DE19927724A1 - Endoscope, microscope or endomicroscope for two-photon excitation of tissue - Google Patents

Abstract

An endoscope, microscope or endomicroscope includes and optic fiber (20) for transmitting a pulsed main beam (14) along an optical path between a femtosecond laser (12) and the tissue sample (44). Reverse chirped Bragg grating fibers (28, 30) act to reverse the dispersion occurring in the optic fiber. An Independent claim is also included for the following: A method of two-photon endoscopy or microscopy using the principles of the described endoscope/microscope.

Description

The invention relates to the field of endoscopy and Mi. microscopy and a. endomicroscopy, and in particular Endoscopy and microscopy (including endomicroscopy) with light waveguides or optical fibers.

In the following we speak of microscopes, should understand that this also applies to endoscopes.

Recently, Ti: sapphire laser with ultra short pulses can produce extremely high optical excellence at the focal point of a lens with high NA, resulting in Produce nonlinear optical effects with high efficiency to let. It has been found that techniques with two-photon Ab Touch point excitation Advantages for use in conventional Have benchtop microscopes.

Although the clinical use of two-photon excitation could potentially offer the ability to take pictures from larger Depth below the surface of a fabric, reduced photo bleaching and the ability to stimulate UV dyes and indicators without the use of unreliable UV lasers and getting special lenses has never been clear like that Two-photon excitation in the field of clinical endomicroscopy pie could be realized sensibly. For example, they are  high cost of the short pulse laser a major disadvantage for one broader use of two-photon scanning point microscopy.

More critical with regard to the invention is that the Use of an optical fiber to deliver the laser light (as in previous endomicroscope designs) the pulse stretches, which significantly reduces the peak intensity. There this reduces the efficiency of the nonlinear changes interaction, which lowers the signal strength. A pulse spread tion in a fiber in the linear range comes from chromati time dispersion, the degree of which is based on the Fourier transform of the pulse envelope or can be predicted using the Heisenberg uncertainty principle. The blue moves as the pulse passes through the fiber End of the Fourier transform slower than the red, and a broad chirp pulse emerges from the fiber.

Thus, these two facts alone seem to conclude any consideration of the two-photon endoscope as clinical diagnostic tool.

Therefore, it is an object of the invention to provide a ver improved endoscope, microscope or endomicroscope under one To provide the set of two-photon excitation of a tissue.

This task is accomplished with the subject according to the claims Chen solved.

The invention thus provides an endoscope or microscope which has:
an optical fiber for transmitting a pulsed main beam over at least a portion of an optical path between a beam source and a tissue or other sample; and
a disperser;
whereby dispersion occurring in the optical fiber is reversed by dispersion generated by the dispersing device.

The dispersing device preferably has a compression optically arranged after the optical fiber Direction to compress the beam after the beam hits the optical fiber leaves to the peak power of the main to reduce the beam within the optical fiber.  

The dispersing device can be optically before or after optical fiber and can be another have optical fiber.

The endoscope or microscope preferably also has via a disper optically arranged in front of the optical fiber Sionseinrichtung for dispersing the main beam before the Main beam enters the optical fiber.

The dispersion device and / or the compression device direction preferably each have a glass block Grid or a prism on.

Preferably, the dispersion device and / or the compression device on highly dispersing glass.

Preferably, the optical fiber has a large core ß diameter and thus lower standardized frequency or V-number to reduce the area power density in the core ren.

The optical fiber can be a pure silicon oxide core and have a fluoride-doped cladding to make nonlinear op minimize table effects within the fiber.

The invention also provides a method for two-phase endoscopy or microscopy with the following steps:
Transmitting a main pulsed laser beam along an optical fiber over at least a portion of an optical path between a beam source and a tissue or other sample; and
Dispersing the beam by means of a dispersing device;
whereby dispersion occurring in the optical fiber is reversed by dispersion generated by the dispersing device.

In a special aspect of the invention, an endoscope or microscope is provided which has:
beam splitter means for splitting a main pulsed laser beam into first and second beams;
a first optical fiber for transmitting the main beam to the beam splitter device;
anti-dispersion means for reducing the dispersion of the first and second beams;
an optical coupler for recombining at least a portion of the respective first and second beams into a recombined beam; and
a second optical fiber for transmitting the recombined beam from the optical coupler to an endoscope or microscope head;
wherein the main beam can be divided by the beam splitter device into the first and second beams, the dispersion of which can subsequently be reduced by the anti-dispersion device and which can then be recombined by the optical coupler device to form a recombined beam for transmission along the second optical fiber To form head.

This allows a pulsed laser beam to enter a two-phi tone beam for use in an endoscope or microscope being transformed. The anti-dispersion device is reduced the dispersion or compresses the first and second Beam. It should be understandable that the compression except half of the anti-dispersion device or in the endoscope or Microscope head or even occur in a tissue sample or can be completed.

The endoscope or microscope preferably has a La source to provide the main beam.

The laser source is preferably an ultrashort pulse and more preferably a femtosecond laser.

The laser source is preferably a titanium sapphire laser or a Cr: LiSrAlF ₆ laser.

The first optical fiber is preferably a monomo the fiber at the wavelength of the main beam.

The beam splitter device is preferably an opti coupler and more preferably a welded double cone taper coupler.

The beam splitter device preferably divides the Main beam roughly equal to the first and second beams.  

The optical coupler device is preferably an op table coupler and more preferably a welded dop Pelkonus taper coupler.

The beam splitter device and the optical coupler device by a single optical Coupler and more preferably by a single ge welded double cone taper coupler.

The anti-dispersion device preferably has a reverse chirp Bragg grating fiber whose more closely spaced Grid optically arranged closer to the beam splitter device are net, and more preferably over a first and a second Reverse chirp Bragg grating fiber, one for receiving the jewei leagues first and second beam, their respective closer grid was optically closer to the beam splitter device are arranged.

