DE10239028B4 - Process for the identification of naturally occurring or synthetically produced melanin types - Google Patents

Abstract

A method for identifying naturally occurring or synthetically produced types of melanin, in particular eumelanin and pheomelanin, comprising the steps of stimulating the material containing the melanin types with laser pulses in the fs range by means of absorption of photons and spectrally resolved detection of the fluorescent light emitted by the taken or synthetically produced material sample , wherein the fluorescent light emitted by the melanin types present in the material sample is also detected in a temporally resolved manner for the spectral detection and then the melanin types present in the material sample examined are determined from the spectral distribution of the fluorescence and from the fluorescence decay function, and in the one to be examined Material sample generated the fluorescence both by means of one-photon absorption and by means of gradual two-photon absorption, the results compared and the melanins locations are determined.

Description

The invention relates to a method to identify naturally occurring or synthetically produced types of melanin.

It is known to do fluorescence studies, for example in the life sciences and in hydrology and hydrogeology to identify a specific substance, because the ability of the substance to emit light after exposure to light, substance-specific is, i.e. each substance emits different amounts of energy.

For example, in J. Invest. Dermatol. 116/4 (April 2001) pp. 629-630 described that the fluorescence spectrum of melanin in malignant melanomas shows characteristic changes compared to healthy skin with gradual two-photon excitation. With 800 nm excitation, the spectral distribution of fluorescence in healthy skin tissue has a maximum in the range around 500 nm and drops sharply into the long-wave range, whereas the fluorescence of the malignant melanoma tissue is more intense, especially in the long-wave range. The emission maximum is around 575 nm and the intensity decreases only slightly up to 700 nm. This behavior is in 1 presented in mentioned publication. In the described method, the tissue to be examined is first excited to fluorescence with laser pulses in the fs range by means of two-photon absorption and the fluorescence light emitted by the tissue is detected in a spectrally resolved manner.

Although this method enables Detection of a change in the examined material, however, a more extensive analysis, e.g. B. the clear determination different types of melanin that are responsible for the change can, not possible. The general problem when characterizing melanin varieties is that the exact structure is unknown, e.g. B. there melanins cannot be crystallized and therefore no crystal X-ray structure analysis possible is. Only certain partial structural blocks are known.

In the US 5,419,323 The solution described enables the identification of different chromophores in the tissue. The tissue to be examined is excited with radiation of different wavelengths, the fluorescent light emitted by the tissue is examined both spectrally and temporally resolved.

A similar procedure is in US 6 272 376 B1 The histological state of human tissue is characterized by means of time-resolved laser-induced fluorescence spectroscopy. Here, too, the excitation of the fluorescence can take place with radiation of different wavelengths.

In addition to the well-known one-photon absorption, in DE 199 39 706 A1 specified a fluorophore for multi-photon laser scanning microscopy, which has a single fluorescent level and real intermediate levels between the basic level and the fluorescent level for realizing a gradual m-photon absorption with m ≥ 2.

The object of the invention is now a method of identifying melanin varieties, in particular Eumelanin and Pheomelanin to specify. Among types of melanin Naturally occurring or synthetically produced in various ways Melanine understood. These synthetically produced melanins are said to also as identical to natural occurring melanins or characterized as different can be.

The task is accomplished through a process according to claim 1 solved.

It has been shown that the fluorescence lifetime is different Melanin types are different. The fluorescence decay behavior is for the melanin varieties very complex (triple exponential), but the Fluorescence from pheomelanin, for example, sounds much faster than that of Eumelanin. So that in the inventive method to identify melanin types in addition to the known change of the fluorescence spectrum also the previously unknown fact of different fluorescence lifetimes different types of melanin, which is a selective, safer and enables clearer identification of special melanin types. Both by means of one-photon absorption and by means of stepwise Two-photon absorption generated fluorescence spectra of the eumelanin sMela, eMela, nMela are shifted against each other, the fluorescence spectra for the Layer pheomelanin themselves in both suggestions however approximately. This allows, for example, a better statement regarding the "purity" of the pheomelanin in made of the examined material sample or this result for a quantitative Analysis of the components pheomelanin / eumelanin can be used.

In a further development of the procedure will be additional also the spatial Distribution of fluorescence in the material sample examined was determined.

