RU2637630C1 - Method for microscopic study of biological samples marked by phosphorescent probes in vitro - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: multiplexed marking of a biological sample with phosphorescent material, their visualization and photoluminescence lifetime determination using laser scanning microscopy are performed. Nanosized anti-Stokes phosphors (NAF) with a size not exceeding 200 nm are used for multiplexed marking of a biological sample. NAFs are excited by a continuous IR laser with a wavelength of 980 nm. The image obtained using a laser scanning microscope is processed in two steps. In the first step, processing is performed along a slow scanning axis. In the second step, processing is performed along a fast scanning axis.

EFFECT: increased sensitivity during visualization of phosphorescent nanoparticles, increased accuracy of determination of localization and of photoluminescence of lifetime discrete phosphorescent nanoparticles and their aggregates deposited on biological samples.

3 dwg

Description

The present invention relates to the study of biological samples using optical microscopy and nanotechnology and can be used to determine the localization of single phosphorescence nanoparticles or their discrete aggregates deposited on biological samples, and their photoluminescence lifetime during multiplex marking.

Currently, microscopic studies of various biological samples are widespread in order to obtain information about the molecular processes occurring in them, for example, such as the movement of individual molecules during the transmission of cellular signals. The general research algorithm includes marking the biosample with phosphorescent labels, visualizing them using laser scanning microscopy, and determining the photoluminescence lifetime. However, it remains relevant to develop new research methods to obtain more accurate and valuable scientific information.

A known method of microscopic examination of biological samples based on the use of fluorescence labeling with molecular specificity, which is usually implemented using organic fluorescent dyes. The use of fluorescent probes allows visualization of cellular and tissue components, such as the extracellular matrix, organelles and biomolecules, studies of protein-protein interactions, and also analyzes the movements of individual molecules during the transmission of cellular signals [Moerner, W.E. Proceedings of the National Academy of Sciences, 2007, 104, 12596].

However, many known molecular fluorophores are very sensitive to the biochemical environment. In this case, interaction with the environment can lead to broadening of the spectra, flickering or quenching of fluorescence. These disadvantages significantly limit the use of such molecular markers [Gomez, D.E .; van Embden, J .; Jasieniak, J .; Smith, T.A .; Mulvaney, P. Small, 2006, 2, 204].

To partially solve these problems, photoluminescent nanoparticles, such as quantum dots and nanodiamonds, are used. However, the applied quantum dots have high quantum efficiency, narrow and tunable spectra, but the emission of quantum dots is characterized by flickering and they themselves have high biological toxicity [Edmonds, A.M .; Sobhan, M.A .; Sreenivasan, V.K.A .; Grebenik, E.A .; Rabeau, J.R .; Goldys, E. M; Zvyagin, A.V. Particle & Particle Systems Characterization, 2013, 30]; nanodiamonds are non-toxic, but their radiation intensity is much weaker than the radiation intensity of quantum dots [Yu, S.J .; Kang, M.W .; Chang, H. C; Chen, K.M .; Yu, Y.S. Journal of the American Chemical Society, 2005, 127, 17604; Sreenivasan, V .; Zvyagin, A .; Goldys, E. Journal of physics. Condensed matter: an Institute of Physics journal, 2013, 25, 194101].

Photoluminescent labeling using molecular fluorophores and nanoparticles is known. The disadvantages of their use include the presence of strong background radiation from cells and biological tissues. It is known that the contribution of autofluorescence can be partially suppressed using spectral filtering. However, this is not enough to study bimolecular processes, such as the movement of individual molecules during the transmission of cellular signals.

(НАФ) для маркирования биологических объектов. В отличие от традиционных молекулярных флуорофоров НАФ имеют практически неограниченную фотостабильность, уникальные фотофизические свойства, позволяющие преодолеть шумовой вклад автофлуоресценции за счет апконверсии - преобразования энергии света средней интенсивности, поглощенного в инфракрасной области, в свет с большей энергией в видимом и ближнем инфракрасном диапазонах. Кроме того, НАФ имеют большие времена жизни фотолюминесценции, превышающие времена жизни флуоресценции органических красителей и эндогенных флуорофоров. Причем время жизни фотолюминесценции НАФ является параметром, зависящим от окружения наночастиц. Это позволяет эффективно использовать НАФ при мультиплексном маркировании [Lu, Y.Q.; Zhao, J.В.; Zhang, R; Liu, Y.J.; Liu, D.M; Goldys, E.M; Yang, X.S.; Xi, P.; Sunna, A.; Lu, J.; Shi, Y.; Leif, R.C; Huo, Y.J.; Shen, J.; Piper, J.A.; Robinson, J.P.; Jin, D.Y. Nature Photonics, 2014, 8, 33].Known for the use of nanoscale anti-stokes

(NAF) for marking biological objects. In contrast to traditional molecular fluorophores, NAFs have almost unlimited photostability, unique photophysical properties that make it possible to overcome the noise contribution of autofluorescence due to up-conversion - the conversion of medium-intensity light energy absorbed in the infrared region into light with higher energy in the visible and near infrared ranges. In addition, NAFs have large photoluminescence lifetimes that exceed the fluorescence lifetimes of organic dyes and endogenous fluorophores. Moreover, the NAF photoluminescence lifetime is a parameter that depends on the environment of the nanoparticles. This allows the efficient use of NAF in multiplex marking [Lu, YQ; Zhao, J. B .; Zhang, R; Liu, YJ; Liu, DM; Goldys, EM; Yang, XS; Xi, P .; Sunna, A .; Lu, J .; Shi, Y .; Leif, RC; Huo, YJ; Shen, J .; Piper, JA; Robinson, JP; Jin, DY Nature Photonics, 2014, 8, 33].