This allows Bragg lattice fibers to be used compress the first and second beams. These work also used as a dichroic mirror to select the by one Tissue or other sample emitted fluorescent light through dichroic beam splitting.

The endoscope or microscope preferably also has a large diameter optical fiber and a cladding mode coupler, with the cladding mode coupler the second op table fiber and the large diameter fiber so couples that wide-angle scattered light from the head through the coupler in the we significant in the large diameter fiber for transfer is coupled to a photodetector.

In a preferred embodiment, the endoscope or microscope optically after the second optical fiber arranged compression device for compressing the right combined beam after the recombined beam leaves the second optical fiber, causing the tip line Main beam within the first optical fiber and the recombined beam in the second optical fiber is reduced.

The endoscope or microscope preferably also has a Dis optically arranged in front of the first optical fiber  dispersion device for dispersing the main jet, before the main beam enters the first optical fiber.

The dispersion device and / or the compression device direction preferably have a glass block, a grid or a prism on.

Preferably, the dispersion device and / or Compression device on highly dispersing glass.

In a special embodiment, the first op table fiber and the second optical fiber each have a core with a large diameter and thus a lower V-number to achieve the Reduce area power density in the cores.

In a further special embodiment, the first optical fiber and the second optical fiber pure Si silicon oxide cores and fluoride-doped shells to make nonlinear to minimize optical effects within the fibers.

According to a further aspect of the invention, an endoscope or microscope is provided which has:
a dispenser for temporarily spectroscopically dispersing a pulsed main laser beam; and
an optical fiber for transmitting the dispersed main beam to an endoscope or microscope head and reducing the dispersion by compressing the dispersed main beam.

Instead of first allowing dispersion in an op table fiber etc. occurs and then this dispersion to cor rig, the dispersion can initially be in a counter direction introduced and then in the transmission of the beam be removed through the fiber to the sample.

The optical fiber can pass through the dispersing device essentially reverse the dispersion introduced. Dude natively, the endoscope or microscope head can optically look like the optical fiber arranged compression device for Promote or complete the compression of the dispersed Main beam to show the peak power of the main to reduce the beam within the optical fiber.

The dispersing device is preferably also a Beam splitter device for selecting through a tissue or another sample of fluorescent light emitted.  

The dispersing device preferably has a pair Diffractive or refractive elements, and more preferably one Pair of blaze grids and a prism on.

According to the invention, an endoscope or microscope with a head is also provided, the endoscope or microscope having:
a suction or vacuum source, which is arranged close to the head;
a light interceptor for intercepting light from a sample; and
sample receiving means, coupled to the suction source, for receiving the sample when the sample is pressed thereon by the suction source;
the suction source being used to urge the sample toward the pickup, deforming the sample so that the light interceptor can trap some of the light emitted by the sample away from the head.

The sample receiving device is preferably cup-shaped or alternatively frustoconical.

The light interception device is preferably in use separated from the sample by the sample receiving device, and the sample holder is for the light between the sample and the light interception device essentially permeable.

The light interception device can be an optical fiber exhibit.

Alternatively, the light interception device can play a game have gel. The endoscope or microscope can be a lens to focus light from the mirror.

The light interception device is preferably one of several light interception devices.

The invention also provides a method for two-phase endoscopy or microscopy with the following steps:

• 1) Longitudinal transmission of a pulsed main laser beam an optical fiber;
• 2) Split the main beam into first and second Beam;  
• 3) compressing the first and second beams;
• 4) combining at least a portion of the first and second beam;
• 5) transmitting the first and second beams along one optical fiber to an endoscope or microscope head.

In a specific embodiment, the invention provides an endoscope or microscope system that includes:
an endoscope or microscope with a photodetector;
a lighting device for providing ambient lighting;
a switching device for switching the lighting device on and off; and
a synchronization device for synchronizing the switching device with acquisition periods of the endoscope or microscope;
wherein the synchronization device is operable to control the switching device in order to switch the lighting device off during the detection periods and on between successive detection periods.

The synchronization device is preferably so drivable that it controls the switching device to the loading lighting device to switch so that the lighting device during the return period of the endoscope or the microscope is switched on.

The photodetector can be operated so that it is out is switched on when the lighting device is switched on is.

The photodetector preferably has a power supply, which can be operated in such a way that it is switched off when the Lighting device is switched on.

Preferably, the photodetector has a photomultiplier tube, and the power supply is a high voltage power supply (EHT power supply). In another specific embodiment, the invention provides an endoscope or microscope system that includes:
an endoscope or microscope with a photodetector;
a lighting device for providing ambient lighting; and
a filter device for reducing the detection of the ambient lighting by the photodetector.

The lighting device preferably emits Light that is preferably blocked by the filter device becomes.

Alternatively, the filter device is a first filter device, and the system has a second filter device for absorbing light from the lighting device preferred by the first filter device is transmitted, and transmitting light from the lighting tion device preferred by the filter device is blocked.

The system preferably has a switching device Switching the lighting device on and off and one Synchronization device for synchronizing the switching device with acquisition periods of the endoscope or micro skops, with the synchronization device operating in this way bar is that it controls the switching device to the loading Illumination device off during the acquisition periods between successive acquisition periods ten.

Preferably the system further includes a shutter direction to cover the photodetector, the syn Chronization device is operable so that it ver closing device controls to cover the photodetector, when the lighting device is switched on, and the Expose the photodetector during the acquisition periods.

The light detection device (in general a photomultiplier tube) before exposure to the reverse exercise light protected in the course of non-detection periods, to reduce noise levels.

