DD297247A5 - IMPROVEMENT OF TISSUE DIAGNOSIS USING THE EMISSION OF FLUORESCENT LIGHT - Google Patents

Abstract

A sample (1) is irradiated with a pulse from a laser (2), whereby a fluorescence radiation is formed, which is brought in picture form or otherwise to a recording device (6) from the same sample (1) a plurality of spectral intervals (6 A to 6 D) to obtain intensity values. The radiation can be recorded in image form, with the multiplicity being multiplied. The spectral intervals can be wavelength intervals (5 A to 5 D), which are determined by spectral time intervals, which in turn are controlled by a computer (7), which also performs a numerical evaluation with at least one division. The tissue properties can then be derived from the evaluation. Fig. 12 {irradiation of a sample with a laser pulse; Bring resulting fluorescence radiation to a recording device; Determination of tissue properties}

Description

For this 14 pages drawings

Field of application of the invention The invention relates to an improved determination of tissue properties by means of induced fluorescence.

Characteristic doi known prior art

From US-A-4682 694 and US-A-4785806, which are hereby incorporated by reference, it is known to irradiate an optical fiber into a blood vessel, such as an artery, with laser light and to accept the induced fluorescent light. This makes it possible to detect atherosclerotic plaque, which can then be destroyed and eliminated by irradiation with power lasers by dioselbo optical fiber. The patents mentioned include means for excitation, spectral analysis and power irradiation, including beam splitting, couplers and laser devices.

EP-A-85905342.3 describes a fluorescence image recorder which records images of a sample irradiated with an excitation wavelength which has been decomposed into a plurality of images by means of a beam splitting device, the images being recorded before striking a matrix detector (US Pat. CCD detector) have been filtered. For each image, one obtained a set of the corresponding pixel intensity values that can be arithmetically manipulated to obtain combined pixel values. The combined pixel values are used to create an image with improved contrast. For a fluorescence examination of the tissue, the fluorescence behavior of the different types of tissue is exploited. In some cases, the contrast may be increased by administration of substances such. As hematoporphyrin derivatives are improved. A disadvantage, however, is that the patient is then hypersensitive to sunlight for long periods of time.

Object of the invention

The invention provides a method for improving tissue diagnosis by means of the emission of fluorescent light, and a device for determining the tissue properties by means of fluorescence.

Explanation of the essence of the invention

The invention has for its object to provide a method for improving tissue diagnosis by means of the emission of fluorescent light, as well as a device for determining the tissue properties by means of fluorescence, wherein the recording contrast is increased and the fluorescence recording allows maximum contrast.

Furthermore, it is an object of the invention in the in vivo diagnosis of various tissue conditions and variations, e.g. As to determine atherosclerotic plaque and various malignant tumors to improve the detection possibilities. Another object is to improve the treatment options in the removal or destruction of tissue by irradiation by means of improved examination options before, during and after the treatment.

Preventing the destruction of tissue that should remain undamaged is another goal.

A special object of the invention is the detection even in the presence of blood. The existing as an absorbent blood acts as a differential absorber of fluorescent radiation, reduces the otherwise achievable contrast and destroys the diagnostic information to a large extent. By the invention it is possible to substantially eliminate the influence of this destruction factor.

According to an important aspect of the invention, the objectives of the invention are achieved by a combination of laser light sources for fluorescence excitation, a light detector, a light guide such as optical fibers, couplers for the entrance of the laser light into one end of the light guide and for the removal of the light for the detector , diagnostic devices coupled to the detector for the study of fluorescence light in a variety of spectral range channels and by comparing these channels. This combination can be suitably completed by means for the power irradiation over the same light pipe, so that, for example, the destruction and disposal can be carried out with subsequent investigation steps under control. The same laser can be used for both diagnosis and high power radiation when the laser energy is adjustable accordingly.

According to a further aspect of the invention, the system can be extended to an image system, wherein a multi-pixel system allows two-dimensional image acquisition, in which one receives a signal for the variety of spectral fluorescence intervals for each pixel.

