DE102014017006A1 - Method for the determination and evaluation of time-resolved fluorescence or reflection images on extended three-dimensional surfaces - Google Patents

Abstract

The present invention relates to a method for determining and evaluating time-resolved fluorescence or reflection images on extended three-dimensional surfaces which are scanned by pulsed radiation (pointwise optical scanning), for example for tissue characterization and diagnosis in medicine or for examination on mechanically difficult to access technical objects (such as in an explosion-proof environment). The object of the invention to provide a method for determining and evaluating time-resolved fluorescence or reflection images on extended three-dimensional surfaces is achieved in that for the determination and evaluation of parameters of time-resolved fluorescence or reflection images on a three-dimensional surface (230) of objects in which a pointwise pulsed scanning of the three-dimensional surface (230) by means of illumination and detection beam, wherein different distances of the illumination beam exit and detection beam entry point (210) from the pulsed illuminated points of the three-dimensional object (230) are present and a lit first object point (231) and a lit. second object point (232), which is located at a different distance to the illumination beam exit and detection beam entry point (210), in the duration of the excitation and detection beam (221) of the first Obj ectpunkts (231) and the illumination and detection beam (222) of the second object point (232) and a pixel-by-pixel modeling of the different maturities takes place, with different maturities in the determination of the fluorescence lifetime from the different distances of the illumination beam exit point or Detection beam entry point and between different illuminated surface points result, to be considered in a model function.

Description

The present invention relates to a method for determining and evaluating time-resolved fluorescence or reflection images on extended three-dimensional surfaces which are scanned by pulsed radiation (pointwise optical scanning), for example for tissue characterization and diagnosis in medicine or for examination on mechanically difficult to access technical objects (such as in an explosion-proof environment).

The fluorescence lifetime (time-resolved fluorescence) is an intrinsic substance property. It indicates the average residence time of an atom / molecule in the excited state and is usually in the ps to ns range. The fluorescence lifetime can be influenced by the environment of the fluorescent substance, u. a. by the solvent, binding to a protein or collisions with other molecules. The fluorescence lifetime thus provides more information than the fluorescence intensity alone and can therefore be used for tissue characterization and diagnosis, for example in the field of medicine.

The measurement of time-resolved fluorescence in medicine enables the detection of early, functional cellular changes, as a result of which a patient-specific therapy can be used.

Of particular importance is the measurement of time-resolved fluorescence on medical objects, for example on the ocular fundus, the measurement of object surfaces in hard-to-reach areas, such as explosion-proof chambers or ovens during industrial processes or in satellite-based remote sensing.

Diabetic retinopathy and age-related macular degeneration are diseases that affect millions of patients in Germany and can lead to blindness.

Since there is no cure for these diseases, the earliest possible diagnosis is crucial for the patient. The measurement of fluorescence lifetimes can ideally provide such an early diagnosis. However, the assignment of the fluorescence signals to anatomical structures of the fundus is not sufficiently accurate with the current methods, since all previous methods assume a two-dimensional structure. Thus, the diagnostic statement is severely limited.

Early diagnosis is crucial in diabetic retinopathy and age-related macular degeneration. Fluorescence signals mainly contain information on metabolic pathological changes. Fluorescence signals, in particular time-resolved fluorescence, therefore indicate the development of the diseases much earlier than any other current diagnostic method, which usually recognizes only morphological damage. While it is no longer possible to treat the disease by conventional diagnosis, the pathological changes are still reversible at an early stage. If the fluorescence signals can be assigned to certain causative structures and processes in the fundus, there is the possibility of cause-related therapy. However, this requires a three-dimensional assignment of the signals acquired by fluorescence lifetime microscopy.

Fluorescence lifetime microscopy is a microscopic imaging technique that relies on fluorescence (spontaneous emission of light shortly after excitation of a material, where the emitted light is lower in energy than the previously absorbed light).