To clarify the invention in the following be preferred embodiments of the invention as examples of the accompanying drawings. Show it:  

Fig. 1A is a schematic view of an optical two-photon Faserendomikroskops according to a preferred exporting approximately of the invention;

Fig. 1B is a schematic view of an optical two-photon Faserendomikroskops according to an alternative before ferred embodiment of the invention;

Fig. 2A is a schematic view of an optical two-photon Faserendomikroskops according to another preferred embodiment of the invention;

Fig. 2B is a closer view with the head of the endomicroscope of Fig. 2A;

Fig. 3A is a schematic view of an optical two-photon Faserendomikroskops according to yet another before ferred embodiment of the invention;

Figure 3B is an enlarged view of a detail of Figure 3A;

Fig. 4 is a view of a portion of an endomicroscope according to yet another preferred embodiment of the invention;

Figure 5 is a view of the endomicroscope head assembly for use with the endomicroscope of Figures 2 or 3;

Fig. 6 is a view of another endomicroscope Kopfan arrangement for use with the endomicroscope of Fig. 2 or 3;

Fig. 7 is a view of an endomicroscope head assembly for use with an inventive endomicroscope;

Fig. 8 is a view of another endomicroscope-Kopfan arrangement for use with an endomicroscope according to the invention;

Fig. 9 is a view of still another endomicroscope head assembly for use with an inventive En domikroskop;

Fig. 10 is a view of another endomicroscope Kopfan arrangement for use with an endomicroscope according to the invention, which is a variant of the arrangement of Fig. 9;

Figs. 11A, 11B and 11C are schematic diagrams for Dar provision of a method for using a erfindungsge MAESSEN endomicroscope; and

Fig. 12 is a view of a portion of an endomicroscope according to yet another preferred embodiment of the invention.

An optical two-photon fiber endomicroscope according to a preferred embodiment of the invention is shown schematically at 10 in FIG. 1A. The endomicroscope 10 has a laser source in the form of a femtosecond laser 12 for generating a rapidly pulsed beam 14 , a lens 16 for focusing the beam 14 and an optical fiber 20 with a core 18 . The lens 16 serves to focus the beam 14 into the core 18 . The optical fiber 20 is a single mode fiber at the wavelength of the laser 12 .

Each pulse of the beam 14 passes through the fiber 20 to a beam splitter in the form of a welded double cone Ta perkopplers 22nd The coupler 22 divides the main beam 14 et wa evenly into two beams, so that pulses of each of these beams from the coupler 22 along the two output branches of the coupler, optical fibers 24 and 26 , who transmit the. The optical fibers 24 and 26 each have an anti-dispersion device in the form of reverse chirp Bragg grating fibers 28 and 30 , which are closer to the coupler 22 with the shorter spaced grating elements 28 a and 30 a than with the longer spaced grating elements 28 b and 30 b are arranged. As a result, the pulses of each of these beams hit a grating 28 or 30 after leaving the coupler 22 .

The pulses are reflected from gratings 28 and 30 back to coupler 22 with the blue, shorter wavelength components of each pulse leading the red, longer wavelength components of the pulse. The pulses arrive at the coupler 22 close to one another, but not exactly in phase. No attempt is made to exactly match the fiber distances from the Bragg gratings 28 and 30 to the coupler 22 . Each pulse is redistributed by coupler 22 such that two (a component of each pulse reflected by gratings 28 and 30 ) are passed along fiber 20 back to laser 12 and two pulses (also a component of each of gratings 28 and 30 reflected pulse) are passed along a fiber 32 . The impulses in fiber 32 gradually concentrate as the red components catch up with the blue ones. The pulses leaving a fiber tip 34 are thus almost recombined as one pulse (taking into account the compensation of the last chromatic difference delays in lenses 36 and 38 of the endomicroscope head 40 ).

The pulses guided along the fiber 32 are focused by the lenses 36 and 38 to form a Gaussian constriction 42 within the tissue 44 to be examined. From scanning the Gaussian constriction 42 within the tissue can be achieved by a mechanism 46 which triggers movements of the fiber tip 34 in a grid, or by mirror scanning or other desired scan pattern generation.

Fluorescence generated at the Gaussian constriction 42 by two-photon excitation of fluorophores within the tissue 44 runs back through the lenses 38 and 36 and is focused back into the core of the fiber 32 . The fluorescent light then passes through fiber 32 to coupler 22 . It is then divided by the coupler 22 and passes through the fibers 24 and 26 . The fluorescent light, which has a much shorter wavelength than the main beam 14 , passes through the Bragg mirrors 28 and 30 (which thus act as dichroic beam splitters) to the ends 48 and 50 of the fibers 24 and 26 , from which it leads to the photomultiplier tube 52 runs after it has passed through a BG39 spectral filter 54 . Conventional acrylic-coated fibers and communication couplers are quite suitable for this system.

An optical two-photon fiber endomicroscope according to an alternative preferred embodiment of the invention is shown schematically in FIG. 1B, in which the same reference numbers denote the same components as in FIG. 1A. In contrast to the embodiment of FIG. 1A, however, a dispensing device with a first and second blaze grating 55 a and 55 b and a 90 ° retroreflector prism 56 are arranged optically between the laser 12 and the lens 16 . The locations of maxima in the light that is diffracted by the first grating 55 a are wavelength-dependent, so that the beam 14 is spatially effectively divided into its component frequencies: this is shown schematically with light 14 a of shorter wavelength and light 14 b of longer wavelength. Then the beam is reflected by the second grating 55 b to the prism 56 (or alternatively to a mirror), which returns the light via the second grating 55 b to the first grating 55 a. The combination of the gratings 55 a and 55 b and the prism 56 introduces a path difference between the components 14 a and 14 b of the beam 14 with a shorter and longer wavelength and an offset on the first grating 55 a, so that the beam there after is directed to the lens 16 . The lens 16 focuses the components of the now dispersed beam pulses into an optical fiber 58 , the components of shorter wavelengths leading the components of longer wavelengths. As these components move along fiber 58 , the longer wavelength components gradually catch up with the shorter wavelength components. Thus, the pulses leaving a fiber tip 59 are almost recombined as one pulse (taking into account the compensation of the last chromatic difference delays in the subsequent lenses of the endomicroscope head according to the discussion above).

