Microscopy system and method for operating a microscopy system

A microscopy system for simultaneously observing fluorescent and non-fluorescent regions of an object, and a method for operating the microscopy system are provided. The microscopy system includes a microscopy optical unit configured to image an object plane through an observation beam path onto an image plane, an observation filter arrangeable in the observation beam path, two light sources, one being provided for exciting a fluorescent dye in the object and another being provided for visualizing non-fluorescent regions of the object, and a controller to control the light sources individually. With a suitable configuration of the observation filter, all light sources can be operated with a minimum operating current, which ensures the stability of the light. The color rendering of the non-fluorescent regions can be set by the individual settability of the light sources which can be set such that fluorescent and non-fluorescent regions appear to be approximately equally bright.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to German patent application DE 10 2019 101 773.4, filed Jan. 24, 2019, the entire content of which is incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to a microscopy system and to a method for operating a microscopy system. In particular, the microscopy system and the method serve for simultaneously observing fluorescent and non-fluorescent regions of an object admixed with a fluorescent dye, for example protoporphyrin IX (PpIX) or fluorescein.

BACKGROUND

Microscopy systems for simultaneously or sequentially observing fluorescent and non-fluorescent regions of an object are used for example in the field of tumour surgery. A fluorescent dye is added to a tumorous tissue and binds to the tumorous tissue. However, the fluorescent dye does not bind to non-tumorous tissues. After the fluorescent dye has been excited, the tumorous tissue can be localized by recording an image of the tissue by fluorescent light and identifying the fluorescent regions therein.

In order that the tumorous tissue can be removed from the healthy tissue surrounding the tumorous tissue, the tumorous tissue has to be identified and localized relative to its surroundings. It is therefore necessary also to capture the surroundings, that is to say non-fluorescent regions of the object.

In order to achieve this, in conventional microscopy systems for simultaneously observing fluorescent and non-fluorescent regions of an object, use if made of a white light source having a spectrally approximately homogenous intensity in the visible wavelength range, for example a xenon lamp. The setting of the wavelength-dependent intensity of illumination light that is fed to the object is usually set by way of one or more illumination filters. The light emanating from the object and including both fluorescent light and light reflected at non-fluorescent regions is usually filtered by an observation filter and fed to an observer.

Since the ratio between the intensity of the fluorescent light and the intensity of the light used for exciting the fluorescent dye is very small, the fluorescent light and light reflected at non-fluorescent regions of the object are perceived as having greatly different brightnesses, which makes it more difficult to localize the fluorescent regions and thus the tumorous tissue. Moreover, the intensity of the fluorescent light that emanates from low grade tumors, in comparison with the intensity of the fluorescent light that emanates from high grade tumors, is smaller by a factor of up to 20, which additionally hampers the localization.

Moreover, the color rendering of non-fluorescent regions is often not true color, but rather characterized by a dominant color, with the result that the non-fluorescent region is presented to the surgeon in an unnatural color, which hampers the orientation in the non-fluorescent region. This rendering that is not true color is in part also caused by the fact that better profiles of the wavelength-dependent transmittances of the filters are technically not realizable, for example are not realizable as interference filters.

SUMMARY

It is an object of the present disclosure to provide a microscopy system, a method for operating a microscopy system, and a microscopy method which solves the problems mentioned above.

First Aspect

In accordance with a first aspect of the disclosure, a microscopy system for simultaneously observing fluorescent and non-fluorescent regions of an object includes a microscopy optical unit configured to image an object plane through an observation beam path onto an image plane, an observation filter arrangeable in the observation beam path, at least two (narrowband) light sources, and a controller configured to control one or a plurality of the at least two light sources individually, with the result that the ratio of the intensities of the light generated by the at least two light sources is variable.

An image detector can be arranged in the image plane of the microscopy optical unit in order thus to record an image of an object arranged in the object plane.

Of the at least two light sources, (at least) a first light source is configured to generate first light and to direct it onto the object region, wherein the first light is suitable for exciting a fluorescent dye present in the object. For this purpose, the first light includes light having wavelengths in the absorption range of the fluorescent dye. For PpIX, the first light can include wavelengths in the range of 380 nm to 420 nm.

Of the at least two light sources, (at least) a second light source is configured to generate second light and to direct it onto the object region, wherein the second light serves for visualizing non-fluorescent regions of the object. For this purpose, the second light includes light having wavelengths outside the absorption and emission ranges of the fluorescent dye.

One or a plurality of the light sources can be narrowband. Narrowband light sources generate light in accordance with a spectral intensity distribution, wherein the spectral intensity distribution has a maximum spectral emission intensity at a central wavelength and wherein the difference between an upper limiting wavelength, at which the spectral emission intensity is 1% of the maximum spectral emission intensity and which is larger than the central wavelength, and a lower limiting wavelength, at which the spectral emission intensity is 1% of the maximum spectral emission intensity and which is less than the central wavelength, is at most 150 nm, in particular at most 100 nm, more particularly at most 50 nm or at most 20 nm or at most 10 nm.

One or a plurality of the light sources can be controlled individually by the controller. That is to say that a light source can be controlled independently of the other light sources. The intensity of the light generated by the individually controllable light sources can thus be set individually. The ratio of the intensities of light generated by different light sources can thus be varied. By way of example, the controller is configured to vary the operating current and/or the operating voltage of the individually controllable light sources in order thereby to vary the intensity of the light generated by said light sources. The light sources are light emitting diodes, for example.

The (wavelength-dependent) transmittance of the observation filter is defined—as usual—as the ratio of the intensity of light of one wavelength that is transmitted by the observation filter to the intensity of light of the same wavelength that is directed onto the observation filter.

The transmittance of the observation filter is embodied such that it blocks (does not transmit) the light used for exciting the fluorescent dye, that it transmits to a predetermined degree the light used for visualizing non-fluorescent regions and that it transmits the fluorescent light in the best possible manner.

The following advantages are afforded by the individual settability of the light sources and thus of the spectral intensity components of the light directed onto the object and the suitable choice of the transmittance in the wavelength ranges used for visualizing non-fluorescent regions:

Firstly, the fluorescent regions and the non-fluorescent regions can be perceived as almost equally bright by virtue of the intensity of the light generated by the individual light sources being set accordingly. The first light source can be used to set the intensity of the fluorescent light emanating from the fluorescent regions (substantially independently of the second light source). The second light source can be used to set the intensity of the light reflected from the object at non-fluorescent regions (substantially independently of the first light source). The intensities of the light emanating from fluorescent and non-fluorescent regions can thus be coordinated with one another, with the result that both regions appear to be approximately equally bright.

Furthermore, all light sources can be operated in their emission-stable working ranges. The emission spectrum of light sources is generally not stable at very small operating currents and/or voltages. That is to say that the emission spectrum changes as the operating current changes. In order to avoid this, all light sources can be operated in their emission-stable working ranges, for example by all light sources being operated with at least 0.1% of their respective maximum power consumption. In order that the intensity of the light which is used for visualizing non-fluorescent regions and which is operated by a light source with at least 0.1% of its maximum power consumption is approximately equal to the intensity of the fluorescent light (i.e., fluorescent and non-fluorescent regions appear equally bright), the transmittance of the observation filter in the wavelength ranges used for visualizing non-fluorescent regions can be chosen to be correspondingly small. As a result, all light sources can be operated in their emission-stable working ranges.

Furthermore, an illumination filter is not required.

If at least two light sources and at least two separate passbands in the observation filter are used for visualizing non-fluorescent regions, a further advantage is afforded as a result: The color rendering of non-fluorescent regions can be set by the suitable choice of the intensities of the light generated by the light sources used for visualizing non-fluorescent regions and the transmittances of the passbands correspondingly provided for this purpose in the observation filter. In particular, the color rendering can be set to a true color rendering.

In the exemplary embodiments described below, the transmittance of the observation filter from λ1to λ2is less than W1, from λ3to λ4is larger than W2and from λ5to λ6is larger than W3, wherein λ1, λ2, λ3, λ4, λ5, and λ6are wavelengths for which 350 nm<λ1<λ2≤λ3<λ4≤λ5<λ6<750 nm holds true, and wherein W1, W2, and W3are values for which 0<W1<W2<W3<1 holds true; and a first light source is configured to generate first light having wavelengths of between λ1and λ2, and a second light source is configured to generate second light having wavelengths of between λ3and λ4.