Unlike other fluorescence microscopy methods, fluorescence lifetime microscopy is not based on measuring the intensity of the fluorescence, but on measuring the different lifetimes of the excited states of fluorescent molecules. The fluorescence lifetime is the mean time that a molecule remains in the excited state before it returns to its ground state with the release of a photon. The fluorescence degradation is reflected in an exponential decrease in fluorescence intensity over time. At the same time, the fluorescence lifetime is inversely proportional to the sum of the decay rates for radiative and non-radiative processes ( A. Periasamy, RM Clegg: Flim Microscopy in Biology and Medicine. CRC Press, 2010 ).

Fluorescence lifetime microscopy provides images with the fluorescence lifetime for each pixel of the image. The measurement of the fluorescence lifetime is based either on a pulsed excitation and a measurement of the temporal fluorescence drop, or on an intensity-modulated excitation and a measurement of the phase shift.
( www.olympusfluoview.com/applications/flimintro.html )

The simplest method for determining the fluorescence lifetime is to count released photons using time-correlated single-photon counting after periodic excitation with short light pulses in the picosecond range, ie in a range significantly shorter than the typical fluorescence lifetime, which is in the nanosecond range.

With a sufficiently short excitation, it is possible for most samples to measure the time-dependent exponential decrease in fluorescence intensity from which the fluorescence lifetime can be determined. For this purpose, the excitation intensity is reduced so far that in a series of, for example, 10 excitation pulses only about one photon is detected, the time between excitation pulse and photon being in the range of a few picoseconds.

From a large number of such individual measurements, a histogram can be generated, from which the fluorescence lifetime can be calculated directly (see also "time-correlated single photon counting" (TCPSC) method). This requires a relatively low probability of producing a fluorescence photon (<10%). With n excitation, the histogram of the photons detected in the individual time channels corresponds to the probability density function of the molecular relaxation process.

This method has the advantage that it is independent of fluctuations of the excitation intensity and has a high precision in the fluorescence lifetime determination, with a simultaneously low radiation exposure of the sample (in particular of the eye). In order to extract the fluorescence lifetime information from the TCSPC data, the method of multi-exponential approximation is often used.

The sufficiently short excitation (in the range of a few picoseconds) for measuring the fluorescence lifetime is carried out according to the prior art by a pulsed light source (for example in the form of a laser), whereby the fluorophores to be investigated are excited.

The time measurement is started by the excitation pulse and the photon emitted during the transition from the excited state to the ground state stops the measurement. This measurement is repeated many times and the individual temporally correlated photons are sorted according to their measured time (with respect to the excitation pulse) in the histogram.

This histogram typically has a temporal channel resolution or class width of about 1 to 50 ps and represents the exponential decay of the fluorescence intensity after the excitation.

In fluorescence lifetime measurements, however, temporal differences in the signal response, which are caused by different distances of scanned object points from 3D objects to the detection system, but also by light paths of the detection beam paths of different lengths in the internal optical arrangement of the detection system and additionally by alignment errors of the measurement system to the examination subject become.

On the other hand, the time-resolved measurement of reflection signals allows the determination of the surface structure of hard-to-reach objects.

The following are some examples of the prior art:
The DE 199 20 158 A1 discloses a method and an arrangement for the determination of fluorophores on objects, in particular on the living fundus, in which excitation and / or fluorescence spectra at least partially overlapping fluorophores of objects can be distinguished even at very low fluorescence intensities and can be selected in a two-dimensional representation.

For this purpose, the object (eg the ocular fundus for ophthalmological examinations) is spot-illuminated with a pulsed laser and excited to autofluorescence with a two-dimensional expansion. The fluorescence light which briefly emerges after excitation by each laser pulse is detected in time-correlated single-photon counting. From the time behavior of the fluorescent light determined by the time-correlated single-photon counting for each location of the autofluorescence, the fluorescence decay time constants are calculated and deduced therefrom to the fluorophores excited in the object. The time regime for the time-correlated single-photon count is controlled by the detected pulses of the fluorescence-exciting laser light and of the fluorescent light generated at the object. The local Assignment of the fluorescence with multiple scanning excitation takes place via a synchronized with the scanning system routing unit.