As in the embodiment of Fig. 1A fluorescence produced is in the sample Siert back focus in the core of the fiber 58, the fiber passes through 5 B to the lens 16 and is reflected by it most grid 55 a to the photomultiplier tube 52 after a (not shown) BG39 spectral filter runs through. The returned fluorescence is two-photon induced so that it has a different wavelength range than the beam 14 . Thus, it is diffracted at a different angle than the pulsed beam 14 and can be appropriately detected by the photo multiplier tube 52 without disturbing the optical path of the components 14 a and 14 b of the beam 14 .

In Fig. 2A, an endomicroscope is shown according to another preferred embodiment of the invention at 60. The endomicroscope 60 is similar to the endomicroscope 10 of FIG. 1A and has a laser source 62 , a lens 64 , an optical fiber 66 , a coupler 68 with two output branches (fibers 70 and 72 ), each having a reverse chirp Bragg grating fiber 74 and 76 have, except that the last section of an optical glass cladding fiber 78 , which leads from the coupler 68 to the endomicroscope head 80 , with a material (not shown in the illustration) of low refractive index, e.g. B. silicone rubber low refractive index, is coated, which allows the transmission of cladding modes. Pulsed laser light that emerges from a fiber tip 82 is focused by lenses 84 and 86 to form a Gaussian constriction 88 in the tissue 90 .

As a result of "snake light" 92 (forward scattering of the 2ω-excited fluorescent light at a small angle) which is emitted by the Gaussian constriction region 88 , a large part of the fluorescence misses the core 94 of the fiber 78 during the return, but enters the glass jacket of the fiber 78 . These snake light beams are limited by the silicone rubber jacket with a low refractive index and spread as jacket modes 96 and 98 until they hit a jacket mode coupler 100 .

At this point, endomicroscope 60 , coupled by cladding mode coupler 100 to fiber 78 , has a large diameter fiber 102 . As a result, the Mantelmo the 96 and 98 are largely decoupled into the large diameter fiber 102 and move along this fiber 102 to the photomultiplier tube 104 , which generates a signal for a suitable imaging system (not shown).

FIG. 2B is a closer view of the endomicroscope head 80 of FIG. 2A. Generated two-photon fluorescence 92 is emitted at the laser focal point 88 in the tissue 90 . The two-photon fluorescence of shorter wavelength is subject to greater forward scattering at a small angle ("snake scattering light"), as a result of which, as described, the return light passing through the lenses 86 and 84 of the endomicroscope head 80 does not refocus to the core 94 of the fiber 78 . Instead, the stray light enters the glass cladding 95 and is captured by the clear silicone rubber cladding 106 of low refractive index. A cladding mode coupler 100 takes most of this light into the large diameter fiber and leads it to the photomultiplier tube 104 .

Fig. 3A is a view of yet another preferred embodiment of an endomicroscope 110 according to the invention. In this embodiment, the endomicroscope 110 has a very flat-angled prism 112 which is inserted in the optical path between the lenses 114 and 116 .

Otherwise, the endomicroscope 110 is similar to the endomicroscope 60 of FIGS . 2A and 2B. Thus, the endomicroscope 110 has a laser source 118 , a lens 120 , an optical fiber 122 , a coupler 124 with two output branches (fibers 126 and 128 ), each having a reverse chirp Bragg grating fiber 130 and 132 , and an optical fiber 134 from coupler 124 to an endomicroscope head 136 . The optical fiber 134 has a glass cladding 138 (for the sake of simplicity only shown in the area of a cladding mode coupler 140 and the endomicroscope head 136 ).

In addition, the endomicroscope 110 has the mentioned Man telmodenkoupler 140 and a fiber 142 with a large diameter.

The prism 112 does not have sufficient dispersion to distribute the excitation beam significantly at the Gaussian constriction region 144 , but deflects the shorter fluorescence wavelengths by a sufficient amount so that they are shifted sideways to the side 146 of the core 148 emitting the excitation wavelengths.

This ensures that most or all of the 2ω-excited fluorescence is returned as cladding modes in the light supply fiber 134 a. These modes are coupled into the fiber 142 with a large diam ser through the Mantelmodenkoppler cherröhre 140 and through the fiber 142 to the Photovervielfa out 150th

This would provide a simpler optical return path for the fluorescence, which runs entirely over the fiber of the Mantelmo denkopplers 140 .

Although not shown, a similar effect could be achieved without the need for prism 112 if lenses 114 and 116 were low in chromatic aberration.

Fig. 3B is an enlarged view of the endomicroscope head 136 of FIG. 3A with the fiber 138 of the fiber 142 having a large diameter and the Mantelmodenkoppler 140th In this representation, the lateral shift of the fluorescence beams of shorter wavelength to the side 146 of the core 148 of the fiber 138 is clearer.

Fig. 4 is a schematic view of a Endomikro Skops 160 according to another preferred embodiment of the invention. The endomicroscope 160 has a laser source in the form of a Ti: sapphire laser 162 for providing a laser beam 164 , a focusing lens 166 and an optical fiber 168 . The optical fiber 168 directs the beam 164 to a welded double-cone taper coupler 170 , which divides the beam 164 into two approximately equal beams that are passed through fibers 172 and 174 , respectively. As in other preferred embodiments previously described, fibers 172 and 174 each have a reverse chirp Bragg grating fiber 176 and 178, respectively. These Bragg gratings 176 and 178 are oriented closer to the coupler 170 with the shorter spaced grating elements 176 a and 178 a than with the longer spaced grating elements 176 b and 178 b.