For the simultaneous observation of fluorescent and non-fluorescent regions of an object admixed with PpIX, the observation filter and the light sources can be configured as follows:

First Exemplary Embodiment

For observing the non-fluorescent regions in the blue wavelength range, as is customary and desired in specific applications, the following settings furthermore hold true: 480 nm<λ4<520 nm, and/or wherein the transmittance of the observation filter from λ3to λ4is larger than 10% and less than 60%, and/or wherein the transmittance of the observation filter from λ2+Δ to λ3−Δ and from λ4+Δ to λ5−Δ is less than 0.1 times W1, wherein 3 nm≤Δ≤10 nm holds true, and/or wherein the intensity of the first light above λ3is at most 1% of its maximum spectral intensity, and/or wherein the intensity of the second light below λ2and above λ5is at most 1% of its maximum spectral intensity.

In order to be able to set the color rendering of the non-fluorescent regions, in particular to a true color rendering, the microscopy system further includes a third light source configured to generate third light having wavelengths of between λ7and λ8and to direct it onto the object plane, wherein λ7, λ8are wavelengths and λ2<λ7<λ8<λ5holds true. A description is given below of some configurations used to achieve a true color rendering of the non-fluorescent regions by suitable operation of the light sources.

Second Exemplary Embodiment

The transmittance of the observation filter from λ3to λ6is larger than W3. Typically, the intensity of the first light above λ3is at most 1% of its maximum spectral intensity. Typically, the intensity of the second light below λ2and above λ5is at most 1% of its maximum spectral intensity. Typically, the intensity of the third light below λ2and above λ5is at most 1% of its maximum spectral intensity.

Third Exemplary Embodiment

The transmittance of the observation filter from λ3to λ4is less than 5% and 600 nm<λ4<λ5<620 nm holds true. Typically, the intensity of the first light above λ3is at most 1% of its maximum spectral intensity. Typically, the intensity of the second light below λ2and above λ5is at most 1% of its maximum spectral intensity. Typically, the intensity of the third light below λ2and above λ5is at most 1% of its maximum spectral intensity.

Fourth Exemplary Embodiment

With further preference, the microscopy system according to the third exemplary embodiment further includes an illumination filter arrangeable between the light sources and the object plane, wherein the transmittance of the illumination filter from λ1to λ3′ and from λ4′ to λ4is larger than W3and from λ3′+Δ to λ4′−Δ and from λ4+Δ is less than W1, wherein 3 nm≤Δ≤10 nm holds true, and wherein λ3′ and λ4′ are wavelengths and λ3<λ3′<λ4′<λ4and 480 nm<λ3′<520 nm and 520 nm<λ4′<550 nm and λ4′−λ3′>20 nm hold true. Typically, the intensity of the first light above λ3is at most 1% of its maximum spectral intensity. Typically, the intensity of the second light below λ2and above λ4′ is at most 1% of its maximum spectral intensity. Typically, the intensity of the third light between λ4′ and λ4is significantly high and below λ3′ and above λ5is at most 1% of its maximum spectral intensity.

Fifth Exemplary Embodiment

The transmittance of the observation filter from λ3to λ4is less than 5%; the transmittance of the observation filter from λ7to λ8is larger than W2and less than 5%, and the transmittance of the observation filter from λ4+Δ to λ7−Δ is less than W1, wherein 3 nm<Δ<10 nm holds true, wherein λ7and λ8are wavelengths and wherein λ4<λ7<λ8<λ5and 480 nm<λ4<520 nm and 520 nm<λ7<550 nm and λ7−λ4>20 nm and λ5−λ8<30 nm hold true. Typically, the intensity of the first light above λ3is at most 1% of its maximum spectral intensity. Typically, the intensity of the second light below λ2and above λ7is at most 1% of its maximum spectral intensity. Typically, the intensity of the third light below λ4and above λ5is at most 1% of its maximum spectral intensity.

Sixth Exemplary embodiment

The transmittance of the observation filter from λ3to λ4is less than 5%, the transmittance of the observation filter from λ4+Δ to λ5−Δ is less than W1, wherein 480 nm<λ4<520 nm and 530 nm<λ5<560 nm and λ5−λ4>20 nm hold true. The microscopy system further includes an illumination filter arrangeable between the light sources and the object plane, wherein the transmittance of the illumination filter from λ1to λ4is larger than W3, wherein the transmittance of the illumination filter from λ4+Δ to λ5′−Δ is less than W1, wherein the transmittance of the illumination filter from λ5′ to λ6′ is larger than W2and less than 5%, wherein the transmittance of the illumination filter from λ6′ to λ6is less than W1, wherein 3 nm≤Δ≤10 nm holds true, wherein λ5′ and λ6′ are wavelengths and λ4<λ5′<λ6′<λ6holds true, and wherein 530 nm<λ5′<570 nm and 600 nm<620 nm hold true. Typically, the intensity of the first light above λ3is at most 1% of its maximum spectral intensity. Typically, the intensity of the second light below λ2and above λ5is at most 1% of its maximum spectral intensity. Typically, the intensity of the third light below λ4and above λ5is at most 1% of its maximum spectral intensity.

For the simultaneous observation of fluorescent and non-fluorescent regions of an object admixed with PpIX, the light sources configured as follows are suitable, in particular, for the embodiments described above: The first light source can be a light emitting diode having a spectral emission maximum in the range of 400 nm to 420 nm and a full width at half maximum in the range of 10 nm to 20 nm. The second light source can be a light emitting diode having a spectral emission maximum in the range of 440 nm to 470 nm and a full width at half maximum in the range of 20 nm to 50 nm. The third light source can be a light emitting diode having a spectral emission maximum in the range of 500 nm to 560 nm and a full width at half maximum in the range of 40 nm to 110 nm.

Seventh Exemplary Embodiment

For the simultaneous observation of fluorescent and non-fluorescent regions of an object admixed with fluorescein, the microscopy system furthermore includes a third light source configured to generate third light having wavelengths of between λ5′ and λ6′ and to direct it onto the object plane, an illumination filter arrangeable between the light sources and the object plane, wherein the transmittance of the illumination filter from λ1to λ4is larger than W3, wherein the transmittance of the illumination filter from λ5′ to λ6′ is larger than W2and less than 5%, wherein λ5′, λ6′ are wavelengths and λ5<λ5′<λ6′ holds true, and wherein the transmittance of the observation filter from λ3to λ4is less than 5%.

Typically, the transmittance of the illumination filter from λ4+Δ to λ5′−Δ is less than W1, wherein 3 nm<Δ<10 nm holds true. Typically, the transmittance of the illumination filter from λ6′ to 750 nm is less than W1. Typically, the transmittance of the observation filter from λ4+Δ to λ5−Δ is less than W1, wherein 3 nm<Δ<10 nm holds true.

The observation filter and the light sources can be configured as follows for the simultaneous observation of fluorescent and non-fluorescent regions of an object admixed with fluorescein:

Typically, the intensity of the first light above λ5is at most 1% of its maximum spectral intensity. Typically, the intensity of the second light below λ3and above λ5′ is at most 1% of its maximum spectral intensity. Typically, the intensity of the third light below λ4is at most 1% of its maximum spectral intensity.

For the simultaneous observation of fluorescent and non-fluorescent regions of an object admixed with fluorescein, the light sources configured as follows are suitable, in particular, for the exemplary embodiments described above: The first light source can be a light emitting diode having a spectral emission maximum in the range of 440 nm to 470 nm and a full width at half maximum in the range of 10 nm to 20 nm. The second light source can be a light emitting diode having a spectral emission maximum in the range of 500 nm to 560 nm and a full width at half maximum in the range of 40 nm to 110 nm. The third light source can be a light emitting diode having a spectral emission maximum in the range of 600 nm to 640 nm and a full width at half maximum in the range of 10 nm to 20 nm.

Typically, wavelength-dependent transmittances of the illumination filter and of the observation filter are coordinated with one another such that between 350 nm and 750 nm no wavelength exists at which both the absolute value of the wavelength-dependent gradient of the transmittance of the illumination filter and the absolute value of the wavelength-dependent gradient of the transmittance of the observation filter are larger than 2%/nm. This prevents a large change in the transmittance of the illumination filter and of the observation filter at a wavelength. Production dictated tolerances with regard to the transmittance of the illumination filter and of the observation filter therefore do not significantly affect the trueness of color and the brightness of different spectral ranges which are achieved with the illumination filters and observation filters.

Second Aspect

A second aspect of the present disclosure relates to a method for operating a microscopy system, in particular one of the microscopy systems described herein.