In this disclosure, the spectral fluorescence intervals include both the wavelength and time intervals. In the latter case, the fluorescence-inducing radiation has the form of short pulses or at least one emission which can be stopped within a very short time interval. Then the temporal distribution of the decreasing fluorescence radiation can be recorded, the decrease of which is caused by the different lifetime values of the excited states, which in turn are characteristic of the different substances peculiar to the different tissues.

A particular feature of the invention is the use of division-involving arithmetic operations in the intensity values. By dividing two values of magnitude of intensity, it is possible to obtain related or dimensionless values which compensate for variations in intensity, pitch, angle, or other variables, which, on the other hand, could distort the results.

When using time intervals, the use of a pulse laser or at least one laser with fast turn-off capacity is to be preferred. Although a continuous laser can be used in the determination without the use of time interval windows, the use of a pulse laser in this case may also be of interest in simplifying the elimination of false radiation due to inaccuracies in the spectral resolution.

AusfQhrungsbelsplele

The invention will now be described with reference to examples and exemplary embodiments illustrated in the drawings, which are intended to illustrate and not to limit the invention.

Figures 1a and 1b illustrate the mechanism of fluorescence radiation. Fig. 2: shows the wavelength spectra for a number of tumor labeling reagents. Fig. 3: shows the difference in the wavelength fluorescence spectra between a cancerous and

a healthy tissue

 Fig. 4 :. shows the difference in the wavelength fluorescence spectra between blood vessels with and without

plaque

 Fig. 5: shows the difference in time spectra for a given wavelength for a normal vessel and

a vessel with plaque

 Fig. 6: shows the results of the divisions between tent spectrum signals and at the same wavelength for

healthy and plaque vessels.

Fig. 7: shows the results of the divisions for two wavelengths with and without time-spectral analysis. Fig. 8: illustrates the absorption spectra of blood. Fig. 9: illustrates the efficiency of the different wavelengths in distinguishing between

normal vessel (0) and four ascending damaged, atherosclerotic classes of vessels

(IbIsIV).

Flg. 10: shows the possibilities of separating the time spectra for a specific wavelength. Fig. 11: shows the elimination of blood disorders using temporal analysis (ratio

between late to early fluorescence) at a particular wavelength.

FIG. 12 shows an exemplary embodiment of the image recording according to the invention. FIG. FIGS. 13 to 15 show cancer detection via image acquisition. Fig. 16: shows the different time-spectral behavior of normal tissue and malignant

tumor tissue

 Fig. 1 a: shows the principle of the fluorescence mechanism in large molecules. An irradiated sample

absorbs the radiation and different levels are excited. Some states return

basically go back to the old state (elastic scattering), some go to the internal one

Transformation, lost through collisions and other mechanisms. Some, however, are a source of inspiration Fluorescence radiation, depending on the state distribution of a broad Wavelength distribution results, as can be seen from the schematic intensity spectrum under Fig. 1 a

can see.

Some useful tumor-labeling reagents, such as hematoporphyrin derivatives, result in broader structured fluorescence spectra, as in Fig. 1b, especially when excited in secretory band by 405nm. The fluorescence spectrum shows typical peaks at 630nm and 690nm, which in practice are superimposed on a more unstructured intrinsic fluorescence of the tissue. There are still more known examples of such reagents. Fig. 2 shows fluorescence spectrograms of substances irradiated at 337 nm (N ₂ laser) for OHE (dihematoporphyrin ether / ester), HP (hematoporphyrin) PHE (polyhematoporphyrin ester) and TSPC (tetrasulfated phthalocyanine).

Another example of the difference in wavelength spectra of different tissue types is shown in Fig. 3, in which the spectrum for tonsil cancer due to the endogenous porphyrin clearly differs from that of the normal mucosa.

example 1

For the synchronous pumping of a color laser with coherent radiation and cavity caching was eir. mode-locked argon ion laser (CK-12 coherent radiation). The color laser delivered 6-second pulses at 640nm with a repetition rate of 3MHz. The average power was 10mW. The red pulses were frequency doubled in a KD'P crystal to 320nm, with the efficiency of frequency doubling being about 0.5%. Together with interference filters, the fluorescence light was selected by wavelength in a 0.5 m spectrometer and determined in a photomultiplier with microchannel plate (Hamamatsu 1564 U). The electronics contained a start pulse channel, and appropriate signal amplifiers, constant pitch discriminators and also a time-amplitude converter were used. In a multi-channel analyzer, the temporal histograms were created and the data analysis was performed by means of a program package on an IBM-compatible personal computer. The temporal reaction function of the apparatus was measured with scattered light and resulted in FWHM = 250 ps. This value was used in the computer deconvolution procedure of the fluorescence signal.