An endomicroscope head 180 is provided which is optically connected to the coupler 170 by an optical fiber 182 . Fig. 4 illustrates the head 180 in its arrangement for examining a tissue sample 184th

Fluorescent light is detected by a photomultiplier tube 175 after it has passed through a BG39 spectral filter 177 .

In addition, the endomicroscope 160 has two prism sets 186 and 188 , which are arranged optically between the laser 162 and the focusing lens 166 .

Each prism set 186 and 188 has a pair of closely positioned optical wedge prisms 186 a, b and 188 a, b, respectively.

The prisms 186 a, b and 188 a, b are movable on tracks 187 a, b and 189 a, b, either by one or more actuation motors (not shown) or manually, so that the optical path length traveled by the beam within the Endomi microscope 160 can be adjusted. The prism movement is parallel to the sloping adjacent surfaces of each prism set 186 or 188 . In the illustrated form of this embodiment, all four prisms are movable. For the sake of simplicity, however, it may be preferred that the prisms 186 a and 188 a or 186 b and 188 b are movable.

The prisms 186 a and 186 b consist of quartz glass or germanium-doped silicon oxide (or another material with a relatively linear dispersion) and can be adjusted so that they compensate for the variability of the total fiber length between systems.

The primes 188 a and 188 b are made of flint glass or another material with strongly curved dispersion curves and can be moved in and out to adjust the chirplinearity. Both prism sets 186 and 188 are to be used to compensate for deviations in the optical path length in the glass and also for deviations that could be introduced when the last lenses 190 and 192 of the head 180 are changed.

Then the prism sets 186 and 188 (by means of the Bragg grating fibers 176 and 178 ) allow the over-compensated system to be tuned. In addition, the prism sets 186 and 188 can be used to adjust the optical path length in order to keep the variability between the fiber systems within the optical path distance.

Fig. 5 is a view of the head portion of an Endomi microscope according to another preferred embodiment of the invention in use. In this embodiment, the endomicroscope uses a suction effect to draw tissue to be examined into a well, which is provided adjacent to the optical front surface of the endomicroscope head.

The endomicroscope of this embodiment has a head 200 with lenses 202 and 204 and an optical front plate 206 . Also provided is a suction hose 208 (which is connected to a suitable vacuum or vacuum source, not shown), in order to suck tissue to be examined 210 into a cup 212 , which is formed by a transparent wall 214 and the optical front plate 206 of the endomicroscope head 200 is. The hose 208 is connected to the bowl 212 so that the suction provided by the hose 208 pulls the tissue 210 into the bowl and onto the plate 206 .

One or more optical fibers 216 a, b are arranged around the wall 214 of the cup 212, with their ends have rich for loading, which is examined in the well 212th These fibers 216 capture the fluorescent light from a focal point 218 that does not enter the lens 204 and would not otherwise contribute to the signal. As a result, at least a portion of such light directed toward the wall 214 is received by the optical fibers 216 and transmitted to a photomultiplier tube 220 .

Fig. 6 shows a similar arrangement as Fig. 5, which differs only in that a cup 222 is frustoconical and optical fibers 224 a, b each have tapered "radiation bundle" fiber tips 226 a, b to the diameter of to the fiber bundle leading to the photodetector 220 .

Fig. 7 is a schematic view of an arrangement according to the invention, in which additional fluorescent light is not collected by placing optical fibers adjacent to the focal point of the laser in the examined tissue, but by using one or more mirrors.

An endoscope head 230 , which has lenses 232 and 234 and is attached to an optical fiber 236 , also has mirrors 238 a, b, which are located at the front end 240 of the head 230 . Fluorescent light in the execution form of Fig. Would be captured by the fibers 216, or 224 5 and 6, instead of by the mirrors 238 in fibers 242 a, reflecting b which extend parallel to the main axis of the En domikroskopkopfs 230th Lenses 244 a, b are arranged between the mirrors 238 a, b or fibers 242 a, b in order to focus light into the fibers 242 a, b and thus to capture as much light as possible.

However, these lenses 244 are optional and, as shown in Figure 8, it may be desirable to exclude them (for compactness or light weight of the endomicroscope head or for other reasons). Such an arrangement is shown in FIG. 8, in which reference numerals correspond to those of FIG. 7.

The arrangements of FIGS. 7 and 8 avoid curved fibers which, as in the embodiments of FIGS. 5 and 6, originate from the region surrounding the tip.

However, these arrangements can also use a suction hose and a cup (e.g., as shown in FIGS. 5 and 6), with the mirrors 238 arranged adjacent to and outside of the cup.

Note that in each of the embodiments, un The use of suction to insert tissue into a bowl suck, the light is collected from a solid angle can, which exceeds 2π sterad, d. H. a much higher up trapping efficiency than that of a conventional LSCM-In photon endoscope is possible.

By "cup pulling" due to vacuum or suction the tendency is that the person to be examined The area swells with blood by the microvasculature is artificially dilated. This potentially reduces it the signal level from fluorescence since hemoglobin is strong Ab sorption bands in the blue, green and yellow range. On One way to overcome this problem would be to use a Thread around the edge of the "bowl". By contracting this Thread ring is a "button" compressed from tissue and that Blood squeezed out to pale during contact of the tissue with the optical front surface of the endomicroscope still remains.

An alternative way of realizing one or more of these advantages (larger solid angle when capturing light and excluding additional blood) is to clamp and hold a sample tissue flap on the first optical surface of the endomicroscope. In this case, optical return can also be obtained from the back of the flap. Examples of games for these embodiments are shown in FIGS. 9 and 10.

In one embodiment of the invention (which is shown in FIG. 9), the endomicroscope is provided with a clamping device in the form of transparent plates 250 and 252 for clamping a tissue sample 254 . In addition, the plate 250 serves as a front optical element of an endomicroscope head 256 , which also has lenses 258 and 260 . Head 256 is attached to an optical fiber 262 from which a pulsed laser beam is emitted.