The method includes: generating illumination light and directing the generated illumination light onto an object; generating an observation beam path, which images the object into an image plane, wherein an observation filter is arranged in the observation beam path. The illumination light is generated such that
|{right arrow over (W)}−{right arrow over (f)}|≤0.2   (1)
holds true, wherein {right arrow over (W)} represents a color locus of a white point in the CIE standard chromaticity diagram of the CIE 1931 standard colorimetric system, and {right arrow over (f)} represents a color locus having coordinates xfand yfin the CIE standard chromaticity diagram of the CIE 1931 standard colorimetric system.

The distance |⋅| between a color locus {right arrow over (A)} having coordinates xAand yAin the CIE standard chromaticity diagram of the CIE 1931 standard colorimetric system and a color locus {right arrow over (B)} having coordinates xBand yBand in the CIE standard chromaticity diagram of the CIE 1931 standard colorimetric system is defined as:

The coordinates xfand yfof the color locus {right arrow over (f)} are defined by

xf=XX+Y+Z⁢and⁢yf=YX+Y+Z,(2)
wherein X , Y and Z represent tristimulus values of the CIE 1931 standard colorimetric system which are defined by
X=k∫I(λ)·T(λ)·x(λ)·dλ,
Y=k∫I(λ)·T(λ)·y(λ)·dλ, and
Z=k∫I(λ)·T(λ)·z(λ)·dλ,(3)
wherein I(λ) represents the intensity of the illumination light, T(λ) represents the transmittance41,61,81,101,121,141,161of the observation filter23, {right arrow over (x)}(λ), {right arrow over (y)}(λ), and {right arrow over (z)}(λ) represent the spectral value functions of the TRG909926 CIE 1931 standard colorimetric system, and k is a constant.

The white point can be for example the white point D50 having the coordinates x=0.3457 and y=0.3585 in the CIE standard chromaticity diagram of the CIE 1931 standard colorimetric system. Alternatively, the white point can be for example the white point D65 having the coordinates x=0.3127 and y=0.329 in the CIE standard chromaticity diagram of the CIE 1931 standard colorimetric system. As a further alternative, the white point can correspond to a color valence whose color locus is at a distance of at most 0.2, in particular at most 0.1 or at most 0.05, from the color locus of the white point D50 in the CIE 1976 u′v′ chromaticity diagram.

The distance |⋅| between a color locus {right arrow over (A)} having coordinates u′Aand v′Ain the CIE 1976 u′v′ chromaticity diagram and a color locus {right arrow over (B)} having coordinates u′Band v′Bin the CIE 1976 u′v′ chromaticity diagram is defined as:

Typically, the illumination light is generated such that |{right arrow over (W)}−{right arrow over (f)}|≤0.15 or |{right arrow over (W)}−{right arrow over (f)}|≤0.1 holds true. The integrals over the wavelength λ are integrated from 380 nm to 780 nm.

Equation 1 describes a condition for a maximum color valence distance in the CIE 1931 standard chromaticity diagram. In this case, {right arrow over (f)} represents a color locus in the CIE standard chromativcity diagram of the CIE 1931 standard colorimetric system that is obtained if illumination light of the intensity I(λ) impinges on a white object and the light emanating from the white object is filtered by an observation filter having the transmittance T(λ). The required small distance from the white point in the CIE 1931 standard chromaticity diagram means that the white object can be observed as approximately white. When applied to general objects, this means that the object can be observed as approximately true color. This facilitates the orientation in the operating area for a surgeon.

The illumination light is generated for example by a plurality of light sources that generate light in different wavelength ranges. Generating the illumination light can include: setting, in particular varying, an energy fed to at least one of the plurality of light sources for generating the illumination light.

The method can be carried out by the microscopy systems described herein.

Third Aspect

A third aspect of the present disclosure relates to a microscopy method in which fluorescent regions and non-fluorescent regions can be perceived as approximately equally bright.

For this purpose, the microscopy method includes: generating illumination light and directing the generated illumination light onto an object, generating an observation beam path, which images the object into an image plane, wherein an observation filter is arranged in the observation beam path, wherein a first value, which represents a mean value of the transmittance of the observation filter over a first wavelength range, is less than a second value, which represents a mean value of the transmittance of the observation filter over a second wavelength range, wherein the second value is less than a third value, which represents a mean value of the transmittance of the observation filter over a third wavelength range, wherein a fourth value, which represents a mean value of the intensity of the illumination light over the first wavelength range is larger than a fifth value, which represents a mean value of the intensity of the illumination light over the second wavelength range, wherein the fifth value is larger than a sixth value, which represents a mean value of the intensity of the illumination light over the third wavelength range, wherein the first, second, and third wavelength ranges do not overlap one another and are in each case between 350 nm and 1000 nm.

The first wavelength range corresponds to that wavelength range in which a fluorescent dye present in the object has a significant absorption. In order to be able to excite the fluorescent dye efficiently, the illumination light in the first wavelength range has a high intensity characterized by the fourth value. In order that light in the first wavelength range that is reflected at the object does not swamp the fluorescent light generated substantially exclusively in the third wavelength range, the observation filter has in the first wavelength range a low transmittance characterized by the first value.

In order that the fluorescent light can be observed efficiently, the observation filter has in the third wavelength range a high transmittance characterized by the third value. In order that the fluorescent light is not swamped by illumination light in the third wavelength range that is reflected at the object, the illumination light in the third wavelength range has a low intensity characterized by the sixth value.

The second wavelength range serves for visualizing non-fluorescent regions of the object. For this purpose, the illumination light has a medium intensity in the second wavelength range, said intensity being characterized by the fifth value, and the observation filter has in the second wavelength range an average transmittance characterized by the second value.

The light in the first and second wavelength ranges that is transmitted by the observation filter brings about the visualization of non-fluorescent regions of the object and thus predominantly contributes to the brightness of the non-fluorescent regions in the image plane. The light in the third wavelength range that is transmitted by the observation filter brings about the visualization of fluorescent regions of the object and thus predominantly contributes to the brightness of the fluorescent regions in the image plane. The indicated relation of the first to sixth values with respect to one another has the effect that the non-fluorescent regions and the fluorescent regions of the object appear approximately equally bright. Moreover, they are coordinated with one another such that moreover an approximately true color observation of non-fluorescent regions is made possible.

The first wavelength range can contain wavelengths at which an absorption rate—normalized to their maximum value—of the fluorescent dye present in the object is at least 5%, in particular at least 10%, more particularly at least 50%. Accordingly, the first wavelength range contains those wavelengths at which the fluorescent dye can be excited efficiently.

The third wavelength range can contain wavelengths at which an emission rate—normalized to their maximum value—of the fluorescent dye present in the object is at least 5%, in particular at least 10%, more particularly at least 50%. Accordingly, the third wavelength range contains those wavelengths at which the fluorescent dye can be excited efficiently.

The second wavelength range can contain exclusively wavelengths at which an absorption rate—normalized to their maximum value—of the fluorescent dye present in the object is at most 20%, in particular at most 10%, more particularly at most 5%, and at which an emission rate—normalized to their maximum value—of the fluorescent dye present in the object is at most 20%, in particular at most 10%, more particularly at most 5%. Accordingly, the second wavelength range contains substantially only those wavelengths which lie outside the wavelength ranges in which the fluorescent dye can be excited efficiently and generates fluorescent light.

With the use of the fluorescent dye protoporphyrin IX, the first wavelength range typically includes the wavelength 405 nm and/or wavelengths of 390 nm to 420 nm, the second wavelength range typically includes wavelengths of 450 nm to 600 nm and the third wavelength range typically includes wavelengths of 620 nm to 720 nm.

With the use of the fluorescent dye fluorescein, the first wavelength range typically includes wavelengths of 480 nm to 500 nm, the second wavelength range typically includes wavelengths of 620 nm to 750 nm and the third wavelength range typically includes wavelengths of 550 nm to 600 nm.

Typically, a ratio of the first value to the second value is at most 1/100, in particular at most 1/1000. Typically, a ratio of the second value to the third value is at most 0.9, in particular at most 0.8, more particularly at most 0.5. Typically, a ratio of the fourth value to the fifth value is at least 2, in particular at least 5. Typically, a ratio of the fourth value to the sixth value is at least 1000, in particular at least 10000. Both an approximately equal brightness and an approximately true color rendering are achieved as a result.

The illumination light can be generated using a plurality of (narrowband) light sources, for example using the light sources described with regard to other aspects. At least one of the light sources generates light having wavelengths in the first wavelength range; at least one of the light sources generates light having wavelengths in the second wavelength range. At least one of the light sources can be individually controllable in order thereby to be able to vary the ratio between the intensity of the light having wavelengths in the first wavelength range and the intensity of the light having wavelengths in the second wavelength range.

