DE10065146A1 - Method for non-invasive 3D optical examination of skin and for the therapy of pathological changes ascertained treats melanomas with laser applications - Google Patents

Abstract

An 80MHz femto-second laser (1) has an adapter (2) adjustable in x/y directions with an 800 nm wavelength NIR beam that uses an optical articulated arm (3) with reflective elements to enable effective transmission of laser pulses without dispersing the pulses. On a first reflective element in the arm there is a removable cover (4) for making simple adjustments in the arm. On the end of the arm a hand piece (5) has a scanner (11) with a scanner lens (10) for pinpointing a beam on a desired area of tissue, for scanning a region of interest and for performing a single line scan. An Independent claim is also included for a system for non-invasive 3D optical examination of skin and for the therapy of pathological changes ascertained.

Description

The invention includes a method and an arrangement for non- invasive optical examination of the skin and skin appendages the how. z. B. hair and nails, as well as for therapy pathological changes using laser radiation.

The method and the arrangement are preferably used for in vivo application investigation of cellular and subcellular structures and / or Detection of foreign substances, such as pharmaceuticals or cosmetic Substances in the skin and in skin appendages as well as for the therapy of Melanoma through selective processing of areas containing melanin.

The invention relates to a method and to an arrangement for non-invasive three-dimensional optical representation of cellular and subcellular structures as well as foreign substances in the skin and Skin appendages and for non-invasive optical processing biological structures with high precision by means of laser radiation.

The optical examination of skin and skin appendages for Purpose of diagnostics as well as the study of enrichment and Pharmacokinetics of drugs and cosmetic substances in tissues So far, this has usually been done by a two-dimensional representation using visualization with the eye or camera systems.

A deeper, so to speak, "three-dimensional" investigation Allowing shifts is generally done by a doctor Tissue sample mechanically taken invasively (biopsy) and approved nete cutting devices in thin slices, so-called histological Cuts, disassembled. These are then often determined by the coloring procedure subject. The histological sections are then included observed under the microscope in transmission, reflection or fluorescence. From these observations, usually by an experienced  Pathologists can be made to get sick on type and extent changes are closed. This type of diagnosis generally takes hours and days.

Faster three-dimensional examinations without tissue extraction are made by ultrasound methods, the use of ionizing Radiation or strong magnetic fields and also by optical processes allows.

Optical 3D processes are based on the absorption or scattering of radiation incident on the tissue. The three-dimensional non-invasive optical examination is also called optical tomography or optical biopsy. One such optical method with spatial resolution for non-invasive 3D tissue diagnostics is optical coherence tomography (OCT) (e.g., J. Am. Acad. Dermatol. 37 ( 1997 ) 958; Proc. SPIE 3567 ( 1999 ) 70 ). This imaging method is based on the interference between a weak signal remitted by the object (backscattered or reflected) and a strong reference signal. British patent application No. 9725014.6 describes an OCT apparatus with a tunable depth resolution for the non-invasive optical biopsy of tissues. Depending on the bandwidth of the light source used, the resolution is typically in the range from a few tens of micrometers to a few millimeters. The recording times are in the seconds and minutes range. Another disadvantage is the restriction of the information to the information content of the weak remitted signal.

Another non-invasive imaging technique is based on photoacoustic effects. These are generated by local absorption of optical radiation (e.g. by blood vessels) and the associated low temperature increase and pressure wave generation. The pressure waves can be detected on the tissue surface. Acoustic detectors of small dimensions allow resolutions in the near infrared range (NIR) resolutions in the range of a few hundred micrometers to ten millimeters (e.g. Opt. Lett. 23 ( 1998 ) 648, Appl. Phys. Lett. 75 ( 1999 ) 1058). The method is particularly suitable for studying blood flow phenomena. Sensitive fluorescence measurements, especially for studying the material composition of the tissue with high spatial resolution, are not possible with these methods.