It also shows a histogram of the photon density of the emitted fluorescent light over the wavelength and the time within the fluorescence decay and the examined Material sample created.

Another further development provides that for each individual photon of the emitted fluorescent light, the wavelength and the time within the fluorescence decay and the location within of the examined material areas are determined and the resulting data stream is analyzed on-line or off-line. A surface distribution of different types of melanin can thus be determined.

The procedure can also be used to determine of malignant melanoma tissue can be used in removed skin. If the skin has a malignant degeneration, then the components have of the pigment melanin changed. It can thus be determined whether in the material sample to be examined next to Eumelanin also contains pheomelanin, and in what proportion this Substance also dependent from the location.

The procedure is explained below the drawings closer described.

Show

1 : Fluorescence spectra of healthy skin tissue and malignant melanoma tissue, excited by means of gradual two-photon absorption at 800 nm;

2 : Decay behavior of fluorescence for healthy skin tissue and malignant melanoma tissue, stimulated with 100 fs pulses at 800 nm;

3 : One-photon and two-photon fluorescence spectra for sMela;

4 : One-photon and two-photon fluorescence spectra for eMela;

5 : One-photon and two-photon fluorescence spectra for nMela;

6 : One-photon and two-photon fluorescence spectra for pMela;

7 : an arrangement for the simultaneous measurement of the spectral fluorescence and the fluorescence decay behavior, with which the method can be carried out.

The method is described below using the example of the inherent fluorescence of melanin in human skin. Here, the melanin types contained in the removed skin tissue are excited, for example, by step-by-step absorption of two photons when irradiated with pulses in the fs range in the spectral range around 800 nm. This excitation process is based on the specific spectral absorption behavior of the melanin, which in connection with a fluorophore for multi-photon laser scanning microscopy, in particular melanin as a fluorophore for two-photon laser scanning microscopy, in DE 199 39 706 A1 was reported. Other endogenous fluorophores or foreign substances (absorbed or applied as impurities) are not stimulated. The measured fluorescence spectra, ie the dependence of the intensity on the wavelength, are in 1 for healthy and diseased tissue and show clear differences, as already mentioned.

2 shows the characteristic fluorescence decay behavior for examined healthy skin tissue and malignant melanoma tissue. As can be seen, the fluorescence of the malignant melanoma tissue, which contains more pheomelanin due to the (pathological) change, decays faster than that of healthy skin. In 2 the square of the apparatus function (dashed curve) is also shown, as well as the fluorescence profiles calculated with respect to this function by global data analysis (deconvolution), which are shown as solid curves. A three-fold exponential decay of the fluorescence was determined with the following decay times: τ ₁ = (100 ± 30) ps; τ ₂ = (788 ± 100) ps; τ ₃ = (4.3 ± 0.3) ns. The amplitudes a ₁ = 0.80 are obtained for healthy skin tissue; a ₂ = 0.14; a ₃ = 0.06 and for malignant melanoma tissue the amplitudes a ₁ = 0.95; a ₂ = 0.04; a ₃ = 0.01.

From the measurement results in the 1 and 2 the following becomes clear: The two-photon fluorescence spectra for malignant melanomas, which contain a significant proportion of pheomelanin, show a clear bathochromic shift compared to those of healthy skin, which is coupled with a sharp reduction in the fluorescence decay.

The method of time-correlated single photon counting (described in DD 282 518 A5 in connection with a fast ADC principle (shown in DE 43 39 784 A1 , with multi-channel detector technology ( DE 43 39 787 A1 ) and with a scan method (described in the magazine "Photonics", 3/2000, pp. 16-19).

Of course, the procedure in dependence of the desired Statement can be simplified. So the scanning process can be dispensed with become. The fluorescence spectrum can be recorded on a few (down to two) spectral channels be reduced.

The evaluation of the measurement data obtained can be done in two different ways. Either one Histogram of the photon density over the wavelength, the time and the scanned area generates a data record that is created for each point of the examined area for each spectral channel get a full fluorescence decay function, or it will be for each single detected photon the time within the laser pulse sequence, the spectral channel and location are determined. The first way shown provides a compressed data set that contains all information for later evaluation contains; the second way delivers very large amounts of data, but it can a decision about the State of the material sample can be taken online in the data stream.