Typically, the light sources generate substantially no light having wavelengths in the third wavelength range. This prevents the light generated by the light sources from corrupting the fluorescent light in the third wavelength range.

An alternative microscopy method includes generating illumination light and directing the generated illumination light onto an object, generating an observation beam path, which images the object into an image plane, wherein the illumination light is generated such that a ratio of a first characteristic value to a second characteristic value has a value in the range of 20/1 to 1/20, wherein the first characteristic value is a value of a characteristic variable for the intensity of light of the observation beam path at first locations in the image plane, at which first locations color valences of the light of the observation beam path lie within a first color valence range of a color space, wherein the second characteristic value is a value of the characteristic variable for the intensity of the light of the observation beam path at second locations in the image plane, at which second locations color valences of the light of the observation beam path lie within a second color valence range of the color space.

Typically, the illumination light is generated such that the ratio of the first characteristic value to the second characteristic value has a value in the range of 10/1 to 1/10 or 5/1 to 1/5 or 3/1 to 1/3.

A projected image of the object is generated in the image plane through the observation beam path. If a fluorescent dye present in the object is excited by the illumination light, the projected image generally includes fluorescent regions (first locations) and non-fluorescent regions (second locations). Strongly fluorescent regions (first locations) have in the projected image the color valences typical of the fluorescent dye, which lie within the first color valence range. Weakly fluorescent and non-fluorescent regions (second locations) have in the projected image other color valences, which lie within the second color valence range. Accordingly, the first and second color valence ranges are non-overlapping ranges of the same color space.

Fluorescent regions of the object (first locations) can be distinguished from non-fluorescent regions (second locations) in the image plane on the basis of the color valences typical of the fluorescent dye. In order that both fluorescent and non-fluorescent regions can be distinguished by an observer or a controller, it is advantageous if the fluorescent and the non-fluorescent regions appear approximately equally bright, that is to say have an intensity of approximately equal magnitude or a luminous flux of approximately equal magnitude in the image plane. Luminous flux denotes that proportion of a radiation power which is visible to the human eye, weighted with the luminous efficiency curve of the human eye.

This is achieved in the microscopy method by virtue of the illumination light being generated such that the ratio of the first characteristic value to the second characteristic value has a value in the range of 20/1 to 1/20. The first characteristic value represents for example the mean value of the intensity of the light in the observation beam path in the image plane at the first locations, that is to say those locations in the image plane at which the light in the observation beam path has the color valences typical of the fluorescent dye. In this example, the second characteristic value represents the mean value of the intensity of the light in the observation beam path in the image plane at the second locations, that is to say those locations in the image plane at which the light in the observation beam path does not have the color valences typical of the fluorescent dye.

The first characteristic value and the second characteristic value respectively represent a value of a characteristic variable of the same type. By way of example, if the first characteristic value represents a maximum value, then the second characteristic value likewise represents a maximum value. Alternatively, if the first characteristic value represents a mean value, for example, then the second characteristic value likewise represents a mean value.

The first characteristic value can represent a maximum value or a mean value of the intensity (or of the luminous flux) of the light of the observation beam path at the first locations. The second characteristic value can represent a maximum value or a mean value of the intensity (or of the luminous flux) of the light of the observation beam path at the second locations.

The microscopy method can furthermore include determining the first locations in the image plane, at which first locations the color valences of the light of the observation beam path lie within the first color valence range of the color space, determining the second locations in the image plane, at which second locations the color valences of the light of the observation beam path lie within the second color valence range of the color space, wherein the illumination light is generated depending on the intensity of the light of the observation beam path at the first locations determined and depending on the intensity of the light of the observation beam path at the second locations determined. As a result, the first and second locations in the image plane are identified and the illumination light is generated depending on the intensity at these locations.

The microscopy method can further include determining the intensity of the light in the observation beam path at the first and second locations for determining the luminous flux of the light in the observation beam path at the first and second locations. By way of example, an image detector is arranged in the image plane, and is configured to record a color image of the projected image, based on which firstly the first and second locations can be identified and secondly the intensity of the light in the observation beam path at the first and second locations can be determined.

The microscopy method can furthermore include recording an image of the object in the image plane, wherein the image includes a plurality of image points, comparing color valences at the image points of the image with the first color valence range and/or with the second color valence range, determining first image points from the plurality of image points based on a result of the comparison, wherein the first image points correspond to the first locations in the image plane and wherein color valences at the first image points lie within the first color valence range of the color space, and determining second image points from the plurality of image points on the basis of a result of the comparison, wherein the second image points correspond to the second locations in the image plane and wherein color valences at the second image points lie within the second color valence range of the color space.

The microscopy method can further includes determining the first characteristic value on the basis of the determined intensity of the light of the observation beam path at the first locations determined, and determining the second characteristic value on the basis of the determined intensity of the light of the observation beam path at the second locations determined, wherein the illumination light is generated depending on the first characteristic value determined and depending on the second characteristic value determined. Accordingly, the first and second characteristic values are determined, for example calculated, specifically based on the determined or measured intensity values at the first and second locations that were identified previously on the basis of a recorded image and on the basis of their color valences, in particular by comparison with the first and second color valence ranges. The first and second characteristic values thus obtained are then used for generating the illumination light.

The illumination light is generated for example by a plurality of light sources which generate light in different wavelength ranges. Generating the illumination light can include setting, in particular varying, an energy fed to at least one of the plurality of light sources for generating the illumination light, depending on the first and second characteristic values.

In particular, provision can be made for the illumination light to be generated independently of the intensity of light of the observation beam path at third locations in the image plane, wherein color valences of the light of the observation beam path at the third locations lie outside the first and second color valence ranges. That is to say that exclusively the intensity of the light in the observation beam path at the first and second locations is used for generating the illumination light. The intensity of the light in the observation beam path at other locations in the image plane is not used for generating the illumination light.

The first color valence range and the second color valence range can be chosen such that a smallest color difference between the first color valence range and the second color valence range in the CIE 1976 u′v′ chromaticity diagram is at least 0.01, in particular at least 0.1. Furthermore, the first color valence range can be chosen such that it is limited to color valences which have a color difference of at most 0.1, in particular at most 0.03 or at most 0.01, with respect to reference color valences in the CIE 1976 u′v′ chromaticity diagram, wherein the reference color valences correspond to light in a reference wavelength range containing exclusively wavelengths at which an emission rate—normalized to their maximum value—of a fluorescent dye present in the object is at least 5%, in particular at least 10%, more particularly at least 50%. The reference color valence is for example a color valence which corresponds to the emission spectrum of a fluorescent dye in the object. Accordingly, only such color valences which are separated from said reference color valence by not more than the maximum difference indicated belong to the first color valence range.

If the fluorescent dye is PpIX, it is typical for the first color valence range to include a color valence which corresponds to light having a wavelength of 635 nm. Furthermore, it is typical for the first color valence range to be limited to color valences which have a color difference of at most 0.1, in particular at most 0.03 or at most 0.01 with respect to a reference color valence corresponding to light having a wavelength of 635 nm in the CIE 1976 u′v′ chromaticity diagram.

If the fluorescent dye is fluorescein, it is typical for the first color valence range to comprise a color valence which corresponds to light having a wavelength of 530 nm. Furthermore, it is typical for the first color valence range to be limited to color valences which have a color difference of at most 0.1, in particular at most 0.03 or at most 0.01 with respect to a reference color valence corresponding to light having a wavelength of 530 nm in the CIE 1976 u′v′ chromaticity diagram.

Features of the first, second, and third aspects can be combined with one another.

The observation filters and illumination filters described herein can be embodied for example as interference filters. Interference filters include a plurality of layers of materials having different optical properties, for example different refractive indices. Each layer can have a different thickness. The number of layers can be between a few and several hundred, depending on the requirements made of the filter. The transmittances described herein can be realized by the specific choice of the thickness and the optical property of each layer.

Aids for determining the thicknesses and optical properties of the layers of a filter depending on a predefined wavelength-dependent transmittance are known. Such layer sequences can be designed using simulation programs that use as input data the optical properties (refractive index and absorption depending on the wavelength, dispersion) of the materials to be used and also the desired transmission and/or reflection spectrum. The simulation program outputs the simulated transmission and/or reflection spectrum and also the layer sequence and/or the thickness of the layers and the optical properties and/or materials used. The calculation can be carried out in an iteration method. In this way, even filters with complicated requirements, e.g. multiband filters, can be designed and produced.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

FIG.1shows one exemplary microscopy system1for simultaneously observing fluorescent and non-fluorescent regions of an object.