Optical imaging methods for the analysis of biological tissues with a higher spatial resolution in the range from a few hundred nanometers to a few micrometers have hitherto been based on confocal detection of fluorescent radiation and of backscattered radiation, so-called reflective radiation. In the case of confocal detection, spatial filters, so-called pinholes or "pinholes", are used, which are intended to enable preferential detection of photons from the focal plane. Typically, confocal laser scanning microscopes are used. The journal "Bioimaging" 4 ( 1996 ) 13 reports on the use of laser scanning microscopes with laser wavelengths of 365 nm and 488 nm in order to detect fluorescence in the wavelength range above 515 nm in normal skin. This so-called fluorescence imaging represents a very sensitive method, which offers the possibility, in addition to the imaging of morphological structures ("morphological imaging"), also a functional imaging ("functional imaging") (e.g. functionality of the respiratory chain and the redox behavior through To enable fluorescence imaging of reduced coenzymes). However, the use of short-wave fluorescence excitation radiation is of great disadvantage since this has only a small penetration depth into biological tissue due to its high absorption and scattering coefficients.

In order to obtain information from deeper layers, confocal microscopes with laser radiation in the red or near in the infrared (NIR) spectral range are advantageously used. Since the absorption when using low-intensity radiation is very low and a fluorescence of the cell's own fluorophores cannot be effectively stimulated, only the reflective radiation is detected (J. Invest. Dermatol. 104 ( 1995 ) 946; J. Invest. Dermatol. 113 ( 1999 ) 293; Urology 53 ( 1999 ) 853). Fast recordings in video frame rate are possible (Appl. Opt. 38 ( 1999 ) 2105). However, the detection of the remission radiation allows only a limited information gain, which only results from differences in refractive index within the tissue. Nevertheless, it was observed that melanin, due to increased backscatter, causes a high contrast to the surrounding matrix and can thus be detected (Journal of Investigative Dermatology 104 ( 1996 ) 946).

A general disadvantage of confocal detection of tissue components len is also the small number of photons, which is per unit time through the spatial filter reaches the detector. In addition, due to multiple scatter an exact assignment of the detected photons to the focus level not possible.

A newer method is the so-called multiphoton laser scanning fluorescence microscopy, in which visible fluorescence can be detected in a spatially resolved manner by means of two- and three-photon excitation using laser radiation of high light intensity in the low focus volume of a microscope objective with radiation in the spectral range of the near infrared (NIR). Patent US 5,034,613). In contrast to confocal microscopy, multiphoton microscopy uses a very small volume of fluorescence excitation. The detected fluorescence photons can be assigned to this volume, which eliminates the need for confocal detection. In addition to measuring the fluorescence intensity, the recording of time-resolved two-photon-excited fluorescence on isolated cells was reported on a glass window (J. Microsc. 183 ( 1996 ) 197). In order to achieve the required high light intensities, femtosecond pulses with high peak power are typically used, but more moderate, average power averaged over time. A suitable laser source is the titanium sapphire laser with a high repetition rate. The fluorophores are usually excited via a non-resonant process in which, in the case of two-photon excitation, the fluorescence is induced by simultaneous absorption of two photons, and each photon is half of that provides the necessary excitation energy. In contrast, it was described in studies on synthetic melanin that the pigment could be stimulated by means of a resonant process, a two-stage process, using NIR femtose customer pulses. In addition, a two-photon-excited autofluorescence spectrum of an extracted skin sample using a femtosecond laser and spectrometer was described, which could possibly be based on melanin fluorescence (Photochem. Photobiol. 73 ( 1999 ) 146). However, other cell-specific fluorophores also emit in this area. A spatially resolved measurement does not take place. Autofluorescence is the fluorescence that is based on the stimulation of the body's own (endogenous) fluorophores.

In the publications Biophys. J. 72 ( 1997 ) 2405 and Ann. NY Acad. Sci. 838 ( 1998 ) 58 it was reported that with the aid of a multiphoton laser scanning microscope, based on the coupling of a mode-synchronized titanium-sapphire laser pumped by means of an argon ion laser into an inverted microscope "Axiovert 35 " from Zeiss, the two-photons -excited NADH fluorescence of cells in normal skin is spatially detectable in vivo. Pathological changes in the skin have not been investigated. The arrangement, which is based on the use of a microscope, is not suitable for the in vivo examination of patients for the purpose of diagnosing pathological changes. No processing of biological structures was carried out with this arrangement.