Furthermore, this arrangement has a first mirror 264 , which is arranged behind the plate 252 , a lens 266 and a second mirror 268 . Fluorescent light that is scattered back from the tissue 254 through the plate 252 is reflected by the mirror 264 , focused by the lens 266 and then reflected by the second mirror 268 into an optical fiber 270 , which increases the detected total solid angle.

When the plates 250 and 252 are pressed harder against the tissue 254 , they cause the tissue 254 to become pale as described above, which reduces the excessive blood accumulation in the tissue 254 .

It is clear that the mirrors 264 and 268 could be omitted in this arrangement and light could be focused directly through the lens 266 into the fiber 270 by the lens 266 being oriented at a 90 ° angle with respect to the position of FIG. 9 and the Fiber 270 is arranged directly behind the plate 252 . Because of their greater compactness, the arrangement shown is preferred.

Alternatively, mirrors 264 and 268 , lens 266 and fiber 270 could be eliminated if plate 252 is replaced with a retroreflector plate as shown in FIG. 10.

In this arrangement, the tissue 254 is in turn clamped, but between the plate 250 and a retroreflector plate 272 . A retroreflector plate can reflect light back in parallel to the direction of incidence, but spatially shifted. As a result, light scattered from the tissue 254 back to the plate 272 is reflected back through the tissue 254 and caught by a suitable method, e.g. B. the previously described with reference to Fig. 1, 2A, 3A or 4 be.

Because of the wide angular range in the optical catch, the corner cubes 274 of the retroreflector plate 272 cannot build on total internal reflection, which is why the retroreflector plate 272 is metallized.

A method of facilitating use of the two-photon endomicroscope in situations where a high level of illumination is required in clinical studies will now be described with reference to FIGS . 11A, 11B and 11C.

In confocal single-photon microscopy and endomicroscopy, the return light is spatially filtered through the pinhole, which effectively eliminates interference from ambient light. In the two-photon endomicroscope, it would be desirable to have a larger aperture in order to collect "serpentine" return photons and to amplify the signal. However, this can lead to a higher level of ambient false light, especially if a reasonably high level of illumination in the clinical examination environment was necessary. This effect can be particularly important if additional fibers are used to increase the signal value to the photodetector as in the embodiments of the invention from FIGS. 5 to 8.

Both endoscopes and microscopes (including Endomicroscopes) this effect can be reduced by the head or the objective lens in a case or other Ab cover is included, which covers the tissue under investigation there or is essentially sealable to it. But will um this light effect can still be detected further reduce or eliminate the general or Ambient lighting is supplied by a light source that Light only generated during the system's flyback period e.g. B. a strobe type system. In extreme circumstances could also use the photomultiplier tube voltages (PMT voltages) interrupted during the return lighting period to prevent PMT saturation and overload.

Alternatively or additionally, the room lighting Be light of a color or a range of colors, and the PMT could be provided with a filter that this Far be (n) does not let through significantly. For example, B. the reverse  lighting source, a red light source or a light source behind a red filter, and the PMT could be with be provided with a blue or blue / green filter.

It can also be used to reduce the light level on the PMT hit in non-acquisition periods, each of these tech niken by a closure (e.g. a mechanical ver or an electro-optical shutter) over the PMT be supplemented. This lock would be in non-detection peri oden closed (to protect the PMT from ambient light, that could contribute to noise levels), but in detection pe open periods.

Figure 11A is a graphical representation of the varying position of the scanning mirror of an endomicroscope. The scanning position changes essentially as a sine wave 280 with (in this example) a frequency of 750 Hz.

Image acquisition periods 282 and 284 are shown in FIG. 11B with their timing relative to the Spiegelabtastpo position shown by dashed lines 282 a and 284 a.

As a result, ambient lighting is limited to those periods in which no detection takes place, which is shown in FIG. 11C with hatched areas 286 and 288 .

In some special applications it may be desirable or be necessary to use high beam powers, e.g. B. can be desired with deep tissue imaging up to 500 mW his. However, nonlinear optical effects can occur in the glass the fiber due to self-modulation serious. Lei Power loss and short wavelength generation at peak performance over 50 mW at a pulse rate of 100 fs in a standard optical fiber.

According to the invention, this problem can be solved in several ways Because of reduce.

The first of these arrangements is illustrated in FIG. 12, which shows an endomicroscope 290 similar to FIG. 4. However, it should be clear that these and the further arrangements described below can be used with endomicroscopes according to any of the embodiments of the invention.

According to FIG. 12, an endomicroscope 290 has a laser source 292 , two prism sets 294 and 296 , a focusing lens 298 , a first optical fiber 300 , a double cone taper coupler 302 , fibers 304 and 306 with reverse chirp Bragg grating fibers 308 and 308, respectively 310 , which are oriented closer to the coupler 302 with the shorter spaced grating elements than with the longer spaced grating elements, and an endomicroscope head 312 which is optically coupled to the coupler 302 by a second optical fiber 314 . Fig. 12312 shows the head in the arrangement for examining a tissue sample 316th Fluorescent light emitted from the tissue sample 316 is detected by a photomultiplier tube 318 after it has passed through a BG39 spectral filter 320 .

The endomicroscope 290 works similarly to the endomicroscope 160 of FIG. 4. However, the endomicroscope 290 additionally has a dispersion device in the form of a first block 322 made of highly dispersing glass, which is arranged between the laser source 292 and the pair of prisms 294 , and a compression device in Form of a second block 324 made of highly dispersing glass, which is arranged within the Endomi microscope head 312 between the first lens 326 and the second lens 328 . The lengths of the first and second optical fibers 300 and 314 are reduced so that additional dispersion effects can be introduced through the first and second glass blocks 322 and 324 .