The microscopy system1includes an illumination device3. The illumination device3comprises a plurality of light sources5, an illumination optical unit7and an illumination filter9, which can optionally be arranged in an observation beam path10formed by the illumination optical unit7. This is illustrated by a double headed arrow inFIG.1. By the plurality of light sources5and the illumination filter9, the illumination device3can generate various illumination light spectra and direct them onto an object region11.

The microscopy system1includes a controller12configured to control the plurality of light sources5individually, i.e., independently of one another. By way of example, the light sources are two or more light emitting diodes. Each of the light emitting diodes can be controlled individually by the controller12. By way of example, the controller12controls the operating current and/or the operating voltage fed and/or respectively applied to a light emitting diode in order to generate light. The intensity of the light generated by the light sources5can be set individually as a result.

By the observation beam path10, the illumination device3directs illumination light onto the object region11, in which an object13can be arranged, which can be admixed with a fluorescent dye. The fluorescent dye fed to the object13accumulates greatly in some regions of the object13(fluorescent regions). These regions contain tumor cells, for example, to which the fluorescent dye binds. The fluorescent dye does not accumulate or accumulates only slightly in other regions of the object13(non-fluorescent regions). These regions contain no tumor cells, for example.

The microscopy system1further includes a microscopy optical unit14, which in the present example includes an objective15and further lenses16and17. The microscopy optical unit14is configured to image the object region11, in particular an object plane18, onto an image plane19. In the present example, a detection area of a light detector20of the microscopy system1is arranged in the image plane19. The light detector20can be an image sensor for example. The light detector20outputs a signal representing the intensity of the light impinging on the detection area of the light detector20. The light detector20is connected to the controller12and the controller12receives from the light detector20the signal21output by the latter. The controller can process and display the image generated by the light detector20. Instead of or in addition to the light detector20, an eyepiece can be provided, which, in conjunction with the microscopy optical unit14, is suitable for imaging the object plane18onto the retina of an eye.

An observation filter23can be arranged in the observation beam path22provided by the microscopy optical unit14, said observation beam path imaging the object plane18onto the image plane19. Furthermore, an actuator (not shown) can be provided, which can introduce the observation filter23into the observation beam path22and can withdraw the observation filter23from the observation beam path22. This is illustrated by a double headed arrow inFIG.1.

Depending on the configuration of the plurality of light sources5, of the illumination filter9and of the observation filter23, fluorescent and non-fluorescent regions of the object13can be observed simultaneously. Various exemplary embodiments for the fluorescent dyes protoporphyrin IX (PpIX) and fluorescein are described below.

FIG.2shows the wavelength-dependent absorption spectrum of PpIX as graph A,PpIX, the wavelength-dependent emission spectrum of PpIX as graph EPpIX, the wavelength-dependent absorption spectrum of fluorescein as graph AFL and the wavelength-dependent emission spectrum of fluorescein as graph EFL.

The fluorescent dye PpIX has an absorption spectrum PpIX which between 350 nm and 430 nm has a normalized absorption intensity of more than 0.2. The normalized absorption intensity is normalized to the maximum absorption intensity, i.e., the normalized absorption spectrum only has values of between 0 and 1. The fluorescent dye PpIX can therefore be excited efficiently in the range of 350 nm to 430 nm. The fluorescent dye PpIX has the maximum of the absorption at approximately 405 nm. The fluorescent dye PpIX emits fluorescent light in a spectral range from approximately 600 nm to 750 nm, with a main maximum of the emission intensity lying at 635 nm and a secondary maximum lying at approximately 705 nm.

The fluorescent dye fluorescein has a normalized absorption intensity of more than 0.2 between approximately 450 nm and 530 nm. Therefore, the fluorescent dye fluorescein can be excited well in this range. The absorption spectrum of fluorescein has a maximum at approximately 495 nm. The fluorescent dye fluorescein emits emission light in the range from approximately 490 nm to 650 nm. The maximum of the emission spectrum lies at approximately 520 nm.

Example with Respect to the First Embodiment

FIG.3shows illumination light spectra (spectral intensity distributions)31and32of a first and a second light source in accordance with the first exemplary embodiment. The first light source serves substantially exclusively for exciting the fluorescent dye PpIX, i.e., for visualizing fluorescent regions of the object. The second light source serves substantially exclusively for visualizing non-fluorescent regions of the object.

The first light source generates an illumination light spectrum31having a spectral emission maximum in the range of 400 nm to 410 nm and a full width at half maximum in the range of 10 nm to 20 nm. Below approximately 370 nm and above approximately 450 nm, the intensity of the first light generated by the first light source is at most 1% of its maximum spectral intensity. The first light source is a light-emitting diode, for example.

The second light source generates an illumination light spectrum32having a spectral emission maximum in the range of 440 nm to 470 nm and a full width at half maximum in the range of 20 nm to 50 nm. Below approximately 440 nm and above approximately 500 nm, the intensity of the second light generated by the second light source is at most 1% of its maximum spectral intensity. The second light source is a light-emitting diode, for example.

The first and second light sources are operated such that the maximum spectral intensity of the first light source is at most 20 times and at least 3 times the magnitude of the maximum spectral intensity of the second light source.

FIG.4shows the wavelength-dependent transmittance41of an observation filter23in accordance with the first embodiment. From λ1=360 nm to λ2=430 nm the transmittance41is approximately 0.01%. From λ3=435 nm to λ4=500 nm the transmittance41is approximately 55%. From λ5=600 nm to λ6=750 nm the transmittance41is approximately 95%. In the remaining wavelength ranges between λ1and λ6the transmittance41is at most 0.001%. Furthermore, it holds true that: W1=0.1%; W2=1%; and W3=80%.

Accordingly, the observation filter23suppresses the first light reflected at the object13, said first light having a high intensity substantially only between λ1and λ2. Furthermore, the second light reflected at the object13, said second light having a high intensity substantially only between λ3and λ4, is transmitted by the observation filter23, with the result that non-fluorescent regions of the object13can be observed as bluish. Furthermore, the fluorescent light emanating from the object13, said fluorescent light having a high intensity substantially only between λ5and λ6is transmitted by the observation filter23, with the result that fluorescent regions of the object13can be observed as reddish color valences.

Two major advantages are afforded by the individual settability of the intensities of the first and second light (cf.FIG.3) and the concrete choice of the transmittance41of the observation filter23. Firstly, the fluorescent regions and the non-fluorescent regions can be perceived as almost equally bright by virtue of the intensity of the light sources being set accordingly. Secondly, on account of the concrete choice of the transmittance41between λ3and λ4and also between λ5and λ6, the light sources can be operated here in their stable emission range. Moreover, an illumination filter is not required.

Examples with Respect to the Second to Sixth Exemplary Embodiments

FIG.5shows illumination light spectra (spectral intensity distributions)51,52, and53of a first, a second, and a third light source in accordance with a second to sixth exemplary embodiment. The first light source serves substantially exclusively for exciting the fluorescent dye PpIX, i.e., for visualizing fluorescent regions of the object. The second and third light sources serve substantially exclusively for visualizing non-fluorescent regions of the object.

The first and second light sources correspond to those of the example with respect to the first exemplary embodiment. That is to say that the illumination light spectrum31of the first light source of the first exemplary embodiment as illustrated inFIG.3and the illumination light spectrum51of the first light source of the second to sixth exemplary embodiments as illustrated inFIG.5are identical, and that the illumination light spectrum32of the second light source of the first embodiment as illustrated inFIG.3and the illumination light spectrum52of the second light source of the second to sixth embodiments as illustrated inFIG.5are identical.

The third light source generates an illumination light spectrum53having a spectral emission maximum in the range of 500 nm to 560 nm and a fullwidth at half maximum in the range of 40 nm to 110 nm. Below approximately 470 nm and above approximately 630 nm, the intensity of the third light generated by the third light source is at most 1% of its maximum spectral intensity. The third light source is a light-emitting diode, for example.

The first and third light sources are operated such that the maximum spectral intensity of the first light source is at most 20 times and at least 3 times the magnitude of the maximum spectral intensity of the third light source.

Example with Respect to the Second Exemplary Embodiment

FIG.6shows the wavelength-dependent transmittance61of an observation filter23in accordance with the second exemplary embodiment. From λ1=350 nm to λ2=435 nm the transmittance61is approximately 0.005%. From λ3=440 nm to λ4=λ5=600 nm and from λ5to λ6=750 nm the transmittance61is approximately 95%. Furthermore, it holds true that: W1=0.01%, W2=1%, and W3=80%.