From the DE 101 45 823 A1 For example, a method for determining the two-dimensional distribution of various fluorophores is known, which is also applicable in the case of extremely weak fluorescence signals. The time-dependent fluorescence behavior for each location is approximated by a multi-exponential decay behavior, where the decay times are constants and only the pre-exponential factors are determined. The decay time constants to be used are determined by the objects to be examined by a multiexponentielle approximation of combined image areas. The relative proportions of individual fluorophores in total fluorescence are each determined from the product of pre-exponential factor and decay time constant of a fluorophore divided by the sum of all products of pre-exponential factors and decay time constants.

The DE 10 2008 045 886 A1 discloses a method for evaluating the fluorescence in a layer system (for example an ocular fundus for ophthalmological examinations), with which the overall decay behavior of the fluorescence can be evaluated very precisely and with which the origin of the individual fluorescences of the layer system can be deduced.

In each case, the time of origin of the fluorescence in the individual layers of the layer system are determined by determining layer-specific time-dependent parameters for the respective fluorescences, which respectively indicate the time of onset of fluorescence in the respective layer and are taken into account in the model function for calculating the overall decay behavior of the fluorescence become.

From the DE 10 2004 017 956 A1 a microscope is known for investigating the lifetime of excited states in a sample which generates excitation light with at least one light source and with at least one detector receives the detection light emanating from the sample.

In this case, the microscope contains the light source in the form of a semiconductor laser which emits pulsed excitation light, an adjustment device being provided for setting the pulse repetition rate to the specific service life properties of the sample.

The WO 2008/094794 A1 discloses a device for time-resolved fluorescence imaging, which is based on a two-dimensional detector, which has the disadvantage that thereby different maturities of the excitation and detection beam can not be considered. In addition, a modeling of the different maturities is not disclosed.

From the WO 2006/135769 A1 For example, a device and a method are known for the time-resolved determination of the three-dimensional distribution of the fluorescence in a sample by means of a software-based evaluation based on Green's function. The different transit times of the individual excitation and detection beams are neither detected nor evaluated.

It is also known, for example, from the DE 10 2004 017 956 A1 a microscope which realizes an adjustment device for adjusting the pulse repetition rate with respect to the specific fluorescence lifetime properties of the sample. However, this technical solution does not take into account the different distances, as they occur in particular in an extended 3D object.

mit

I(t, x, y):

Fluoreszenz zum Zeitpunkt t am Bildpunkt (x, y),

IRF:

Instrumental Response Function,

i:

Komponente, welche idealerweise einen fluoreszierenden Stoff repräsentiert,

τ_i:

Abklingzeit der Komponente i,

α_i:

präexponentieller Faktor für Komponente i,

b:

Hintergrundintensität,

wobei ein summarisches Abklingverhalten der Fluoreszenz beschrieben wird.Also known is a multi-exponential modeling of the time-resolved fluorescence measurement data by the (in DE 101 45 823 A1 ) disclosed equation:

With

I (t, x, y):

Fluorescence at time t at the pixel (x, y),

IRF:

Instrumental Response Function,

i:

Component which ideally represents a fluorescent substance,

τ _i :

Cooldown of component i,

α _i :

pre-exponential factor for component i,

b:

Background intensity,

a summary decay of the fluorescence is described.

By a three-dimensional extended surface or by the oblique adjustment of the measuring system to the normal vector of the surface, there are runtime differences of the excitation and detection beams between the object points. These differences in transit time can lead to errors in determining the fluorescence lifetime according to equation (1).

In addition, artefacts can occur with very short fluorescence lifetimes and relatively long propagation delays. These artefacts are expressed in a superposition of the overall picture, which is ramped. The differences in transit time caused by skew adjustment of the measurement system are modeled by the fluorescence lifetime increasing over the site, in which example the true value of the fluorescence lifetime over the site is constant. In the same way, different lengths of light paths act in the optical system of the detection device

The determined according to equation (1) fluorescence lifetimes have large deviations from the true value and are thus for z. Fluorescence lifetime based diagnostic methods are poorly suited.