Up to now, laser processing of biological structures has generally been carried out with systems of high or medium laser power and with continuously emitting (cw) lasers or pulsed systems with a low repetition rate and a pulse duration of nanoseconds or longer. Typically, tissue processing is based on thermal processes or on a surface ablation with a laser with a low penetration depth, such as the CO ₂ laser or the excimer laser working in the ultraviolet range. Although through the choice of a laser wavelength and the pulse duration depending on the absorption capacity and the target size, a selective destruction of deep-lying targets, e.g. B. blood vessels and melanosomes can be achieved, even with this so-called selective thermolysis (Science 220 ( 1983 ) 524) high precision is not given. In addition, the combination with 3D fluorescence diagnostics for the purpose of localizing the target is not known.

A high-precision laser surgical method for processing intra-cellular structures, in particular for the dissection of chromosomes, using ultrashort pulse lasers in the NIR range, on the other hand, is described in DE 19 19 345 A1 and in the publication Cell. Mol Biol. 45 ( 1999 ) 195 described the. This procedure involves the processing of biological nanostructures within a cell and does not cover the diagnostic area.

An arrangement for optical micromanipulation, analysis and processing Treatment of objects by means of visible and NIR radiation was described in DE 197 19 344 A1 described. It is characterized by a switchover from continuously emitting laser operation (cw mode) in the pulsed Laser operation off and is not pa for examination and processing proposed changes in theology.

The object of the invention is to provide a laser arrangement and a method for non-invasive, three-dimensional optical sub search with subcellular resolution for the purpose of diagnostics and Therapy control and for non-invasive high-precision processing of pathological changes in the skin and appendages, the is suitable for use on patients and which also the sub search for the enrichment and pharmacokinetics of drugs and kos Metic substances in biological tissues allowed and in particular the three-dimensional localization of melanoma and its targeted non-invasive destruction without affecting neighboring non- pigmented tissue areas.

This object is achieved by the He described in the claims solution solved.

The inventive method and the inventive arrangement are suitable for a non-invasive three-dimensional "optical biopsy" resolution for the purpose of diagnostics and therapy control create as well as the enrichment and pharmacokinetics of drugs and investigate cosmetic substances in tissue. They are suitable also for the detection as well as the exact localization and extension  of pathological changes in the skin and skin appendages, such as B. melanoma, psoriasis, pigment disorders, foreign body storage genes, wound diseases, acne, fungal and bacterial diseases. to the arrangement and the method enable a non-invasive High precision tissue processing including inactivation cellular multicellular tissue areas and single cells. The processing device can also be applied to the interior of tissues without impairment gender tissue sections and without destroying the tissue surface The method and the arrangement are particularly suitable, Me lanomas of small extent can be recognized and with a high spatial localize solution as well as selectively, immediately and irreversibly damage.

According to the invention for non-invasive optical 3D diagnostics and Treatment of pathological tissue changes focused radiation in the spectral range from 700 nm to 1200 nm, consisting of Femtosekun the pulses or picosecond pulses depending on the Tissue depth, the degree of pigmentation and the radiation target (Diagnostics or processing) suitably changeable pulse power, a set at light intensities in the order of gigawatts per Square centimeters of non-resonant and resonant multiphoton Fluorescence excitations of specific endogenous fluorophores, in particular Melanin, which in combination with a scanning unit and adjustable focal plane a three-dimensional imaging of tissue enable, and based on the spatial arrangement of the autofluorescence A distinction of intensity and the autofluorescence lifespan certain pathological and healthy tissue as well as the effect of pharmaceuticals and cosmetic substances enable a Fluorescence excitation of certain substances in pharmaceuticals and cos cause metallic substances, as well as by increasing pulse power and an intensity on the order of terawatts per square centimeter selective selective destruction of individual cells and individual cells Tissue areas allows to irreversible without surrounding tissue areas damage.  