Accordingly, the observation filter23suppresses the first light reflected at the object13, said first light having a high intensity substantially only between λ1and λ2. Furthermore, the second and third light reflected at the object13, said second and third light having a high intensity substantially only between λ3and λ4is transmitted by the observation filter23, with the result that non-fluorescent regions of the object13can be observed as approximately true color. Furthermore, the fluorescent light emanating from the object13, said fluorescent light having a high intensity substantially only between λ5and λ6is transmitted by the observation filter23, with the result that fluorescent regions of the object13can be observed as reddish color valences.

Three major advantages are afforded by the individual settability of the intensities of the first, second and third light (cf.FIG.5) and the concrete choice of the transmittance61of the observation filter23. Firstly, the fluorescent regions and the non-fluorescent regions can be perceived as almost equally bright by virtue of the intensity of the light sources being set accordingly. Furthermore, on account of the concrete choice of the transmittance61between λ3and λ4and also between λ5and λ6, the light sources can be operated here in their stable emission range. Finally, as a result of the individual settability of the intensities of the second and third light, the color rendering of the non-fluorescent regions can be set such that the non-fluorescent regions can be observed as approximately true color. Moreover, an illumination filter is not required.

FIG.7shows the CIE 1931 xy standard chromaticity diagram including the spectrum locus S and the purple boundary P. A hollow circle represents the D50 white point at x=0.3457, y=0.3585. A cross at approximately x=0.26, y=0.45 represents a color valence with which a white object is perceived by the normal observer using the microscopy system in accordance with the second exemplary embodiment if the observation filter is configured as shown inFIG.6, the maximum spectral intensity of the light generated by the first light source is in each case approximately 20 times the magnitude of the maximum spectral intensity of the light generated by the second and third light source, respectively. Non-fluorescent regions of the object can therefore be perceived as approximately true color. In addition, this configuration results in an approximately equal brightness of fluorescent and non-fluorescent regions of the object.

Example with Respect to the Third Exemplary Embodiment

FIG.8shows the wavelength-dependent transmittance81of an observation filter23in accordance with the third exemplary embodiment. From λ1=350 nm to λ2=435 nm the transmittance81is approximately 0.005%. From λ3=440 nm to λ4=610 nm the transmittance81is approximately 2%. From λ5=620 nm to λ6=750 nm the transmittance81is approximately 95%. Furthermore, it holds true that W1=0.01%, W2=1%, and W3=80%.

Accordingly, the observation filter23suppresses the first light reflected at the object13, said first light having a high intensity substantially only between λ1and λ2. Furthermore, the second and third light reflected at the object13, said second and third light having a high intensity substantially only between λ3and λ4is transmitted by the observation filter23, with the result that non-fluorescent regions of the object13can be observed as approximately true color. Furthermore, the fluorescent light emanating from the object13, said fluorescent light having a high intensity substantially only between λ5and λ6is transmitted by the observation filter23, with the result that fluorescent regions of the object13can be observed as reddish color valences.

The advantages of the example of the second exemplary embodiment can be afforded by the individual settability of the intensities of the first, second and third light (cf.FIG.5), wherein a better, trueness of the observation of the non-fluorescent regions is achieved on account of the lower transmittance81between λ3and λ4in comparison with the second exemplary embodiment. In addition, the second and third light sources can be operated with a higher power in comparison with the second exemplary embodiment, which in turn improves the stability of the illumination light spectra52,53generated. Moreover, an illumination filter is not required.

FIG.9shows the CIE 1931 xy standard chromaticity diagram including the spectrum locus S and the purple boundary P. A hollow circle represents the D50 white point at x=0.3457, y=0.3585. A cross at approximately x=0.33, y=0.38 represents a color valence with which a white object is perceived by the normal observer using the microscopy system in accordance with the third embodiment if the observation filter is configured as shown inFIG.8, the maximum spectral intensity of the light generated by the first light source is in each case approximately 7 times the magnitude of the maximum spectral intensity of the light generated by the second light source and the maximum spectral intensity of the light generated by the first light source is approximately 5 times the magnitude of the maximum spectral intensity of the light generated by the third light source. Non-fluorescent regions of the object can therefore be perceived as approximately true color. In addition, this configuration results in an approximately equal brightness of fluorescent and non-fluorescent regions of the object.

Example with respect to the fourth exemplary embodiment

FIG.10shows the wavelength-dependent transmittance101of an observation filter23(solid line) and the wavelength-dependent transmittance102of an illumination filter9(dashed line) in accordance with the fourth embodiment. The observation filter of the example with respect to the fourth exemplary embodiment corresponds to the observation filter of the example with respect to the third exemplary embodiment (λ1=350 nm, λ2=435 nm, λ3=440 nm, λ4=610 nm, λ5=620 nm, and λ6=750 nm).

From 360 nm to λ3′=500 nm the transmittance102of the illumination filter9is approximately 95%. From approximately λ3′ to approximately λ4′=560 nm the transmittance102of the illumination filter9is approximately 0.005%. From λ4′ to approximately λ4the transmittance102of the illumination filter9is approximately 95%. From approximately λ4to λ6the transmittance102of the illumination filter9is approximately 0.005%. Furthermore, it holds true that W1=0.01%, W2=1%, and W3=80%.

The illumination filter and the observation filter together have substantially the same effect as the observation filter of the example with respect to the fifth exemplary embodiment. The observation filter suppresses the first light reflected at the object13, said first light having a high intensity substantially only between λ1and λ2and passing through the illumination filter substantially without being damped. Furthermore, the second light reflected at the object13is restricted by the illumination filter and the observation filter to wavelengths lying substantially only between λ3and λ3′. Furthermore, the third light reflected at the object13is restricted by the illumination filter and the observation filter to wavelengths lying substantially only between λ4′ and λ4. Light in the wavelength range from λ3′ to λ4′ is effectively suppressed by the illumination filter and the observation filter. Non-fluorescent regions of the object13can therefore be observed as approximately true color. Furthermore, the fluorescent light emanating from the object13and having a high intensity substantially only between λ5and λ6is transmitted by the observation filter23, with the result that fluorescent regions of the object13can be observed with reddish color valences.

FIG.11shows the CIE 1931 xy standard chromaticity diagram including the spectrum locus S and the purple boundary P. A hollow circle represents the D50 white point at x=0.3457, y=0.3585. A cross at approximately x=0.35, y=0.36 represents a color valence with which a white object is perceived by the normal observer using the microscopy system in accordance with the fourth exemplary embodiment if the observation filter and the illumination filter are configured as shown inFIG.10, the maximum spectral intensity of the light generated by the first light source is in each case approximately 10 times the magnitude of the maximum spectral intensity of the light generated by the second light source and the maximum spectral intensity of the light generated by the first light source is approximately 5 times the magnitude of the maximum spectral intensity of the light generated by the third light source. Non-fluorescent regions of the object can therefore be perceived as approximately true color. In addition, this configuration results in an approximately equal brightness of fluorescent and non-fluorescent regions of the object.

Three major advantages are afforded by the individual settability of the intensity52of the second light and the intensity53of the third light (cf.FIG.5) and the concrete choice of the transmittance101of the observation filter23and the transmittance102of the illumination filter9. Firstly, the fluorescent regions and the non-fluorescent regions can be perceived as almost equally bright by virtue of the intensity of the light sources being set accordingly. Furthermore, the light sources here can be operated in their stable emission range since the light generated by the second and third light sources is damped to a sufficiently great extent by the observation filter23and the illumination filter9. Finally, as a result of the individual settability of the intensities of the second and third light, the color rendering of the non-fluorescent regions can be set such that the non-fluorescent regions can be observed as approximately true color.

The dashed line depicted inFIG.11represents the colors of the non-fluorescent regions of the object which can be obtained by variation of the ratio between (a maximum of) the intensity52of the second light and (a maximum of) the intensity53of the third light. In this way, typically, a white point different from D50 can also be set.

Example with Respect to the Fifth Exemplary Embodiment

FIG.12shows the wavelength-dependent transmittance121of an observation filter23in accordance with the fifth exemplary embodiment. From λ1=350 nm to λ2=435 nm the transmittance121is approximately 0.005%. From λ3=440 nm to λ4=500 nm the transmittance121is approximately 2%. From approximately λ4to approximately λ7the transmittance121is approximately 0.005%. From λ7=535 nm to λ8=610 nm the transmittance121is approximately 2%. From λ5=620 nm to λ6=750 nm the transmittance121is approximately 95%. Furthermore, it holds true that W1=0.01%, W2=1%, and W3=80%.