The invention is therefore based on the object to provide a method which avoids the disadvantages of the prior art and allows determination and evaluation of time-resolved fluorescence images on extended three-dimensional surfaces which are scanned by pulsed radiation.

According to the invention this object is achieved by the characterizing features of the first claim. Further favorable embodiments of the invention are specified in the subordinate claims.

The method for determining parameters of time-resolved fluorescence or reflection images on extended three-dimensional surfaces is based on the point-by-point scanning of a three-dimensional object and a pixel-by-pixel modeling of different radiation propagation times.

For this purpose, the extended three-dimensional surface of the object is detected by means of an illumination and detection beam, wherein different distances of the illumination beam exit point and the detection beam entry point of pulse-shaped illuminated object points of a three-dimensional object are present. In this case, an arbitrary, illuminated first object point and an arbitrary, illuminated second object point, which is located at a different distance to the illumination beam exit point and detection beam entry point than the first object point, differ in the transit time of the excitation and detection beam, wherein a pixel-by-pixel modeling of the different transit times takes place ,

The invention will be explained in more detail below with reference to the embodiments and the figures. Showing:

1a : Schematic representation of the embodiment of an excitation and detection beam of the fluorescence of two object points with different distances to the excitation beam exit point or detection beam entry point, respectively,

1b : Schematic structure of the embodiment of an arrangement for measuring the time-resolved fluorescence on an object with a three-dimensional surface,

2 : Comparison of time-resolved fluorescence signals from two different object points of a three-dimensional surface and

3 : Course of the fluorescence lifetime of a fluorescent surface whose normal vector is not parallel to the direction of the excitation beam, in multi-exponential modeling according to equation (1) and according to equation (3).

As in 1a is shown in the method for the determination and evaluation of parameters of time-resolved fluorescence or reflection images on three-dimensional surfaces ( 230 ) a pointwise scanning of a three-dimensional object by means of illumination and detection beam (pointwise detection by pulsed radiation), wherein different distances of the illumination beam exit point and detection beam entry point ( 210 ) of pulsed illuminated object points of a three-dimensional object ( 230 ) and any illuminated, first object point I ( 231 ) and any illuminated, second object point ( 232 ) located at a different distance from the illumination beam exit point and detection beam entry point (FIG. 110 ) is in the duration of the excitation and detection beam ( 221 ) of the first object point ( 231 ) and the illumination and detection beam ( 222 ) of the second object point ( 232 ) and a pixel-by-pixel modeling of the different maturities takes place.

mit

I(t, x, y):

Intensität zum Zeitpunkt t am Bildpunkt (x, y)

i:

Komponente

αi(x, y):

präexponentieller Faktor für Komponente i am Bildpunkt (x, y)

t_K(x, y):

Zeit, die ein Photon vom Anregungsstrahlaustrittspunkt zur dreidimensionalen Oberfläche und zurück zum Detektor benötigt

b(x, y):

Hintergrundintensität

f(t, x, y):

Modellfunktion der Komponente i

In this case, the optical signal detected by each point of the surface is approximated by a model function according to equation (2):

With

I (t, x, y):

Intensity at the time t at the pixel (x, y)

i:

component

αi (x, y):

pre-exponential factor for component i at the pixel (x, y)

t _K (x, y):

Time a photon takes from the excitation beam exit point to the three-dimensional surface and back to the detector

b (x, y):

Background intensity

f (t, x, y):

Model function of component i

Here, t _K (x, y) = t _b (x, y) + t _f (x, y) represents the time required for a photon from the excitation beam exit point to the three-dimensional surface and back to the detector from the three-dimensional surface. t _b (x, y) is the time required for a photon from the excitation beam exit point to the three-dimensional surface. t _f (x, y) is the time required for a photon from the three-dimensional surface to the detector.

mit

IRF:

Instrumental Response Function

τ(x, y):

Abklingzeit am Bildpunkt (x, y)

The evaluation of images of the time-resolved fluorescence of three-dimensionally oriented in space-oriented extended surfaces with exponential adjustment of the time course of normalized fluorescence according to the invention according to equation (3):