According to the invention there is an arrangement for diagnosis and processing used from a compact femtosecond laser or pico second laser with a laser wavelength in the range 700 nm to 1200 nm, a beam guidance system in the form of an optical articulated arm with reflective elements, a special handpiece with scanner and focusing optics of the numerical aperture greater than 0.4 and moto rically adjustable focal plane, one depending on the tissue depth, the degree of pigmentation and the radiation target (diagnosis or Machining) motor-adjustable beam attenuator, one by foot switch-operated switching element, various detectors and opti ken as well as a control, control and evaluation unit.

Surprisingly, it was found in our own research that through Multiphoton excitation using an 800 nanometer femtosecond laser pulsing of endogenous (cellular) fluorophores in normal and pathological tissues significant differences occur for a non-invasive diagnostics of high spatial resolution can be used can. In particular, the three-dimensional optical sub Search for melanoma, the pigment melanin by multiphotons excited autofluorescence with high resolution even in tissue depth be selectively detected. In addition, the detection of pharmaceuticals and of cosmetic products in the skin and their effects possible in tissue with subcellular resolution.

Interestingly, it was observed that by a suitable increase the laser intensity at the same wavelength and pulse duration that could only have destructive effects in the melanin-containing tissues areas occurred, but not in the surrounding non-pigmented areas Cells. With a suitable light intensity setting, Com combination with a scanning device that scans the tissue with the focused laser beam, on an extracted Me lanom be demonstrated that a selective cell toxic effect in Tissue interior could be reached, with pigmented cells immediately was destroyed and non-pigmented cells were left undamaged the. If the laser intensity was increased further, non-pigmentation could also occur cells immediately irreversibly damaged and cell material removed  (ablated). If the beam was only on one cell within one directed a tissue bandage, this could selectively destroy a cell without irreversibly damaging surrounding cells.

The process of destruction is a so-called optical breakthrough and Underlying plasma formation in which free by multiphoton ionization Electrons and biomolecule ion cores are generated. This process can be used for material ablation. Because when using Focusing optics with high numerical aperture only in the central part of the Submicron lighting spots sufficient laser intensities for Selective high-precision machining can be available without Impairment of adjacent tissue areas are made possible. Because of The threshold for the optical breakthrough of pigmentation can be opened depends on the degree, melanin-containing cells can also be selectively destroyed by scanning the tissue. So a selekti ves, non-invasive knocking-out of individual melanoma cells possible.

The invention will be explained in more detail below using exemplary embodiments. . In the accompanying illustrations or drawings, Figures 1 to 6 illustrate the method of the invention and its effectiveness, as well as Figures 7 and 8 show two embodiments of the arrangement according to the invention.:

Figures 1A and 1B show autofluorescence images of normal skin of a healthy subject from 15 µm (A) and 45 µm (B) tissue depth with submicron resolution.

Fig. 2 shows autofluorescence images of the skin of a patient with a fungal disease on the forearm.

Figures 3A and 3B show the autofluorescence of a melanoma at different tissue depths.

Fig. 4A and 4B of cryostat sections of a melanoma in transmission (A) and autofluorescence (B) clearly demonstrate the dominant melanin fluorescence in the pigmented areas compared to the autofluorescence of pigment-free tissue areas.

Fig. 5 shows an example of fluorescence decay melaninhaltigem tissue as compared to pigment-free tissue.

Figures 6A and 6B show the irreversible damage to tissue areas containing melanin due to intense femtosecond laser pulses.

Fig. 7 shows an embodiment of the inventive arrangement.

FIG. 8 shows a modification of the embodiment of FIG. 7.

Figures 1 and 2 illustrate that it is possible to differentiate between healthy tissue and pathological tissue based on the spatial distribution of the autofluorescence intensity. The autofluorescence in the visible spectral range was achieved by irradiation of 170 femtosecond laser pulses of the NIR wavelength of 800 nm, a pulse repetition frequency of 80 MHz and an average power on the skin surface in the range of 1 mW to 2 mW on the surface and up to 50 mW in the tissue depth initially stimulated on the skin and appendages of a healthy subject and displayed in three dimensions with an image acquisition time of 16 seconds. The radiation was focused by focusing optics with a working distance greater than 150 µm in different layers of the skin and the pulse power increased with increasing tissue depth. As can be seen from Fig. 1, which shows the autofluorescence of normal skin in a tissue depth of 15 microns ( Fig. 1A) and 45 microns ( Fig. 1B), individual cells and fluorescent cell components in different layers of the can with subcellular resolution normal skin can be detected. In particular, the fluorescence of the coenzymes NAD (P) H and Flavin was stimulated. The individual cells including the non-fluorescent cell nuclei are clearly visible. Typical fluorophores excited by 800 nm are the reduced coenzymes NADH and NADPH, as well as flavine, collagen, elastin, keratin, lipofuscin, metal-free and zinc-containing porphyrins. While the cell boundaries show a clear autofluorescence in the stratum corneum, mainly cell organs, especially mitochondria, and connective tissue components fluoresce in the layers below.