In the 3 to 6 Fluorescence spectra for different types of melanin (sMela, eMela, nMela, pMela) are shown in comparison, which can be examined by single-photon absorption (excitation wavelength: 400 nm) and by two-photon absorption (excitation wavelength: 800 nm) the material sample was generated. It can be seen that only in the case of pheomelanin (pMela) do the spectra coincide approximately. This gives a clear determination of this type of melanin.

In the 7 an arrangement is now shown with which the inventive method can be implemented.

A laser L generates a high-frequency sequence of fs pulses in the near infrared. The light from the laser L is guided via a dichroic mirror S to two scan mirrors S _x , S _y , which deflect the beam in the x and y directions. The axis of rotation of the scan mirrors S _x , S _y is imaged on an objective O, which focuses the laser L on the measurement object M. A rectangular field is scanned on the measurement object M by the movement of the two scan mirrors S _X , S _y . A lens Li arranged between the laser L and the dichroic mirror S widens the beam, so that the effective numerical aperture of the image in the measurement object M is enlarged and thus the diffraction disk is reduced in focus. In order to hold and fix the measurement object M in the focal plane of the objective O, it is expediently pressed onto the back of a glass plate GP. The fluorescence of the melanin excited in the focus of the laser L is collected by the objective O and goes back via the scanning unit SE and through the dichroic mirror S. Behind the dichroic mirror S there is a fixed focus which leads into the input slit of a polychromator P. becomes. The light from the polychromator P is directed to a multi-channel photomultiplier PMT. If a channel of the photomultiplier PMT registers a photon, a pulse of a few ns duration is generated at the relevant output. The pulses of the channels of the photomultiplier PMT are fed to a router R. This combines the pulses of all input lines into a single signal and also generates a digital signal that indicates in which channel a pulse has occurred. This signal is used as wavelength information.

The combined pulses are received by a constant fraction discriminator CFD ₁ . Another CFD ₂ receives reference pulses from the laser L. A circuit for time measurement (either a time-to-amplitude converter, TAC, with a subsequent analog-to-digital converter, ADC, or a time-to-digital converter circuit, TDC) determines the time difference between the photon pulses and the reference pulses from the laser L. The information generated is the time t of the respective photon within the fluorescence decay function.

The scanning unit SE for distraction of the laser beam delivers several synchronization signals, i.e. one Transition impulse to the next Pixels, a pulse at the beginning of each new line and a pulse at the beginning of a new picture. An X-Y circuit XY consisting of a combination of several counters, generates the current x-y position within the scanned Area. The x-y information can also be generated directly by the scan unit SE are omitted in this case the X-Y circuit XY.

In the variant with histogram formation the sizes x, y, t and λ for Addressing a memory SP used. The value of the addressed storage space is increased by one unit for each photon, so that the Photon density over x, y, t and λ built becomes.

Another option is at the sizes x, y, t and λ in enter the memory SP. The memory SP is then called a FIFO (first in first out) Storage configured. It is used to buffer the irregularly incoming photon data. The memory SP is read continuously, so that a data stream with the information of the individual photons.

The described embodiment can be varied depending on the application.

So the entire scan arrangement SE including of the X-Y complex XY can be omitted. A single point measurement is then obtained Intensity over t and λ.

Becomes a highly sensitive measurement of two-photon fluorescence images in the different wavelength ranges desired so the time measurement can be omitted.

Another variant results if the detector is replaced by a single-channel photomultiplier PMT the route R is then omitted. The result is Fluorescence images for different times after the laser pulse or an array of pixels, which each contain a full fluorescence decay function.

To increase the data throughput, the entire electronics part (router, CFDs, time measurement, memory) several times accomplished become.