With

IRF:

Instrumental Response Function

τ (x, y):

Cooldown on the pixel (x, y)

For nearly identical optical path lengths of excitation beam exit point and detection beam entry point, t _K / 2 is equivalent to the distance d between the three-dimensional surface and detector for each individual pixel. By means of the refractive index n and the speed of light c, t _K / 2 can be converted into the distance according to equation (4):

The time resolution of t _K (x, y) thus directly determines the depth resolution at the three-dimensionally oriented surface of the object to be examined. Therefore, a modeling of t _K (x, y) with a higher time resolution than the time resolution of the measured data is advantageous.

Preferably, the distance calculation is realized as a difference to a reference point. The parameter estimation in equation (3) is usually done by a nonlinear optimization algorithm.

The modeling according to the invention enables the exact determination of very short fluorescence lifetimes, since thereby the parameter estimation becomes more exact, in particular at fluorescence signal sections with strong gradients.

Preferably, nearly identical optical path lengths of excitation beam exit point and detection beam entry point to the three-dimensional surface are used.

This is advantageously used in determinations of time-resolved fluorescence on objects in which propagation time differences of the radiation are present at different locations of an object in the order of the time width of a time channel.

g(t, x, y):

Pulsform der Beleuchtung

For the evaluation of time-resolved reflection images of three-dimensionally space-oriented surfaces, equation (2) is modified according to equation (5): f (t, x, y) = g (t, x, y) (5) With

g (t, x, y):

Pulse shape of the lighting

The pulse shape of the illumination is application specific and can be inter alia Gaussian, triangular or rectangular pronounced.

The technical implementation of the method can first be carried out by a quasi point-like emerging and the three-dimensional surface punctiform sequentially scanning laser beam with a quasi point-shaped detector. Furthermore, the simultaneous areal illumination of the three-dimensional surface is provided by a pulsed laser beam issuing quasi point-like and the signal detection with a detector array.

In the example of the detection of time-resolved fluorescence, the time-resolved fluorescence signals per pixel are analyzed and the temporal offset to the excitation beam exit point or detection beam entry point is determined. The data analysis is based on an approximation of the measurement data using the model function of equation (2) and equation (3).

The schematic structure for the precise determination of the fluorescence lifetime of an object with a three-dimensionally oriented surface is shown in FIG 1b shown. A pulsed laser ( 210 ) excites after passing through a dichroic filter ( 220 ) and by a laser scanner system ( 230 ) an object ( 240 ) to the fluorescence whose surface ( 241 ) is oriented three-dimensionally in space. Here are the points ( 242 ) and ( 243 ) at different distances from the excitation and / or detection site, so that a transit time difference exists between the excited fluorescence.

After passing through the laser scanner system ( 230 ) the fluorescence radiation from the dichroic filter ( 220 ) to a detector ( 250 ), before which a block filter ( 260 ), which separates the fluorescence radiation from the excitation radiation.

The time-dependent course of the detector ( 250 ) registered fluorescence radiation is in a computer ( 270 ) determined by the TCSPC method. In the computer ( 270 ) the calculation of the exact fluorescence lifetime taking into account different durations of the fluorescence signals from the surface locations ( 242 ) and ( 243 ) performed.

In 2 the transit time difference of the detected fluorescence of two locations of the fundus at different distances from the excitation and / or detection site is shown.

The calculation of the fluorescence lifetime takes place in one or more steps. Here the procedure is described in 2 steps:

In step 1, starting from the corner points of a rectangular field of the surface ( 241 ) of the object ( 240 ) the temporal behavior of the normalized fluorescence according to equation (2) and equation (3) adapted.

In contrast to time t, which is divided into time channels of constant duration t _z , the transit time t _K can assume arbitrarily small values. As a result of this calculation, for each point x, y of the surface ( 241 ) the exact values for t _K. The fluorescence lifetimes πi calculated in the first step are discarded.

In step 2, the time-dependent behavior of the fluorescence is also calculated according to equation (2) and equation (3), wherein the values for t _K calculated in the first step at each location of the surface ( 241 ) are kept constant and the fluorescence lifetimes πi newly calculated exactly.