Accordingly, the observation filter23suppresses the first light reflected at the object13, said first light having a high intensity substantially only between λ1and λ2. Furthermore, the fluorescent light emanating from the object13, said second light having a high intensity substantially only between λ3and λ4, is transmitted by the observation filter23. Furthermore, the third light reflected at the object13is transmitted efficiently by the observation filter23substantially only between λ7and λ8. Non-fluorescent regions of the object13can therefore be observed as approximately true color. Furthermore, the fluorescent light emanating from the object13, said fluorescent light having a high intensity substantially only between λ5and λ6is transmitted by the observation filter23, with the result that fluorescent regions of the object13can be observed as reddish color valences.

The advantages of the example with respect to the third exemplary embodiment can be afforded by the individual settability of the intensities of the first, second, and third light (cf.FIG.5), wherein an even better settability of the true color rendering of the non-fluorescent regions is achieved on account of the more pronounced spectral separation of second and third light reflected at the object13in comparison with the third exemplary embodiment. In addition, the second and third light sources can be operated with a higher power in comparison with the example with respect to the second exemplary embodiment, which in turn improves the stability of the light spectra generated. Moreover, an illumination filter is not required.

FIG.13shows the CIE 1931 xy standard chromaticity diagram including the spectrum locus S and the purple boundary P. A hollow circle represents the D50 white point at x=0.3457, y=0.3585. A cross at approximately x=0.34, y=0.35 represents a color valence with which a white object is perceived by the normal observer using the microscopy system in accordance with the fifth exemplary embodiment if the observation filter is configured as shown inFIG.12, the maximum spectral intensity of the light generated by the first light source is in each case approximately10times the magnitude of the maximum spectral intensity of the light generated by the second light source and the maximum spectral intensity of the light generated by the first light source is approximately 6 times the magnitude of the maximum spectral intensity of the light generated by the third light source. Non-fluorescent regions of the object can therefore be perceived as approximately true color. In addition, this configuration results in an approximately equal brightness of fluorescent and non-fluorescent regions of the object.

The dashed line depicted inFIG.13represents the colors of the non-fluorescent regions of the object which can be obtained by variation of the ratio between (a maximum of) the intensity52of the second light and (a maximum of) the intensity53of the third light. In this way, typically, a white point different from D50 can also be set.

Example with Respect to the Sixth Exemplary Embodiment

FIG.14shows the wavelength-dependent transmittance141of an observation filter23(solid line) and the wavelength-dependent transmittance142of an illumination filter9(dashed line) in accordance with the sixth exemplary embodiment.

From λ1=350 nm to λ2=430 nm the transmittance141of the observation filter23is approximately 0.005%. From λ3=435 nm to λ4=500 nm the transmittance141of the observation filter23is approximately 2%. From approximately λ4to λ5=560 nm the transmittance141of the observation filter23is approximately 0.005%. From λ5to λ6=750 nm the transmittance141of the observation filter23is approximately 95%. Furthermore, it holds true that: W1=0.01%; W2=1%; and W3=80%.

From 360 nm to approximately λ4the transmittance142of the illumination filter9is approximately 95%. From approximately λ4to approximately λ5′=565 nm the transmittance142of the illumination filter9is approximately 0.005%. From λ5′ to approximately λ6′=620 nm the transmittance142of the illumination filter9is approximately 2%. From approximately λ6′ to λ6the transmittance142of the illumination filter9is approximately 0.005%.

The illumination filter and the observation filter of the example with respect to the sixth exemplary embodiment together have substantially the same effect as the illumination filter and the observation filter of the example with respect to the fifth exemplary embodiment. The observation filter23suppresses the first light reflected at the object13, said first light having a high intensity substantially only between λ1and λ2and passing through the illumination filter9substantially without being damped. Furthermore, the second light reflected at the object13is restricted by the illumination filter and the observation filter to wavelengths lying substantially only between λ3and λ4. Furthermore, the third light reflected at the object13is restricted by the illumination filter and the observation filter to wavelengths lying substantially only between λ5′ and λ6′. Light in the wavelength range from approximately λ4to λ5is effectively suppressed by the illumination filter and the observation filter. Non-fluorescent regions of the object13can therefore be observed as approximately true color. Furthermore, the fluorescent light emanating from the object13and having a high intensity substantially only between λ6′ and λ6is transmitted by the observation filter23, with the result that fluorescent regions of the object13can be observed with reddish color valences.

Example with Respect to the Seventh Exemplary Embodiment

FIG.15shows illumination light spectra of a first, a second, and a third light source according to a seventh exemplary embodiment.

The first light source serves substantially exclusively for exciting the fluorescent dye fluorescein, i.e. for visualizing fluorescent regions of the object. The second and third light sources serve substantially exclusively for visualizing non-fluorescent regions of the object.

The first light source generates an illumination light spectrum151having a spectral emission maximum in the range of 440 nm to 470 nm and a full width at half maximum in the range of 20 nm to 50 nm. Below approximately 430 nm and above approximately 500 nm, the intensity of the first light generated by the first light source is at most 1% of its maximum spectral intensity. The first light source is a light-emitting diode, for example.

The second light source generates an illumination light spectrum152having a spectral emission maximum in the range of 500 nm to 560 nm and a full width at half maximum in the range of 40 nm to 110 nm. Below approximately 430 nm and above approximately 650 nm, the intensity of the second light generated by the second light source is at most 1% of its maximum spectral intensity. The second light source is a light-emitting diode, for example.

The third light source generates an illumination light spectrum153having a spectral emission maximum in the range of 610 nm to 640 nm and a full width at half maximum in the range of 10 nm to 20 nm. The third light source is a light-emitting diode, for example.

FIG.16shows the wavelength-dependent transmittance161of an observation filter23(solid line) and the wavelength-dependent transmittance162of an illumination filter9(dashed line) in accordance with the seventh exemplary embodiment.

From λ1=420 nm to λ2=480 nm the transmittance161of the observation filter23is approximately 0.005%. From λ3=490 nm to λ4=500 nm the transmittance161of the observation filter23is approximately 2%. From approximately λ4to approximately λ5=540 nm the transmittance161of the observation filter23is approximately 0.005%. From λ5to λ6=700 nm the transmittance161of the observation filter23is approximately 95%. From approximately λ6to 750 nm the transmittance161of the observation filter23is approximately 0.005%. Furthermore, it holds true that W1=0.01%, W2=0.5%, and W3=80%.

From 350 nm to approximately λ1the transmittance162of the illumination filter9is approximately 0.005%. From λ1to approximately λ4the transmittance162of the illumination filter9is approximately 95%. From approximately λ4to approximately λ5′=630 nm the transmittance162of the illumination filter9is approximately 0.005%. From approximately λ5′ to λ6′=690 nm the transmittance162of the illumination filter9is approximately 0.08%. From approximately λ6′ to 750 nm the transmittance162of the illumination filter9is approximately 0.005%.

Accordingly, the observation filter23suppresses the first light reflected at the object13, said first light having a high intensity substantially only between λ1and λ2. Furthermore, the second light is suppressed by the illumination filter9above λ4. The proportion of the second light between λ3and λ4is reflected at the object13and transmitted by the observation filter23and thus contributes to the visualization of the non-fluorescent regions. Furthermore, the third light is suppressed by the illumination filter9below λ5′. The proportion of the third light between λ5′ and λ6′ is reflected at the object13and transmitted by the observation filter23and thus contributes to the visualization of the non-fluorescent regions. Furthermore, the fluorescent light emanating from the object13and having a high intensity substantially only between λ5and λ5′ is transmitted by the observation filter23, with the result that fluorescent regions of the object13can be observed with yellowish color valences.

Three major advantages are afforded by the individual settability of the intensities of the first, second, and third light (cf.FIG.15) and the concrete choice of the transmittance161of the observation filter23and the transmittance162of the illumination filter9. Firstly, the fluorescent regions and the non-fluorescent regions can be perceived as almost equally bright by virtue of the intensity of the light sources being set accordingly. Furthermore, the light sources here can be operated in their stable emission range since the light generated by the second and third light sources is damped to a sufficiently great extent by the observation filter23and the illumination filter9. Finally, as a result of the individual settability of the intensities of the second and third light, the color rendering of the non-fluorescent regions can be set such that the non-fluorescent regions can be observed as approximately true color.