• – Das Verfahren gestattet eine eindeutige Identifizierung ausschließlich unterschiedlicher Melaninsorten.
• – Die Messdaten des Verfahrens liegen in Form von Bildern vor. Bei medizinische Anwendungen finden Bilder eine weitaus höhere Akzeptanz als Einzelpunkt-Messungen.
• – Jedes vom Detektor empfangene Photon wird verarbeitet, wodurch eine maximale Empfindlichkeit realisiert wird.
• – Durch die hohe Empfindlichkeit in Verbindung mit einer hohen Laser-Repetitionsrate wird mit relativ geringer Impulsenergie gearbeitet. Verglichen mit niedrig repetierenden Lasern hat das Verfahren deshalb einen größeren Sicherheitsabstand zu der Intensitätsschwelle, an der gefährliche Effekte, wie Drei- und Vier-Photonenprozesse oder sogar Plasma-Erzeugung, auftreten.
• – Die Zwei-Photonen-Anregung findet während des erfindungsgemäßen Verfahrens in nennenswertem Umfang nur im Fokus statt. Das erlaubt die Aufnahme von Schnittbildern in verschiedenen Tiefen der untersuchten Materialprobe.
• – Die Zeitauflösung wird nur von der Laufzeitstreuung in Detektor bestimmt. Diese ist gewöhnlich etwa 10 mal kleiner als die Impulsbreite der Photonenimpulse. Die Auflösung ist deshalb 10 mal besser als beim Einsatz des gleichen Detektors im Analog-Betrieb.
• – Durch Variation der Scan-Parameter kann die Anzahl der Bildpunkte beliebig verändert werden. Gleichzeitig kann die Auflösung der Zeitmessung verändert werden, indem Zeitkanäle zusammengefasst werden. Auch benachbarte Wellenlängen-Kanäle können beliebig zusammengefasst werden. Dadurch sind sowohl hochaufgelöste Bilder mit moderater Zeitauflösung als auch zeitliche Präzisionsmessungen einer geringeren Anzahl von Bildpunkten möglich.
• – Durch die Aufzeichnung des vollen x-y-t-λ-Datensatzes können die Fluoreszenz-Komponenten unterschiedlicher Fluorophore anhand ihrer unterschiedlichen Spektren und Abklingzeiten gut getrennt werden.

In summary, the method according to the invention for identifying melanin types has the following advantages:

• - The method allows a clear identification of only different types of melanin.
• - The measurement data of the method are available in the form of images. In medical applications, images are much more accepted than single-point measurements.
• - Every photon received by the detector is processed, whereby a maximum sensitivity is realized.
• - Due to the high sensitivity in connection with a high laser repetition rate, relatively low pulse energy is used. Compared to low repeating lasers, the method therefore has a greater safety margin from the intensity threshold at which dangerous effects such as three and four photon processes or even plasma generation occur.
• - The two-photon excitation takes place only to a significant extent during the process according to the invention. This allows the recording of sectional images at different depths of the examined material sample.
• - The time resolution is only determined by the runtime spread in the detector. This is usually about 10 times smaller than the pulse width of the photon pulses. The resolution is therefore 10 times better than when using the same detector in analog operation.
• - The number of pixels can be changed as desired by varying the scan parameters. At the same time, the resolution of the time measurement can be changed by combining time channels. Adjacent wavelength channels can also be combined as required. This enables high-resolution images with moderate time resolution as well as temporal precision measurements of a smaller number of pixels.
• - By recording the full xyt-λ data set, the fluorescence components of different fluorophores can be separated well on the basis of their different spectra and decay times.

Claims (5)

Process for the identification of naturally occurring or synthetically produced types of melanin, in particular of eumelanin and pheomelanin, comprising the process steps of stimulating the Material containing melanin types with laser pulses in the fs range by means of absorption of photons and spectrally resolved detection of the material sample taken or synthetically produced emitted fluorescent light, which is different from that in the material sample existing melanin types emitted fluorescent light simultaneously for spectral detection is also detected in a temporally resolved manner and then off the spectral distribution of the fluorescence and from the fluorescence decay function the melanin types present in the material sample examined be determined and in the material sample to be examined fluorescence using both single-photon absorption and gradual two-photon absorption generated, the results compared and the types of melanin are determined. The method of claim 1, wherein additionally spatial Distribution of the fluorescence of the material sample examined existing melanin types is determined. The method of claim 1, wherein a histogram of the Photon density of the emitted fluorescent light above the wavelength and the time within the fluorescence decay and those examined material surface is created. A method according to claim 1, characterized in that for each individual photon of the emitted fluorescent light has the wavelength and the time within the fluorescence decay function and the location within the examined material area be determined and the resulting data stream on-line or off-line is analyzed. Use of the method according to one of claims 1 to 4 for the determination of malignant melanoma tissue in removed skin.