In the exemplary embodiment, a nonlinear optimization algorithm according to Nelder-Mead is used to determine the model parameters. For this purpose, a computer ( 270 ) used.

The 3 shows the faulty course of the fluorescence lifetime ( 101 ) of an ocular fundus when the measuring system is misaligned to the eye when calculating the fluorescence lifetime according to the prior art equation (1). This erroneous ramp-shaped course of the fluorescence lifetime ( 101 ) is avoided if the calculation is carried out according to the inventive equation (2). The course of the fluorescence lifetime calculated according to the invention ( 102 ) is constant over the place.

In ophthalmic applications, preferably three exponential functions are used in parameter approximation based on equation (2).

In patients with early age-related macular degeneration, significant prolongations of the fluorescence lifetimes of the mean exponential function can be observed in comparison with the control group of ophthalmologists in the same age group.

The advantage of this method is that in particular the time-resolved fluorescence of extended three-dimensional objects and structures can be determined and evaluated with high accuracy.

Thus, by this method, for example, the errors occurring in measurements on the fundus fluorescence lifetime due to different maturities of the fluorescence signal by incorrect adjustment of the measuring arrangement to the patient's eye and different lengths of light paths in the internal optical system of the measuring device in practical application eliminated.

The method can thereby be used in the time-resolved fluorescence spectrometric diagnosis of other eye diseases such as the examination of vascular occlusions or the therapy control after laser coagulation.

In addition, the use of the method in all clinical and technical applications leads to the determination of exact fluorescence lifetime in which the time-resolved fluorescence of an extended three-dimensional object is measured.

A combination of the measurement of time-resolved fluorescence with therapy devices is also within the scope of the invention. Thus, the method can, for example. In diagnostic equipment, especially multimodal diagnostic equipment, be integrated - z. Structural: Ultrasound (US), Magnetic Resonance Imaging (MRI), Computed Tomography (CT), Optical Imaging, Cameras, etc. - eg. Functional: vessels, metabolism, molecular imaging, electrical and magnetic conduction, electroencephalography (EEG), magnetoencephalography (MEG), functional magnetic resonance imaging (fMRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), near-infrared spectroscopy ( NIRS), etc.

Through the representation of time-resolved reflection images, the method also allows the measurement of the three-dimensionally oriented in space surface of objects that are in mechanically or electrically difficult to access environment, for example. In hazardous areas.

Thus, the wide range of applications of the method in the field of investigation of three-dimensional biological objects in medicine, for example. Imaging of time-resolved fluorescence on the eye, as well as time-resolved reflection measurements on three-dimensional technical objects in manufacturing technology, eg. The method can also be used to measure the three-dimensional surface of a mechanically or hard to reach object.

All features illustrated in the description, the following exemplary embodiments, the claims and drawings can be essential to the invention both individually and in any desired combination.

LIST OF REFERENCE NUMBERS

101

Approximated fluorescence lifetime according to equation (1)

102

Approximated fluorescence lifetime according to equation (2)

210

Excitation beam exit point and detection beam entry point

221

Excitation and detection beam I

222

Excitation and detection beam II

230

fluorescent object with three-dimensional surface

231

excited object point I

232

excited object point II

310

pulse laser

321

dichroic beam splitter

322

Scanner System

323

detection filters

330

fluorescent object with three-dimensional surface

341

Unit for time-resolved detection of fluorescence light

342

Computer system for the evaluation of fluorescence data

401

Time-resolved fluorescence signal of an object point

402

Time-resolved fluorescence signal of an object point at a different distance to the excitation beam exit point or detection beam entry point

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 19920158 A1 [0020]
• DE 10145823 A1 [0022, 0030]
• DE 102008045886 A1 [0023]
• DE 102004017956 A1 [0025, 0029]
• WO 2008/094794 A1 [0027]
• WO 2006/135769 A1 [0028]

Cited non-patent literature

• A. Periasamy, RM Clegg: Flim Microscopy in Biology and Medicine. CRC Press, 2010 [0009]
• www.olympusfluoview.com/applications/flimintro.html [0010]