Pathological changes lead to clearly different autofluorescence images, as shown in Fig. 2 using a patient with a fungal disease on the forearm. In addition to the known morphological changes in the stratum corneum, it was surprising to see a conspicuous autofluorescence in the central part of the cells in this case, which made it possible to clearly differentiate from surrounding normal tissue.

Not just morphological changes to tissue structures and a Different biosynthesis of endogenous fluorophores can be used for different autofluorescence from certain pathological Tissues and healthy tissue may be responsible, but also Un differences in metabolic state and intracellular redox behavior. This can be done, for example, by detection of the respiratory metabolism significantly involved coenzymes NADH and NADPH are detected, the only in the reduced, but not in the oxidized state (NAD, NADP) fluoresce and their detection enable "functional imaging".

The intense NIR fluorescence excitation radiation in the femtosecond range rich can also be used for the investigation of hair roots, fungal attack by Nails, the detection of porphyrin-producing bacteria - such as B. Propionibacterium acnes and abnormal accumulation of Kera tin, collagen and elastin can be used.

When using a wavelength of 800 nm, intracellular melanin could be excited to fluoresce with significantly lower pulse powers than other endogenous fluorophores such as NADH and Flavine. Appropriate adjustment of the pulse power enabled an almost selective display of the melanin in the pigmented tissue. Using 3D imaging of the fluorescence intensity, the pigmented area could be clearly differentiated from the non-pigmented tissue areas with subcellular resolution. FIGS. 3A and 3B show fluorescent melanin clusters in different tissue depths of a melanoma (SSM = superficial spreading melanoma), which were created by excitation with 170 femtosecond laser pulses with an average wavelength of 800 nm.

Fig. 4, which shows cryosections of this extracted melanoma in transmission (A) and fluorescence (B), shows that the non-linearly excited fluorescence preferably comes from the melanin-containing tissue areas and differs significantly from the surrounding autofluorescence of pigment-free cells.

Another way of distinguishing between pathological and healthy tissue is given by the 3D representation of the fluorescence lifetime. The fluorescence lifetime describes the mean time that the molecule remains in the electronically excited state after light absorption. Since a large number of molecules are excited, the course of the fluorescence intensity over time after absorption of the laser pulse describes a typical statistical decay behavior from which the fluorescence lifetime can be determined. With the help of the so-called time-correlated single photon counting (TCSPC), in which the individual NEN fluorescence photons are registered as a function of the time after the laser pulse is hit by a fast detector, melanomas were measured in comparison to the nominal tissue. Interestingly, as can be seen in FIG. 5, tissue containing melanin has a significantly different decay behavior than non-pigmented tissue. Due to the spatially resolved recording of the fluorescence decay curves or the spatially resolved representation of the average lifespan, the melanin-containing areas can also be clearly distinguished from the pigment-free areas with this contrast method.

The application of fluorescent pharmaceuticals and cosmetic sub punching was also detected in the tissue with high resolution and enabled the detection of the diffusion behavior and the intracellular attachment behavior. So was using 800 nm femtose customer pulses the 3D distribution of the topically administered DNA marker Hoechst 33342 and the biosynthesis of the fluorophore protoporphyrin IX  after topical application of aminolevulinic acid in the tumor tissue captured a mouse. But also non-fluorescent substances could indirectly, z. B. due to their effect on cell metabolism or by mor phological cells are detected using 3D autofluorescence. Moisturizing the skin leads to swelling of volume structures in the stratum corneum. The detectable autofluorescence Changes in the individual cell layers show in particular vertical cell enlargement in the stratum corneum. This volume Changes could be recorded time-dependently using autofluorescence images become.

