravity ensures that the tissue sample lies flat on the laser-transparent plate, allowing good cross-sectional images to be captured.

[0031] It is also possible to provide a set with a previously described laser microscope which additionally comprises a sample holder, wherein the sample holder has a laser-transparent plate with a thickness of at least 0.25 mm, in particular between 0.4 mm and 2 mm, in particular between 0.5 and 1.8 mm, in particular between 0.6 mm and 1.2 mm, preferably 1 mm. In this case, the sample holder serves merely to hold the sample holder. The sample holder can have a through-opening, wherein the sample holder is arranged and in particular fastened on the sample holder in such a way that the laser-transparent plate of the sample holder spans the through-opening. The laser light can thus strike the laser-transparent plate of the sample holder directly through the through-opening.Alternatively, the sample holder and the sample holder can each comprise a laser-transparent plate, wherein the sum of the thickness of the laser-transparent plate of the sample holder and the thickness of the laser-transparent plate of the sample holder is preferably more than 170 μm, in particular at least 0.25 mm, in particular between 0.4 mm and 2 mm, in particular between 0.5 and 1.8 mm, in particular between 0.6 mm and 1.2 mm, preferably 1 mm.

[0032] The invention further relates to a method for the histological examination of a dye-enriched tissue sample using a laser microscope, preferably a previously described laser microscope. The method according to the invention preferably comprises the following steps:

[0033] Providing a tissue sample stained with hematoxylin and eosin on a sample holder,

[0034] Scanning the tissue sample using a laser light with an excitation wavelength between 1050 nm and 1070 nm, so that the eosin is excited to emit fluorescent light with a fluorescence wavelength, whereby THG light with a THG wavelength is generated by the hematoxylin,

[0035] Separating the fluorescence wavelength and the THG wavelength using an optical filter,

[0036] Detecting the fluorescence wavelength and the THG wavelength by a measurement signal acquisition module, in particular a photomultiplier, and

[0037] Generating a virtual cross-sectional image, in particular an H&E cross-sectional image, of the scanned tissue sample using an image processing module.

[0038] The advantages and preferred embodiments mentioned in connection with the laser microscope explained above also apply analogously to the method described here.

[0039] The virtual cross-sectional image preferably mimics the appearance of a frozen and / or paraffin section stained with hematoxylin, eosin, and / or an immunohistological stain. This makes it easier for experienced pathologists, in particular, to evaluate the virtual cross-sectional image, as they can draw on their previous knowledge acquired from conventional frozen and / or paraffin sections. In this respect, the assessment of the virtual cross-sectional image is no different from that of conventional frozen and / or paraffin sections.

[0040] The particular advantage of the method according to the invention is that the histological examination of a tissue sample can be carried out extremely quickly and reliably.

[0041] A preferred application is the examination of a dye-enriched tissue sample to identify tumor cells. During surgery to remove a tumor, it is often necessary to obtain tissue samples from the surgical margins to determine whether the tumor has been completely removed. The tissue samples are then sent to a pathology laboratory during the surgical procedure for manual examination. During the pathological or histological examination, waiting time occurs in the operating room that remains unused. Depending on local conditions and the workload of the respective pathology laboratory, the waiting time during the surgical procedure can be between 40 and 60 minutes.However, with the methods according to the invention, this waiting time can be reduced to less than 15 minutes, since the use of the laser microscope allows a virtual cross-sectional image to be created in a short period of time, which can be digitally transmitted to a pathologist for evaluation. This results in significant time savings and a reduction in waiting times during tumor surgery.

[0042] In a preferred development of the method according to the invention, the virtual cross-sectional image is color-coded, with a first color representing cell nuclei identified by the detection of the THG light, and / or at least one further color representing a counterstain and / or immunostain and / or molecular pathological stain. The first color is preferably purple or blue. The further color can be pink or red, in particular, to represent a counterstain. To represent the immunostain, brown or red is preferably provided as a further color. The aforementioned color codings correspond to the colors used in conventional tissue sections for tumor cell detection or histological examination of tissue sections. These are familiar to experienced pathologists, so that the interpretation of virtual cross-sectional images can be carried out without special training.

[0043] Furthermore, the laser light during scanning of the tissue sample, as well as the fluorescent light and the THG light, can be guided through the sample holder. In particular, it is provided that the fluorescent light and the THG light are captured via a laser lens, with the sample holder located between the laser lens and the tissue sample. The capture of the fluorescent light and the THG light thus occurs in the reverse direction, in particular not in transmission.

[0044] The fluorescence wavelength can be between 500 nm and 700 nm, in particular between 550 nm and 650 nm, and / or the THG wavelength can be one third of the excitation wavelength.