The data were recorded in the scan series, which began with a normal blood vessel and proceeded to an atherosclerotic plaque area. The sample is moved on a micrometer-controlled slide to allow reproducible positioning of the sample in a time-resolved scan at different fluorescence wavelengths. At a count frequency of approximately 1000Hz, the decay curves were normally recorded for 2 minutes. The characteristic, time-integrated fluorescence structures of a normal vessel wall and of plaque are shown in FIG.

For plaque and a normal tissue wall, the plots of sample fluorescence at 400nm versus time are shown in FIG. There are clear differences in temporal behavior. Both plaque and normal vessels were found to have three different lifetimes of approximately 8ns, 2ns and less than 200ps.

In Flg. Figure 6 shows plaque, calcified plaque and normal vessel wall data. The monochromator was tuned to 400nm and 480nm according to the characteristic wavelengths. The fluorescence intensities at 400 nm and 480 nm were designated a and c, respectively. Hler wuro .. each from 5ns to 15ns integrated signal divided by that obtained in the first 5 ns of decay. At a fast decay this ratio obviously has a low Wen, whereas higher values indicate a slower decay. In the measurement of the a-signal, a plaque limit ratio of 1.6: 1 is obtained, whereas in the c-signal the limit ratio is lower. This property can be integrated into a suitable limit criterion. Using this time response to form the dimensionless limit function a (5 to 15 ns) / c (0 to 5 ns) instead of currently integrated variables gives a threshold improvement of 1.6. Scanning through a plaque area is shown in Figure 7 for both the in-tent and tent-resolution boundary criteria and shows a threshold improvement of 2.8 to 4.5.

This example shows the value of resolution in two dimensions (wavelength and time) of fluorescence detection to improve resolution of tissue differences.

Figure 8 shows the transmission spectrum through a 0.2 mm thick layer of arterial (top) and venous (bottom) blood boids were diluted with saline to a concentration of 20%. Performing fluorescence spectroscopy over such an absorber, which is always present in in vivo studies (except for temporary replacement with other fluids), presents great difficulties. Therefore, according to one aspect of the invention, at least one pair of different wavelengths having the same absorption factor used to detect the sample is selected. In the drawings, two such pairs are shown.

Example 2

Five classes of plaque-identified pathological specimens in which 0 was normal arteries and I to IV were increasingly advanced disease were measured for fluorescence at various wavelengths. As shown in Fig. 9, the intensities were divided in pairs. As can be seen from this figure, the degree of correlation varied greatly depending on the wavelength selected. F1 to F4 are affected by blood reabsorption, F5 and F6 will not. F5 and Fβ show that there are real spectral differences between these types of tissue, not just variations in the amount of blood. Furthermore, the relative uncertainties (referred to the standard deviation) are smaller for the blood-compensated pairs F5 and F6.