Fig. 6 demonstrates the "knocking out" of a single tumor cell within a tissue bandage using intensive femtosecond pulses in the range of 3 TW / cm ² -4 TW / cm ² . First, the melanin fluorescence was displayed three-dimensionally in individual tumor cells at a lower intensity by two-photon excitation, then the laser beam was "parked" on the target cell to be destroyed and the melanin-containing cells within the tissue association were immediately and irreversibly destroyed by increasing the power. The destruction was then visible through multiphoton-excited fluorescence imaging. Surrounding cells showed no change in fluorescence behavior or morphological changes.

According to the invention, an arrangement for non-invasive optical in vivo 3D diagnosis and processing of pathological tissue changes gene used, which radiation in a spectral range from 700 nm to 1200 nm and a pulse duration in femtosecond or picosecond Use area for excitation of multiphoton effects, an efficient Transmission of laser radiation without the disadvantage of pulse broadening through the use of an optical articulated arm with reflective optics allows a switching device to increase the pulse power with increasing tissue depth as well as when changing from the diagnostic mode in the mode of tissue processing provides a focusing unit the numerical aperture larger than 0.4 and motor-adjustable focus plane as well as a beam guidance system that is both a targeted punctual radiation as well as the scanning of the to be examined and  tissue areas to be processed, and fluorescence detectors includes a spatially high-resolution representation of the fluorescence allow intensity and / or fluorescence life. In the opposite set on the use of light guides and lens systems that are used in Transmission of ultrashort pulses due to optical dispersion too significant The use of opti enables pulse broadening articulated arms with reflective elements an efficient over can be carried in all spatial directions without significant pulse broadening.

An embodiment of the arrangement according to the invention is explained in more detail below with the aid of the schematic FIG. 7.

The arrangement according to the invention consists of a compact 80 MHz femtosecond laser 1 with an adapter 2 adjustable in the x and y directions, the NIR radiation, preferably radiation of wavelength 800 nm, via an optical articulated arm 3 with reflective elements, an efficient transmission of the It enables laser pulses without pulse spreading. On the first reflective element of the articulated arm there is a removable cap 4 , which enables simple adjustment of the articulated arm by visualizing the small beam portion transmitted through the reflective element.

At the end of the articulated arm 3 , a handpiece 5 is mounted, which contains a scanner 11 with a scanner optics 10 , which optionally punk tulle radiation ("parking" the beam on a desired tissue real), a scanning of a region of interest (ROI) and allows scanning along a single line ("line scan"). Other components of the handpiece are a beam expander 6 , a motor-adjustable beam attenuator 9 , for example in the form of a rotatable polarizer, a focusing optics 14 with a numerical aperture greater than 0.4, preferably 1, that can be motorized along the optical axis by means of piezo element 13 , 3 and a first and a second beam splitter 12 and 19 . These beam splitters 12 and 19 are, for example, dichroic mirrors which each reflect NIR radiation and allow visible radiation to be transmitted, and thus a measurement of the fluorescence by transmission through both beam splitters 12 and 19 on a detector 25 and a measurement of small portions of the remission radiation allow transmitted by the beam splitter 12 , but reflected on the beam splitter 19 reflected radiation at a detector 20 . The detector 25 with special optics 23 and an insert 24 for spectral filters is, for example, a sensitive photomultiplier for measuring the fluorescence intensity and the fluorescence decay kinetics in a spectral range determined by a given spectral filter in the insert 24 , while a photodiode is advantageously used as the detector 20 , Both detectors 20 and 25 also enable the measurement of the plasma glow that occurs during processing. In addition, there is a mechanically insertable beam splitter 21 and a miniaturized white light source 22 including optics in the handpiece 5 in order to enable images by means of white light. Furthermore, the handpiece 5 contains a measuring diode 29 for detecting the current Laserlei stung by measuring a very small portion of the same reflected on a glass plate 28 and for triggering the photomultiplier 25th Immediately below the focusing optics 14 is a mechanical device 15 with a ring 16 for receiving a round special glass window, against which the tissue area to be examined is pressed. The special glass with a preferred thickness of 0.17 mm is easily exchangeable and also serves as optical protection. The organ to be examined or treated as a target is advantageously fixed below the device 15 with a special target holder (for example an arm holder).