[0045] The invention is explained in more detail below using an exemplary embodiment with reference to the attached schematic drawings. In the drawings:

[0046] Fig. 1 is a process diagram of a process for the histological examination of a tissue sample according to the prior art;

[0047] Fig. 2 is a process diagram of a method according to the invention for the histological examination of a tissue sample; and

[0048] Fig. 3 shows an exemplary structure of a laser microscope according to the invention.

[0049] Fig. 1 shows the previously common procedure for pathological diagnosis during tumor surgery. During the tumor surgery, the surgical team 10 takes a tissue sample 14, particularly from the area of ​​the tumor edges. The tissue sample 14 is then transported to pathology 11. In pathology 11, frozen sections 15 are created from the tissue sample 14, which may usually only be a few micrometers thick. The frozen sections 15 are placed on a sample holder 16 under a light microscope 12 and evaluated by a pathologist 13. In particular, it is determined or diagnosed whether the frozen section 15 still contains tumor cells. If tumor cells are detected in the frozen section 15, the surgical team 10 receives feedback that tumor tissue is still present in the patient. The surgical team 10 can then decide whether further tissue removal is necessary and responsible.If further tissue is removed, another tissue sample 14 is taken from the area of ​​the cut edge and transported back to Pathology 1 1, where the additional tissue sample is examined again.

[0050] Fig. 1 illustrates that the entire conventional or classic procedure for histological examination of a tissue sample during tumor surgery is very time-consuming. It usually takes more than 40 minutes for the surgical team 10 to be informed of the results of the pathological evaluation. During this time, the tumor surgery is halted.

[0051] Fig. 2 shows the process flow of an exemplary examination method of the invention. During a tumor operation, a surgical team 10 takes a tissue sample 14. The tissue sample 14 is placed, preferably still in or at least near the operating room, into the laser microscope 20 according to the invention, in particular the sample holder 30 or a sample holder 16 insertable into the sample holder 30. The laser microscope 20 scans the tissue sample 14 in one or more tissue layers and creates one or more virtual cross-sectional images. The virtual cross-sectional images are transmitted electronically via a communications network 17 to a pathologist 13, who makes the diagnosis. The process flow according to Fig. 3 takes an average of less than 15 minutes, thus saving valuable operating room time.

[0052] Fig. 3 shows an exemplary structure of a laser microscope 20 according to the invention. The laser microscope 20 comprises a laser objective 21 which passes through a housing 29 of an optical unit of the laser microscope 20. The laser objective 21 is directed from below onto a sample holder 30, on which a tissue sample 14 can be arranged. The tissue sample 14 can be placed directly onto a laser-transparent plate of the sample holder 30. In this case, the sample holder 30 comprises a laser-transparent object glass 31 which is transparent in particular to ultraviolet and / or infrared light. However, it is preferred if the tissue sample 14 is placed in or on a sample holder 16, for example a glass plate, and the sample holder 16 is then placed on or in the sample holder 30. In this case, the sample holder 30 can have an opening orhave a through-opening. In general, the object glass 31 is preferably transparent to ultraviolet and / or infrared light.

[0053] Advantageously, the specimen glass 31 and / or the laser-transparent plate of the specimen holder 16 is transparent to light with a wavelength between 300 nm and 1100 nm, in particular between 340 nm and 360 nm and / or between 500 nm and 700 nm and between 1000 nm and 1200 nm. The specimen glass 31 is surrounded by a frame 32, which defines a receiving space 33 for receiving the tissue sample 14.

[0054] Below the laser lens 21 and within the housing 29, an arrangement of optical filters 22, lens assemblies 23a, 23b, beam deflection units 24 and deflection mirrors 25 and / or shutter diaphragms 26 is provided, which define a light path for the laser light L. The laser light L originates from a laser light source 27, in particular a Yb-doped fiber laser with a central excitation wavelength of 1050 nm.

[0055] The laser light source 27 generates laser light L, which reaches a deflection mirror 25, which deflects the laser light L from a horizontal direction to a vertical direction. It is also conceivable to dispense with the deflection mirror 25, particularly if the laser light source 27 is configured such that the laser light L is directed toward the laser lens 21, particularly upwards.

[0056] From the deflection mirror 25, the laser light L reaches a first lens arrangement 23a, which is preferably designed as a telescope. A telescope is an arrangement of at least two lenses that cause the laser light beam to expand, shrink, or focus.

[0057] The expanded or focused laser light beam is then directed onto a beam deflection unit 24, in particular one or more scanning mirrors. The beam deflection unit 24 preferably has a galvanometric drive or a galvanometer, with which the angular orientation of the scanning mirror can be changed in order to move the laser light L across the tissue sample 14 in a scanning manner. Furthermore, a resonant scanner can be provided, or one of the scanning mirrors can be designed as such. The resonant scanner comprises a mirror and several magnets, which are jointly arranged on a torsion spring, which is specifically set into oscillation by the magnets.