Claims (8)

Method for determining and evaluating parameters of time-resolved fluorescence or reflection images on an extended three-dimensional surface ( 230 ) of objects in which a pointwise pulse-shaped scanning of the extended three-dimensional surface ( 230 ) by means of illumination and detection beam, wherein different distances of the illumination beam exit and detection beam entry point ( 210 ) of the pulsed illuminated points of the extended three-dimensional object ( 230 ) and an illuminated first object point ( 231 ) and an illuminated second object point ( 232 ) located at a different distance from the illumination beam exit and detection beam entry point (FIG. 210 ) is in the duration of the excitation and detection beam ( 221 ) of the first object point ( 231 ) and the illumination and detection beam ( 222 ) of the second object point ( 232 ) and a pixel-by-pixel modeling of the different transit times, characterized in that from each point of the surface ( 230 ) detected optical signal with the equation

where I (t, x, y) is the intensity at time t at the pixel (x, y), i is the component, α _i (x, y) is the pre-exponential factor for component i at the pixel (x, y), t _K (x, y): the time taken by a photon from the excitation beam exit point to the three-dimensional surface and back to the detector, b (x, y) is the background intensity and f (t, x, y): model function of component i and Evaluation of fluorescence images of the extended three-dimensional surface ( 230 ) with exponential adaptation of the time course of the normalized radiation according to the equation

where IRF is the instrumental response function and τ (x, y) is the decay time at the pixel (x, y), or the evaluation of time-resolved reflection images of the extended three-dimensional surface (FIG. 230 ) with the modification f (t, x, y) = g (t, x, y), where g (t, x, y) is the pulse shape of the illumination. A method according to claim 1, characterized in that the optical path length of the illumination and detection beam is set to be identical, wherein t _K / 2 equivalent to the distance d between the three-dimensional surface ( 230 ) and the detector for each individual pixel, so that by the time resolution of t _K (x, y) the depth resolution at the three-dimensional surface ( 230 ) is determined immediately. A method according to claim 1 or 2, characterized in that the shape of the pulse used Gaussian, triangular or rectangular is set. Use of the method according to one or more of the preceding claims 1 to 3 for determining the time-resolved fluorescence on objects in which transit time differences of the radiation are present at different locations of an object in the order of the time width of a time channel. Use of the method according to one or more of the preceding claims 1 to 3 for the determination of the exact fluorescence lifetime. Use of the method according to one or more of the preceding claims 1 to 3 for mapping the three-dimensional surface. Use of the method according to one or more of the preceding claims 1 to 3 for measuring three-dimensional surfaces of objects which are located in a mechanically or electrically difficult to reach environment. Arrangement for carrying out a method according to one or more of Claims 1 to 7, comprising a pulse laser ( 210 ), a dichroic filter ( 220 ), a laser scanner system ( 230 ), a detector ( 250 ), a block filter ( 260 ) and a computer ( 270 ), whereby by the with the pulse laser ( 210 ) generated radiation after passing through the dichroic filter ( 220 ) and by the laser scanner system ( 230 ) an object ( 240 ) is excitable to fluorescence whose surface ( 241 ) is extensively oriented three-dimensionally in space and points ( 242 ) and ( 243 ), which are located at different distances from the excitation and / or detection site, so that there is a transit time difference between the excited fluorescence, and after the passage of the radiation through the laser scanner system (FIG. 230 ) the fluorescence radiation from the dichroic filter ( 220 ) to a detector ( 250 ) is deflectable, wherein in front of the detector ( 250 ) the block filter ( 260 ), which separates the fluorescence radiation from the excitation radiation, and the time-dependent profile of the signal from the detector ( 250 ) registered fluorescence radiation in the computer ( 270 ) is determinable by the TCSPC method, in which in the computer ( 270 ) with software the calculation of the exact fluorescence lifetime taking into account different transit times of the fluorescence signals from the surface locations ( 242 ) and ( 243 ) is vornehmbar.

Images