If the pulsed beam from the laser source 292 passes through the first glass block 322 , the beam is subjected to an initial dispersion before it enters the first optical fiber 300 . This reduces the peak power of the beam in the first optical fiber 300 from its value without using the glass block 322 , thereby reducing nonlinear optical effects in the glass of the first and second optical fibers 300 and 314 .

In contrast to the endomicroscope 160 , in which the recompression of the beam is practically completed by the time the beam emerges from the second optical fiber 182 , in the endomicroscope 290 the beam leaves the second optical fiber 314 without recompression being completed, so that the peak power in the second optical fiber 314 remains at a reduced value. The second glass block 324 provides the additional required recompression only after the beam has left the second optical fiber 314 , and the problem of nonlinear optical effects within the second fiber 314 is avoided.

The glass blocks 322 and 324 normally consist of highly dispersing lead glass, although other highly dispersing elements are also suitable, including grids or other prism shapes.

As previously mentioned, the lengths of the first and second optical fibers 300 and 314 are reduced to compensate for the introduction of the glass blocks 322 and 324 . However, it should be noted that the length of the glass blocks 322 and 324 need not be the same, ie any inequality in their lengths can be adjusted by adjusting the individual lengths of the first optical fiber 300 and / or second optical fiber 314 or by adjusting the gratings 308 and 310 can be compensated. In some applications, it may be very desirable to minimize the length of the second glass block 324 so that minimal weight and length are added to the endomicroscope head 312 . In some applications, it may actually be acceptable or desirable to completely remove the first glass block 322 .

A second arrangement according to the invention, by means of which the problem of nonlinear optical effects in the optical fibers can be reduced, consists in using monomode fibers with a core with a large diameter for the first and second optical fibers (for example fibers 168 and 182 in the embodiment of FIG. 4 or fibers 300 and 314 in the embodiment of FIG. 12). The first and second fibers would therefore have a low V number and thus an effectively low NA. In principle, such single-mode fibers with a core with a large diameter can also be used between the coupler and the Bragg gratings, but this is not critical since the beam is highly dispersed in the optical path at this point.

Therefore the area power density is reduced in the core, what the problem of nonlinear effects at the core of the first and second optical fiber eliminated. A disadvantage of this type  order is that due to the larger diameter of the optical fibers only gradual curves or curves in the Have fiber made. However, in some applications this is may not be inappropriate.

A third method for reducing the problem of non-linear optical effects in the optical fibers would be to use optical fibers with pure silicon oxide cores and fluoride-doped shells, in which fibers non-linear effects are reduced in comparison with conventional GeO ₂ -doped cores with pure silicon oxide shells.

Finally, a combination of any of these techniques can be used. For example, a pair of particularly short glass blocks can be used in the manner shown with the endomicroscope 290 of FIG. 12, the length of the blocks being kept to a minimum taking space and weight into account, but single-mode optical fibers with a large diameter core for them first and second optical fibers are used. The combined effect of these two modifications can then be used to keep the peak power of the beam in the first and second optical fibers at an acceptable level.

Variations within the Grundge thanks and scope of the invention easily made become. Therefore, it should be understood that the invention is not limited to the specific embodiments, which were previously described as examples.

Claims (41)