Example 3

It has been demonstrated that the fluorescence is long lived to provide a short pulse nitrogen laser (PRA model LN 250, tp = 3 ns) in conjunction with a dual channel boxcar integrator (Stanford Instruments, Model SK 250) to distinguish between To use "early" and "later" fluorescence at 400nm. The excitement was made at 337 nm. A photomultiplier of Hamamats ···, Model R105, was used. The one detection channel was adjusted to 0 to 5ns and the second overline 5ns to 15ns, which is shown in Fig. 10, where a decay curve obtained with the pico-secondary system is also inserted. In the boxcar system, the ratio of "later" to "early" fluorescence is formed (having all the properties of a dimensionless size) and displayed on a strip chart recorder. In Fig. 10, the signal is displayed when the schematically represented glass fiber probe is guided pointwise over an arterial sample and determines the Plnquebereiche. As can be seen, a threshold above which the plaque criterion is met and control signals for a plaque ablation laser can be provided. The data of Figure 10 yielded a specimen that was rinsed free of blood to allow clear visual inspection of the atherosclerotic and normal wall areas. A second study was performed on two selected typical sites where the recordings were made through a blood field. The results are shown in FIG. 11. Again, the late-early fluorescence ratio was recorded. As can be seen, the ratios remain constant and separated up to a blood layer thickness of 0.3 mm (60 mm of undiluted blood). For thicker layers, the individual signals are so small that you can no longer obtain a useful signal-to-noise ratio. In the right part of the picture are also the individual signals of the Boxcar channel for a blood-free normal vessel wall shown. From Fig. 8, it is clear that these signals become very weak at 400 nm when the layer of undiluted blood is thicker than a few tenths of a micrometer. Thus, the optical diagnosis must rely on the fiber held close to the sample, or the observation field must be salted in a closed perlpheric artery. In both cases, the diagnostic system must clearly indicate if the signal is too low. On the other hand, a system based on the above given principle provides a reliable guideline regardless of the blood when the fiber tip has first been brought into a sufficient proximity of the arterial wall, and the system switches itself to the data recording mode.

The above examples demonstrate that reliable artery diagnostics are also possible in the presence of blood, using a variety of spectral fluorescence intervals, (1) a zero-differential absorption wavelength pair, and / or (2) two lateral spectral intervals.

The teachings of the invention have heretofore been presented in single-channel applications such as spacing - one can say in one-pixel applications. However, the same principles can also be applied in multiple pixel application forms, i. H. at spatial resolution, to be applied.

Example 4 FIG. 12 shows a schematic overview of the apparatus, as known in principle from EP-A 85905342.3. A sample in

an object plane 1 is irradiated by means of a UV pulse source 2. The sample is imaged by a mirror 3 which is subdivided and angled differently to produce four separate images, 6A to 6D, on a CCD image intensifier camera 6 coupled to the computer 7 after reflection on a Cassegrain mirror 4. The radiation to each of the

Mirror segments 3 are passed through four filters, 5 A to 5 D. Thus, pictures 6A to 6D represent pictures in four different ones Wavelength ranges.

- The detector is a modern microchannel plate image amplifier. The amp can be triggered down to 5ns.

An exemplary result for a malignant rat tumor is shown in FIGS. 13-15. Fig. 13 shows four "black and white" images of 470nm, 600nm, 630nm and 890nm wavelengths. The significance of these wavelengths with respect to the fluorescence of this type of tumor is from the spectrum before) Flg. 15 can be seen. The four colors were assembled on a monitor (not shown) to a false color image. In Fig. 14 is a sketch of the image, as one sees ee represented. The real size of the recorded area was about 10mm.

In this example, Delll Delti image intensification CCD camera system and iSM compatible computer with a DT 7020 vector processor were used for data transmission.

It was experimentally observed that the 630 nm fluorescent band in tumor tissue (due to the porphyrins) is much more durable than the background fluorescence at the same wavelength, as shown in FIG. The temporal differences in plaque and normal vessels have already been discussed in connection with FIG. B and FIG. It is clearly possible to further improve the recording efficiency even in the image pickup device by using a triggered pickup tube, as already noted, and dividing the "late" fluorescence images by the "early" ones.

drawings

Fig. 1: Energy = energy; Intensity = intensity; Wavelength = wavelength; Absorption = absorption; Elastic scattering = elastic scattering; Internal conversion = Inner transformation; and colllslans = and collisions; Vibrational relaxation = vibration relaxation; Fluorescence radiation = fluorescence radiation; HPD hemathoporphyrin derivative.

Fig. 2: fluorescence intensity = fluorescence intensity; Wavelength, nm - wavelength in nm.

3: fluorescence intensity (rol.units) = fluorescence intensity (rel.); Wavelength (nm) = wavelength (nm); Tonsil cancer = tonsil cancer; Normal mucosa = normal mucosa.