FIG.17shows the CIE 1931 xy standard chromaticity diagram including the spectrum locus S and the purple boundary P. A hollow circle represents the D50 white point at x=0.3457 and y=0.3585. A cross at approximately x=0.35 and y=0.33 represents a color valence with which a white object is perceived by the normal observer using the microscopy system in accordance with the seventh exemplary embodiment if the observation filter and the illumination filter are configured as shown inFIG.16, the maximum spectral intensity of the light generated by the first light source is in each case approximately 3 times the magnitude of the maximum spectral intensity of the light generated by the second light source and the maximum spectral intensity of the light generated by the first light source is approximately 5 times the magnitude of the maximum spectral intensity of the light generated by the third light source. Non-fluorescent regions of the object can therefore be perceived as approximately true color. In addition, this configuration results in an approximately equal brightness of fluorescent and non-fluorescent regions of the object.

Third Aspect

One example of a microscopy method in accordance with the third aspect is described with reference toFIGS.18and19. The method can be carried out by the microscopy system in accordance withFIG.1and includes generating illumination light, directing the illumination light generated onto an object13and generating an observation beam path22, which images the object13into an image plane19, wherein an observation filter23is arranged in the observation beam path22.

FIG.18shows one exemplary configuration of a microscopy system for carrying out the microscopy method for protoporphyrin IX. A diagram181inFIG.18shows the wavelength-dependent absorption spectrum184of PpIX and the wavelength-dependent emission spectrum185of PpIX, in each case normalized to their respective maximum values; also see the description concerningFIG.2. A diagram182inFIG.18shows the wavelength-dependent transmittance186of the observation filter23for the same wavelength portion as in diagram181. A diagram183shows the wavelength-dependent intensity187of the illumination light directed onto the object13for the same wavelength portion as in diagram181.

A first value W1represents a mean value of the transmittance186of the observation filter23over a first wavelength range WLB1. A second value W2represents a mean value of the transmittance186of the observation filter23over a second wavelength range WLB2. A third value W3represents a mean value of the transmittance186of the observation filter23over a third wavelength range WLB3. The first value W1is less than the second value W2. The second value W2is less than the third value W3.

A fourth value W4represents a mean value of the intensity187of the illumination light over the first wavelength range WLB1. A fifth value W5represents a mean value of the intensity187of the illumination light over the second wavelength range WLB2. A sixth value W6represents a mean value of the intensity187of the illumination light over the third wavelength range WLB3. The fourth value W4is larger than the fifth value W5. The fifth value W5is larger than the sixth value W6.

The first, second, and third wavelength ranges do not overlap one another and lie in each case between 350 nm and 1000 nm. In the example shown, the first wavelength range WLB1extends from approximately 370 nm to 430 nm, the second wavelength range WLB2extends from approximately 470 nm to 580 nm, and the third wavelength range WLB3extends from approximately 600 nm to 750 nm.

What is achieved with the values for the ratios between the first to sixth values as indicated with regard to the third aspect is that non-fluorescent regions of the object can be observed by light in the first wavelength range WLB1and in the second wavelength range WLB2, while fluorescent light can be observed by light in the third wavelength range WLB3. Moreover, what is achieved on the basis of the indicated ratios between the first to sixth values is that non-fluorescent regions and fluorescent regions can be perceived as approximately equally bright. What is furthermore achieved is that the non-fluorescent regions can be perceived as approximately true color.

FIG.19shows one exemplary configuration of a microscopy system for carrying out the microscopy method for fluorescein. A diagram191inFIG.19shows the wavelength-dependent absorption spectrum194of fluorescein and the wavelength-dependent emission spectrum195of Fluorescein, in each case normalized to their respective maximum values; also see the description concerningFIG.2. A diagram192inFIG.19shows the wavelength-dependent transmittance196of the observation filter23for the same wavelength portion as in diagram191. A diagram193shows the wavelength-dependent intensity197of the illumination light directed onto the object13for the same wavelength portion as in diagram191.

In the example shown, the first wavelength range WLB1extends from approximately 440 nm to 500 nm, the second wavelength range WLB2extends from approximately 640 nm to 750 nm and the third wavelength range WLB3extends from approximately 520 nm to 620 nm.

One example of an alternative microscopy method in accordance with the third aspect is described with reference toFIGS.20to23. The method can be carried out by the microscopy system in accordance withFIG.1and includes generating illumination light, directing the illumination light generated onto an object13and generating an observation beam path22, which images the object13into an image plane19, wherein an observation filter23is arranged in the observation beam path22.

FIG.20shows a schematic illustration of a projected image of the object13into the image plane19. The projected image of the object13includes first locations201, second locations202, and third locations203. The light in the observation beam path22which generates the projected image in the image plane19has at the first locations201color valences which lie in a first color valence range211. The light in the observation beam path22which generates the projected image in the image plane19has at the second locations202color valences which lie in a second color valence range212. The light in the observation beam path22which generates the projected image in the image plane19has at the third locations203color valences which lie outside the first color valence range211and outside the second color valence range212.

FIG.22shows a CIE 1931 standard chromaticity diagram for elucidating the first color valence range211, the second color valence range212and the third color valence range213. The first color valence range211includes red color valences, that is to say the color valences of strong fluorescence of the fluorescent dye PpIX. The second color valence range212includes blue color valences, that is to say the color valences at which the fluorescent dye PpIX can be excited efficiently. The third color valence range213encompasses the remaining color valences of the visible color valences which are not contained in the first and second color valence ranges. An arrow214shows the smallest color difference between the first color valence range211and the second color valence range212.

In accordance with the alternative microscopy method, the illumination light is generated such that a ratio of a first characteristic value to a second characteristic value has a value within a predetermined delimited value range. The first characteristic value represents a value of a characteristic variable for the intensity (or the brightness or the luminous flux or the like) of the light in the observation beam path22at the first locations201. The second characteristic value represents the same characteristic variable in relation to the light in the observation beam path22at the second locations202.

By way of example, the first characteristic value represents a mean value of the intensity of the light in the observation beam path22at the first locations201, and the second characteristic value represents the mean value of the intensity of the light in the observation beam path22at the second locations202. If the illumination light is generated such that the ratio of the first characteristic value to the second characteristic value lies in the value range of 20/1 to 1/20 or a narrower value range, the first locations201, that is to say the fluorescent regions of the object13, and the second locations202, that is to say the non-fluorescent regions of the object13, can be perceived as approximately equally bright.

One example of the microscopy method is explained below with reference toFIG.23. The example relates to a method for controlling the microscopy system fromFIG.1, wherein the brightness of fluorescent and non-fluorescent regions of the object13is matched.

In step S1, an image of the object13in the image plane19is recorded by the color image detector20. The image is an image of the projected image of the object13generated through the observation beam path22in the image plane19.

FIG.21shows a schematic illustration of an image205recorded in step S1, which is an image of the projected image of the object13in the image plane19shown inFIG.20. The image205includes a plurality of image points206.

In step S2, following step Si, first image points207are determined from the image points206. The first image points207correspond to the first locations201. The image205has at the first image points207color valences which lie within the first color valence range211. The first image points207are determined for example by comparing the color valences at the image points206of the image205with the predetermined first color valence range211.

In step S3, following step S1, second image points208are determined from the image points206. The second image points208correspond to the second locations202. The image205has at the second image points208color valences which lie within the second color valence range212. The second image points208are determined for example by comparing the color valences at the image points206of the image205with the predetermined second color valence range212.

In step S4, following step S2, the first characteristic value is determined on the basis of the intensity of the image205at the first image points207. By way of example, the mean value or the maximum value of the intensity at the first image points207is determined.

In step S5, following step S3, the second characteristic value is determined on the basis of the intensity of the image205at the second image points208. By way of example, the mean value or the maximum value of the intensity at the second image points208is determined.

In step S6, which follows when steps S4and S5are concluded, the illumination light is set depending on the first and second characteristic values or depending on the intensity of the light in the observation beam path22in the image plane19at the first and second locations201,202or depending on the intensity at the first and second image points207and208. That is to say that the wavelength-dependent intensity distribution of the illumination light is changed. In this case, the illumination light is generated such that the above-described ratio between the first and second characteristic values has a value within the predetermined value range.

The method can be carried out repeatedly, which is illustrated by an arrow from step S6to step S1inFIG.23.

It is understood that the foregoing description is that of the exemplary embodiments of the disclosure and that various changes and modifications may be made thereto without departing from the spirit and scope of the disclosure as defined in the appended claims.