[0058] From the beam deflection unit 24, the laser light L passes to a second lens arrangement 23b. The second lens arrangement 23b serves to correctly direct the laser light L onto the laser lens 21 during the scanning movement, which is adjusted by the beam deflection unit 24, in particular to expand the laser light L.

[0059] Between the second lens arrangement 23b and the laser objective 21, the laser light L passes through an optical filter 22, which filters out unwanted wavelength components and, in particular, redirects the fluorescent light XF and / or higher harmonic order light, particularly second harmonic order (S HG light) and / or third harmonic order (THG light XT), reflected from the specimen toward the measurement signal acquisition module 28. The optical filter 22 thus forms a beam splitter. The laser light L originating from the laser light source 27 passes through the optical filter 22 largely unchanged.

[0060] As can be clearly seen in Fig. 3, the laser light L passes through the laser lens 26 from below to the tissue sample 14 (not shown here), which is located on the sample holder 12. In this respect, the laser microscope shown is an inverted multiphoton microscope.

[0061] The laser light L emitted by the laser objective is deflected by the beam deflection unit 24 such that individual tissue layers of the tissue sample 14 are scanned line by line. This leads to the excitation of the dye contained in the tissue sample 14, in particular eosin, thereby generating the fluorescent light XF. Furthermore, a THG light XT is generated by means of the hematoxylin contained in the tissue sample 14. The fluorescent light XF and the THG light XT are captured in the reverse direction by the laser objective 21. Since the fluorescent light XF has a fluorescence wavelength and the THG light XT has a THG wavelength that differ from the excitation wavelength of the laser light L, the fluorescent light XF and the THG light XT can be effectively separated via the optical filter 22 and fed to a measurement signal acquisition module 28.The measurement signal acquisition module 28 can have optical spectral filters that separate the fluorescent light XF from the THG light XT and feed them to different measurement sensors. The spectral filters can be designed similarly to the optical filter 22. The measurement sensors each generate analog voltage signals, which are converted into digital signals in an analog-to-digital converter channel of the measurement signal acquisition module 28. An image processing module then creates a virtual cross-sectional image of the tissue sample 14 from these digital signals.

[0062] List of reference symbols

[0063] 10 surgical team

[0064] 1 1 Pathology

[0065] 12 Microscope

[0066] 13 Pathologist

[0067] 14 Tissue sample

[0068] 15 frozen sections

[0069] 16 sample holders

[0070] 17 Communication network

[0071] 20 laser microscope

[0072] 21 Laser lens

[0073] 22 optical filters

[0074] 23a first lens arrangement

[0075] 23b second lens arrangement

[0076] 24 beam deflection unit

[0077] 25 deflecting mirrors

[0078] 26 Shutter

[0079] 27 Laser light source

[0080] 28 Measurement signal acquisition module

[0081] 29 housings

[0082] 30 Sample collection

[0083] 31 Object glass

[0084] 32 frames

[0085] 33 Recording Room

[0086] L Laser light

[0087] XF fluorescent light

[0088] XT THG light

Claims

Patent claims 1. Laser microscope (20), in particular a multiphoton microscope, for the histological examination of a tissue sample (14) with a laser light source (27), a laser objective (21), a measurement signal acquisition module (28) and a sample holder (30) for receiving a tissue sample (14) and / or a sample holder for a tissue sample (14), wherein the laser light source (27) is configured to generate laser light L with an excitation wavelength between 950 nm and 1200 nm, in particular between 1000 nm and 1100 nm, in particular between 1030 nm and 1070 nm, preferably 1050 nm, and the measurement signal acquisition module (28) is arranged and configured to detect fluorescent light (XF) reflected back from the tissue sample (14) into the laser objective (21), in particular with a fluorescence wavelength between 500 nm and 700 nm, preferably between 550 nm and 650 nm, and / or ultraviolet GHG light (XT), in particular with a GHG wavelength between 300 nm and 400 nm,in particular between 330 nm and 370 nm, in particular between 340 nm and 360 nm, preferably 350 nm, characterized in that the sample holder (30) is arranged above the laser objective (21) in such a way that a laser light (L) emitted by the laser objective (21) strikes the tissue sample (14), in particular only through the sample holder (30), in particular wherein the sample holder (30) is freely accessible on a side opposite the laser objective (21).

2. Laser microscope (20) according to at least the preamble of claim 1, characterized in that the measurement signal acquisition module (28) has at least one detection channel for detecting the THG wavelength and at least one further detection channel for detecting a wavelength in the range from 400 nm to 800 nm, wherein the further detection channel is configurable or configured for detecting a counterstain, in particular eosin, and / or an immunostain and / or a molecular pathological stain.