1. Endoscope or microscope with:
an optical fiber for transmitting a pulsed main beam over at least a portion of an optical path between a beam source and a tissue or other sample; and
a disperser;
whereby dispersion occurring in the optical fiber is reversed by dispersion which is generated in the dispersing device. 2. Endoscope or microscope according to claim 1, wherein the dis pergiereinrichtung an optically after the optical fiber arranged compression device for compressing the Beam after the beam has the optical fiber leaves to the peak power of the main beam inside to reduce half of the optical fiber. 3. Endoscope or microscope according to claim 1 or 2 with one Disper optically arranged in front of the optical fiber sionseinrichtung for dispersing the main beam, be before the main beam enters the optical fiber. 4. Endoscope or microscope according to one of the preceding An say, the optical fiber is a core with large Diameter and therefore has a low V-number to the To reduce area power density in the core. 5. Endoscope or microscope according to one of the preceding An say, the optical fiber is a pure silicon oxide core and has a fluoride-doped cladding nonlinear optical effects within the fiber to mi nim.   6. Endoscope or microscope according to one of the preceding An sayings, the dispersing device another has optical fiber. 7. Procedure for two-photon endoscopy or microscopy with the following steps:
Transmitting a main pulsed laser beam along an optical fiber over at least a portion of an optical path between a beam source and a tissue or other sample; and
Dispersing the beam by means of a dispersing device;
whereby dispersion occurring in the optical fiber is reversed by dispersion generated by the disperser. 8. Endoscope or microscope with:
beam splitter means for splitting a pulsed main laser beam into first and second beams;
a first optical fiber for transmitting the main beam to the beam splitter device;
an anti-dispersion device for reducing the dispersion of the first and second beams;
an optical coupler for recombining at least a portion of the respective first and second beams into a recombined beam; and
a second optical fiber for transmitting the recombined beam from the optical coupler to an endoscope or microscope head;
wherein the main beam can be divided by the beam splitter device into the first and second beams, the dispersion of which can subsequently be reduced by the anti-dispersion device and which can then be recombined by the optical coupler device in order to transmit a recombined two-photon beam for transmission along the second optical fiber Form your head. 9. Endoscope or microscope according to claim 8, wherein the endo skop or microscope has a laser source to the To provide main beam. 10. Endoscope or microscope according to claim 9, wherein the La is an ultrashort pulse laser. 11. Endoscope or microscope according to one of claims 8 to 10, wherein the first optical fiber is a single mode fiber is at the wavelength of the main beam. 12. Endoscope or microscope according to one of claims 8 to 11, the beam splitter device and / or the opti cal coupler device each an optical coupler is. 13. Endoscope or microscope according to one of claims 8 to 11, wherein the beam splitter device and the optical Coupler device through a single optical coupler educated. 14. Endoscope or microscope according to one of claims 8 to 13, wherein the beam splitter means the main beam divides approximately equally into the first and second beams. 15. Endoscope or microscope according to one of claims 8 to 14, the anti-dispersion device reversing Chirp Bragg grating fiber with more closely spaced grids has that optically closer to the beam splitter device are arranged. 16. Endoscope or microscope according to one of claims 8 to 14, the anti-dispersion device being a first and second reverse chirp Bragg grating fiber, one for Receiving the respective first and second beams, each because with closer spaced grids that are optically closer are arranged on the beam splitter device.   17. Endoscope or microscope according to one of claims 8 to 16, wherein the endoscope or microscope also an opti large diameter fiber and a cladding mode Coupler has, the cladding mode coupler the two te optical fiber and the large diameter fiber couples so that wide-angle scattered light from the head through the Couplers essentially into the fiber with great throughput Coupled knife for transmission to a photodetector pelt is. 18. Endoscope or microscope according to one of claims 8 to 17, the endoscope or microscope being optically based the second optical fiber arranged compression direction for compressing the recombined beam after the recombined beam has the second optical fiber leaves, causing the peak performance of the Main beam within the first optical fiber and of the recombined beam in the second optical company is reduced. 19. Endoscope or microscope with:
a dispenser for temporarily spectroscopically dispersing a pulsed main laser beam;
and
an optical fiber for transmitting the dispersed main beam to an endoscope or microscope head and reducing the dispersion by compressing the dispersed main beam. 20. Endoscope or microscope according to claim 19, wherein the op table fiber turned on by the disperser led dispersion essentially reversed. 21. Endoscope or microscope according to claim 19, wherein the En doskop or microscope has one head and the head one Compress optically arranged after the optical fiber ons device for conveying or closing the compresses  sion of the dispersed main beam to the Peak power of the main beam within the optical Reduce fiber. 22. Endoscope or microscope according to one of claims 19 to 21, the dispersing device also being a blasting unit lereinrichtung for selecting the by a tissue or is another sample of fluorescent light emitted. 23. Endoscope or microscope according to one of claims 19 to 22, wherein the disperser is a pair of diffraction or Has refractive elements. 24. Endoscope or microscope according to one of claims 19 to 23, the dispersing device being a pair of Blaze-Git ter and has a prism. 25. Endoscope or microscope with a head, the endoscope or microscope having:
a suction or vacuum source, which is arranged close to the head;
a light interceptor for intercepting light from a sample; and
sample receiving means, coupled to the suction source, for receiving the sample when the sample is pressed thereon by the suction source;
the suction source being used to urge the sample toward the receiver, deforming the sample so that the light interceptor can capture a portion of the light emitted by the sample from the head. 26. Endoscope or microscope according to claim 25, wherein the Pro benaufnahmeeinrichtung cup-shaped or frustoconical is. 27. Endoscope or microscope according to claim 25 or 26, wherein the light interception device in use by the Pro  benaufnahmeeinrichtung is separated from the sample and the sample receiving device for the light between the Sample and the light interception device essentially is permeable. 28. Endoscope or microscope according to one of claims 25 to 27, the light interception device being an optical Fa ser has. 29. Endoscope or microscope according to one of claims 25 to 28, wherein the light interception device has a mirror points. 30. Endoscope or microscope according to claim 29 with a lens to focus light from the mirror. 31. Endoscope or microscope according to one of claims 25 to 30, wherein the light interception device one of several Light interception devices is. 32. Method for two-photon endoscopy or microscopy with the following steps:

• 1) transmitting a pulsed main laser beam along an optical fiber;
• 2) splitting the main beam into first and second beams;
• 3) compressing the first and second beams;
• 4) combining at least a portion of the first and second beams;
• 5) Transfer the first and second beams along an optical fiber to an endoscope or microscope head.

33. Endoscope or microscope system with:
an endoscope or microscope with a photodetector;
a lighting device for providing ambient lighting;
a switching device for switching the lighting device on and off; and
a synchronization device for synchronizing the switching device with detection periods of the endoscope or microscope;
wherein the synchronization device is operable to control the switching device in order to switch the lighting device off during the detection periods and on between successive detection periods. 34. Endoscope or microscope system according to claim 33, wherein the synchronization device can be operated in such a way that it controls the switching device to the lighting switch device so that the lighting device during the retraction period of the endoscope or the Microscope is turned on. 35. endoscope or microscope system according to claim 33 or 34, wherein the photodetector is operable to be out is switched on when the lighting device is switched on is switched. 36. Endoscope or microscope system according to one of the claims 33 to 35, the photodetector being a power supply which can be operated in such a way that it is switched off, when the lighting device is switched on. 37. Endoscope or microscope system with:
an endoscope or microscope with a photodetector;
a lighting device for providing ambient lighting; and
a filter device for reducing the detection of the ambient lighting by the photodetector. 38. Endoscope or microscope system according to claim 37, wherein the lighting device emits light through the filter device is preferably blocked.   39. Endoscope or microscope system according to claim 37, wherein the filter device is a first filter device and the system comprises: a second filter device for absorbing light from the lighting device device preferred by the first filter device is transmitted and transmitting light from the loading lighting device by the filter device is preferably blocked. 40. endoscope or microscope system according to one of the claims 37 to 39 with a switching device for on and off switch the lighting device and a synchro nization device for synchronizing the switch direction with acquisition periods of the endoscope or Mi kroskops, the synchronization device so be is drivable that it controls the switching device to the lighting device during the detection period ode out and between successive acquisition switch on periods. 41. Endoscope or microscope system according to one of the claims 37 to 40 with a closure device for covering of the photodetector, the Synchronisationseinrich tion is so operable that it the closure device controls to cover the photodetector when loading lighting device is turned on, and the photo to expose the detector during the detection periods.