Flg. 4: Intensity (rel. Units) "Intensity (rel.); Wavelength (nm) = Wavelength (nm); Plaque = Plaque; Normal vessel = Normal vessel.

Flg. 5: intensity (rel. Units) = intensity (rel.); Time (ns) = time (ns); Plaque = plaque; Normal vessel = normal vessel. Fig. 6: Calcified plaque = calcified plaque; Normal vessel = normal vessel; Plaque = plaque. Fig.7: Normalized unite = related units; Calcified plaque = calcified plaque, thin plaque - thin plaque. Fig.8: Transmission = transmission; Arterial blood = Arterial blood; Venous blood = venous blood. Fig. 10: Time = time; Plaque = plaque, threshold = threshold; Intensity = intensity. Fig. 11: intensity = intensity; Blood-fres = bloodless; Blood = blood, plaque = plaque. Fig. 12: False color image = false color image; Image Intensified CCD camera; CCD image intensification camera; Plotter

Plotter; Split mirror Cassegrainian arrangement = Cassegrain arrangement with split mirror; Individually adjustable mirror Segments = Individually adjustable mirror segments; Pulsed UV source = source of UV radiation; Quartz lenses = Quirzglasoptik; Dichroic beam splitter = dichroic beam splitter; Fiber bundle or microscope adapter = fiber optic bundle or microcircuit adapter; Object plane = object plane; Passband filters = bandpasses.

Fig. 13: Tumor fluorescence images = fluorescence tumor images. Fig. 14: sketch = sketch; muscle tumor = muscle tumor; Needle = needle. Fig. 15; Spectrum = spectrum. Fig. 16: intensity = intensity; Malignant tumor = malignant tumor; Normal tissue - normal tissue; Time = time.

Claims (11)

1. A method for determining the tissue properties by means of fluorescence, characterized in that it comprises the following steps: I. Excitation of the tissue with a laser wavelength of less than 500 nm and here especially below, II. Recording the fluorescence intensity within a plurality of predetermined spectral intervals to obtain a plurality of numerical intensity values. III. Performing an arithmetic operation with these numerical intensity values including at least one division operation, 2. Method according to claim 1, characterized in that the predetermined spectral intervals contain wavelength intervals. A method according to claim 1, characterized in that the predetermined spectral intervals include time intervals which begin and end at a predetermined ratio to the end of the period of a pulse of the laser. A method according to claim 1, characterized in that the predetermined spectral intervals include a plurality of wavelength intervals, each wavelength interval being recorded in a predetermined time interval starting and ending at a predetermined ratio to the end of the period of an impulse of the laser. A method according to claim 1, characterized in that at least two of the predetermined spectral intervals contain spectral intervals around wavelengths having substantially equal absorption values for blood. 6. The method according to claim 1, characterized in that the detection step also simultaneously includes the determination of the fluorescence intensity for the plurality of spectral intervals of the many surface elements in a sample which has been irradiated with the laser pulse. 7. Device for determining the tissue properties by means of fluorescence, characterized in that it contains: I. a laser light source, II. Recording means for recording the fluorescent light from a sample which has been irradiated with the source at a plurality of predetermined spectral fluorescence intervals for the purpose of obtaining a plurality of intensity values, III. Arithmetic and / or logical means for performing an operation on the plurality of intensity values, in which at least one division operation is included, to obtain numerical values characteristic of the tissue properties of the sample. 8. Apparatus according to claim 7, characterized in that it contains means for spectral resolution for the previously determined recording wavelength intervals for the plurality of spectral intervals. 9. Apparatus according to claim 7 or 8, characterized in that the laser light source is a pulse source and has time resolution means for the predetermined temporal spectral intervals of the fluorescence, which begin and stop in the predetermined ratio to the pulses of the source. 10. A source according to claim 1, characterized in that the wavelength of at least two of the predetermined spectral intervals is stored around a wavelength value pair, which basically have the same absorption values in blood. 11. Apparatus according to claim 7, characterized in that the recording means multi-channel character for a simultaneous recording of the fluorescence intensities of the plurality of spectral intervals of the many surface elements in a sample which has been irradiated with the laser light source.