The approach of the tissue area to the ring 16 with the window is checked by means of a miniature illumination source 17 and a miniature CCD camera 18 .

The variation in the pulse power can be achieved both by individual control of the attenuator 9 , in particular in the case of material processing, but preferably by automatic control depending on the position of the focusing optics 14 and the signal level of the remitted signal at the detector 20 for the depth-resolved 3D diagnosis Localization and processing of the target take place. Both the position of the motor-controlled focusing optics 14 and the signal level enable conclusions to be drawn about the focal plane within the tissue area to be examined or processed. Typically, the measurement of autofluorescence with a high signal-to-noise ratio at a depth of 100 µm requires an approximately 10 times higher pulse power than on the surface.

The control of the laser 1 , the scanner 11 , the piezo element 13 , the filter insert 24 , the detectors 20 and 25 or the CCD camera 18 and the signal acquisition is carried out by means of a PC 26 with special hardware and software, which in the case of the time-resolved measurement also contains a time-amplitude converter (TAC) using the so-called time-correlated single-photon counting (TCSPC). The signal is processed using suitable software. In principle, this can also enable an automatic comparison of the measured image with a stored library that contains autofluorescence images of normal and pathologically changed skin, thus automatically locating abnormal cell and tissue changes. The special software enables signal storage and signal evaluation, in particular 3D image evaluation, the creation of fluorescence decay characteristics and fluorescence lifetime calculations. The software also creates 3D animations. On a flat screen 27 , after processing the image, the fluorescence signals and the plasma lights occurring during the optical processing and, if necessary, the remitted beam components are shown. A foot switch 8 ensures the release of a shutter 7, the laser operation for safety reasons only when the same.

Fig. 8 shows a modification of the arrangement of FIG. 7, wherein while maintaining the respective function, a variety of optical and optical optical elements, such as the beam expander 6 , the motor-adjustable beam attenuator 9 , the scanner 11 with the scanner optics 10 , the Optical shutter 7 , the glass plate 28 and the measuring diode 29 are integrated in the optical articulated arm 3 , while the detectors 20 and 25 , the beam splitters 12 , 19 and 21 , the special optics 23 , the filter insert 24 and the white light source 22 are now expediently 30 designated handpiece remain.

LIST OF REFERENCE NUMBERS

1

compact laser

2

Adapter with x, y adjustment

3

optical articulated arm with reflective elements

4

removable cap

5

handpiece

6

beam

7

optical shutter to release the laser radiation

8th

footswitch

9

motor-adjustable beam attenuator

10

scanner optics

11

scanner

12

first beam splitter

13

piezo element

14

focusing optics

15

mechanical device

16

Ring for holding a special glass window

17

Miniature light source

18

Miniature CCD camera

19

second beam splitter

20

Remission detector (photodiode)

21

mechanically insertable beam splitter

22

White light source

23

special optics

24

Insert for spectral filters

25

Detector for measuring fluorescence and plasma lighting (photomultiplier)

26

PC with special hardware and software

27

flat

28

glass plate

29

measuring diode

30

handpiece

Claims (17)