3. Laser microscope (20) according to claim 1 or 2, characterized in that the measurement signal detection module (28) has an optical spectral filter for spectral separation, which is configured to effect a spectral separation of the wavelength detected with a further detection channel, in particular the at least one further detection channel according to claim 2, in a range between 520 nm and 590 nm, in particular between 540 nm and 580 nm, in particular between 570 nm and 580 nm, preferably at 575 nm.

4. Laser microscope (20) according to one of the preceding claims, characterized in that the laser light source (27) comprises a fiber laser, in particular with an ytterbium-doped core fiber.

5. Laser microscope (20) according to one of the preceding claims, characterized in that the laser light source (27) is configured for pulsed operation, wherein a laser pulse rate of at most 60 MHz, in particular between 10 MHz and 50 MHz, in particular between 10 MHz and 40 MHz, in particular between 15 MHz and 30 MHz, preferably 20 MHz, is adjustable or set.

6. Laser microscope (20) according to one of the preceding claims, characterized in that the measurement signal acquisition module (28) is designed and / or configured to acquire at least 2, in particular at least 3, in particular at least 5, in particular at least 10, in particular at least 20, in particular at least 50, preferably at least 100, data points per laser pulse.

7. Laser microscope (20) according to one of the preceding claims, characterized in that an image processing module is provided for processing the measurement signals acquired by the measurement signal acquisition module (28) and for generating a virtual sectional image of the tissue sample (14).

8. Laser microscope (20) according to claim 7, characterized in that the image processing module is configured to display the THG wavelength detected by the measurement signal detection module (28), in particular by the first detection channel, which indicates cell nuclei, in the virtual sectional image in a first color, in particular in purple or blue.

9. Laser microscope (20) according to claim 7 or 8, characterized in that the image processing module is configured to display the counterstaining and / or immunostaining and / or molecular pathological staining detected by the measurement signal detection module (28), in particular by the further detection channel, in the virtual sectional image in a second color, in particular the counterstaining pink or red and / or the immunostaining in brown and / or red.

10. Laser microscope (20) according to one of claims 7 to 9, characterized in that a display is provided for displaying the virtual sectional image generated by the image processing module. 1 1. Laser microscope (20) according to one of the preceding claims, characterized in that the laser objective (21) is linearly movable for focusing on different layers of the tissue sample (14) such that a distance between the sample holder (30) and the laser objective (21) is variable, wherein the laser objective (21) is coupled to a controller which is programmed to automatically focus the laser objective (21) on different layers of the tissue sample (14) during a single image recording process.

12. Laser microscope (20) according to one of the preceding claims, characterized in that the laser objective (21) has a numerical aperture of at most 0.9, in particular between 0.6 and 0.8, preferably 0.

7.

13. Laser microscope (20) according to one of the preceding claims, characterized in that the sample holder (30) for directly receiving a tissue sample (14) has a laser-transparent plate which has a thickness of at least 0.25 mm, in particular between 0.4 mm and 2 mm, in particular between 0.5 and 1.8 mm, in particular between 0.6 mm and 1.2 mm, preferably 1 mm.

14. Set with a laser microscope (20) according to one of the preceding claims and a sample holder (16), wherein the sample holder (16) has a laser-transparent plate with a thickness of at least 0.25 mm, in particular between 0.4 mm and 2 mm, in particular between 0.5 and 1.8 mm, in particular between 0.6 mm and 1.2 mm, preferably 1 mm.

15. A method for the histological examination of a dye-enriched tissue sample (14) with a laser microscope (20), in particular according to one of the preceding claims, wherein the method comprises the following steps: Providing a tissue sample (14) stained with hematoxylin and eosin on a sample holder (30), Scanning the tissue sample (14) by means of a laser light (L) with an excitation wavelength between 1030 nm and 1070 nm, so that the eosin is excited to emit fluorescent light (X) with a fluorescence wavelength, wherein the hematoxylin enhances the generation of THG light (XT) with a THG wavelength, Separating the fluorescence wavelength and the THG wavelength by an optical spectral filter of a measurement signal acquisition module (28), Detecting the fluorescence wavelength and the THG wavelength by a measuring sensor, in particular a photomultiplier, of the measuring signal detection module (28) and Generating a virtual cross-sectional image of the scanned tissue sample (14) by means of an image processing module.

16. The method according to claim 15, characterized in that the virtual sectional image is color-coded, wherein a first color, in particular purple or blue, represents cell nuclei determined by the detection of the THG light (XT) and / or at least one further color represents a counterstain and / or immunostain and / or molecular pathological stain, in particular wherein the counterstain is shown in pink or red and / or the immunostain is shown in brown and / or red.

17. Method according to claim 15 or 16, characterized in that the laser light (L) when scanning the tissue sample (14) as well as the fluorescent light (XF) and the THG light (XT) are guided through the sample holder (30).

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