1. A method for the non-invasive three-dimensional optical examination of the skin and for the therapy of pathological changes detected, preferably for the in vivo examination of cellular and subcellular structures and / or for the detection of foreign substances, such as drugs or cosmetic substances, in the skin and in skin appendages and for the diagnosis and therapy of melanomas by selective processing of melanin-containing areas by means of laser radiation, characterized by the use of pulsed laser radiation in the near infrared range focused in the respective depth of the skin with wavelengths from 700 nm to 1200 nm and with pulse widths of less than 20 picoseconds , wherein the light intensity of the pulses for the examination has an order of magnitude of gigawatts per square centimeter in order to bring about multiphoton excitation of the body's own fluorophores or fluorescent constituents of the foreign substances while it is processing a size order of terawatts per square centimeter in order to achieve material removal as a result of directly acting, destructive multiphoton processes. 2. The method according to claim 1, characterized in that for Purpose of the three-dimensional high-contrast investigation Pulse power increases with the focus of increasing tissue depth becomes. 3. The method according to claim 1 or 2, characterized in that through the choice of the laser wavelength and the light intensity in the loading rich from gigawatts per square centimeter prefers melanic ones Tissue areas by detection of the multiphoton-excited mela nin fluorescence shown and ver for melanoma diagnosis be applied.   4. The method according to any one of claims 1 to 3, that by an appropriate choice of light intensity that is higher than that under claim 3 is used light intensity and in the range of one to twenty terawatts per square centimeter an irreversible tissue area selected by the examination is sibel damaged, but without surrounding, unselected Irreversibly damaging tissue areas. 5. Arrangement for non-invasive three-dimensional optical examination of the skin and for the therapy of pathological changes detected, preferably for the in vivo examination of cellular and subcellular structures and / or for the detection of foreign substances, such as drugs or cosmetic substances, in the skin and in skin appendages and for the diagnosis and therapy of melanomas by selective processing of melanin-containing areas by means of laser radiation, characterized in that they are a compact pulsed laser ( 1 ), an optical articulated arm ( 3 ) with reflective elements for transmitting the laser pulses without noticeable pulse broadening to one handpiece ( 5 , 30 ) to be guided on the skin, motor-driven optics ( 14 ) for focusing the radiation at different depths of the skin or the appendages of the skin and a device ( 9 ) for adjusting the light intensity as a function of the examination or therapy - Modu s and the depth of focus. 6. Arrangement according to claim 5, characterized in that the device for adjusting the light intensity is a motor-controlled beam attenuator ( 9 ). 7. Arrangement according to claim 5 or 6, characterized in that it has a scanner ( 11 ) for scanning the irradiated area in three dimensions. 8. Arrangement according to claim 7, characterized in that the scanner ( 11 ) is set up for selective irradiation, scanning along a line and areal scanning of a tissue area by means of a focused laser beam. 9. Arrangement according to one of claims 5 to 8, characterized in that it further comprises a system of detectors ( 20 , 25 ) and filters ( 24 ) which spatially, spectrally and temporally the fluorescence intensity or the fluorescence decay behavior in a selectable spectral range resolved to detect. 10. Arrangement according to one of claims 6 to 9, characterized in that the motor-focused optics ( 14 ) has a numerical aperture greater than 0.4. 11. The arrangement according to claim 9 or 10, characterized in that a detector ( 25 ) is used which detects a multiphoton excited fluorescent radiation. 12. Arrangement according to one of claims 9 to 11, characterized in that the detector ( 25 ) for time-resolved detection of the fluorescence decay behavior has a time resolution in the picosecond den range. 13. Arrangement according to one of claims 9 to 12, characterized in that the detectors ( 20 , 25 ) are able to detect the plasma lights occurring during the Materialab transfer. 14. Arrangement according to one of claims 5 to 13, characterized in that it has a by means of a foot switch ( 8 ) operable opti's closure ( 7 ) for releasing the laser radiation. 15. Arrangement according to one of claims 5 to 14, characterized by a computer ( 26 ) for signal storage and evaluation. 16. The arrangement according to claim 15, characterized in that the computer ( 26 ) for 3D image evaluation and for the creation of fluorescence decay characteristics and fluorescence lifetime calculations is set up. 17. Arrangement according to the preceding claims 5 to 16, characterized in that while maintaining the respective function ei ne plurality of said optical and optoelectronic elements, such as the beam expander ( 6 ), the motor-adjustable beam attenuator ( 9 ), the scanner ( 11 ) with the scanner optics ( 10 ), the optical shutter ( 7 ), the glass plate ( 28 ) and the measuring diode ( 29 ) are integrated in the optical articulated arm ( 3 ), while the detectors ( 20 ) and ( 25 ) are expediently the beam splitters ( 12 , 19 and 21 ), the special optics ( 23 ), the filter insert ( 24 ) and the white light source ( 22 ) are located in a handpiece ( 30 ).