However, due to the fact that NAFs have very large photoluminescence lifetimes (in millisecond scale), point-to-point recording of a signal when receiving images of samples marked with NAFs requires the accumulation of a photoluminescent signal at each point (pixel) for a time comparable to the lifetime of NAFs .e. on a millisecond scale. This makes the total recording time of the scanned image impractical for a long time. And with faster scanning, the images of nanoparticles look like spots elongated along the fast axis of scanning, as a result of which information content is lost when marking.

Closest to the proposed method for the combination of essential features and the technical result, selected as a prototype of the present invention, is a method for microscopic examination of biological samples labeled with phosphorescent probes in vitro, including multiplex marking of a biological sample with phosphorescent materials, their visualization and determination of photoluminescence lifetime using laser scanning microscopy [Pominova DV, Ryabova AV, Grachev PV, Romanishkin ID, Kuznetsov SV, Ro zhnova J.A., Yasyrkina D.S., Fedorov P.P., Loschenov V.B. Upconversion microparticles as time-resolved luminescent probes for multiphoton microscopy: desired signal extraction from the streaking effect // J. Biomed. Optics 2016. Vol. 21 (9). 096002-1-9].

The known method is as follows.

редкоземельных ионов Yb³⁺-Er³⁺ и Yb³⁺-Tm³⁺, наносят на исследуемый биобразец. Осуществляют визуализацию антистоксовых микрочастиц с использованием лазерного сканирующего микроскопа (LSM-710 NLO, Carl Zeiss, Германия). При этом возбуждение микрочастиц осуществляют импульсным фемтосекундным лазером (Chameleon Ultra II, Coherent, США) на длине волны 980 нм. Далее с использованием воздушного неиммерсионного объектива с 20-кратным увеличением сканируют образец, при скорости сканирования в диапазоне 1.27-3.15 мкс/пикс. Регистрацию фотолюминесцентного сигнала осуществляют с использованием недесканирующих детекторов (NDD) в трех спектральных диапазонах: 400-490 нм, 490-560 нм и 565-700 нм соответственно. Полученное изображение раскладывают на строки с шагом в один пиксель вдоль быстрой оси сканирования. В каждой строке определяют положение микрочастицы как точку с максимальной яркостью и границы ее сигнала. Полученную последовательность (профиль сигнала, заключающийся в указанных пределах) аппроксимируют с использованием функции рассеяния точки (ФРТ) вида:Anti-Stokes microparticles based on doped fluoride matrix

rare-earth ions Yb ³⁺ -Er ³⁺ and Yb ³⁺ -Tm ³⁺ , are applied to the studied biosample. Visualize anti-Stokes microparticles using a laser scanning microscope (LSM-710 NLO, Carl Zeiss, Germany). In this case, microparticles are excited by a pulsed femtosecond laser (Chameleon Ultra II, Coherent, USA) at a wavelength of 980 nm. Next, using a non-immersion air lens with a 20x magnification, a sample is scanned at a scanning speed in the range of 1.27-3.15 μs / pixel. Registration of the photoluminescent signal is carried out using non-scanning detectors (NDD) in three spectral ranges: 400-490 nm, 490-560 nm and 565-700 nm, respectively. The resulting image is laid out on lines in increments of one pixel along the fast scan axis. In each line, the position of the microparticle is determined as a point with maximum brightness and the boundaries of its signal. The resulting sequence (signal profile, which lies within the specified limits) is approximated using the point spread function (PSF) of the form:

h (x, y) = (- Aexp (-y / vτ _r ) + Bexp (-y / vτ _d )) ∫δ (x) dx,

where v is the scanning speed along the fast scanning axis, τ _r and τ _d are the growth and decay times of anti-Stokes photoluminescence, A and B are the coefficients, δ (x) is the Dirac delta function. The parameters of the PSF are determined, then the image is restored using the Lucy-Richardson method.

However, the known method has the following disadvantages:

- the large size of anti-Stokes microparticles complicates and / or prevents their penetration into the cell;

- the sensitivity of the known method is limited, on the one hand, due to the increased contribution of autofluorescence excited by a femtosecond laser used to excite anti-Stokes luminescence, on the other hand, the low-aperture air lens used in the known method has a low resolution that does not allow to observe single nanoscale and even submicron particles;

- the resulting images have a high noise level, since in the known method, scanning is performed at a speed of 1.27-3.15 μs / pixel, exceeding the optimal speed used to achieve the optimal signal to noise ratio (about 10 μs / pixel).

The above disadvantages reduce the accuracy and reliability of determining the lifetime of photoluminescence and localization of nanoparticles.

The objective of the invention is the creation of a method for microscopic examination of biological samples labeled with phosphorescent probes in vitro, devoid of the disadvantages of the prototype.

The technical result of the proposed method is:

- an increase in sensitivity in the visualization of phosphorescent nanoparticles deposited on biological samples;

- reducing the contribution of the noise component (increasing the signal-to-noise ratio);

- increasing the accuracy of determining the localization of discrete phosphorescent nanoparticles and their aggregates on biological samples;

- increasing the accuracy of determining the photoluminescence lifetime of discrete phosphorescent nanoparticles and their aggregates deposited on biological samples from the image.

(НАФ), имеющие размер, не превышающий 200 нм, которые возбуждают непрерывным ИК-лазером с длиной волны 980 нм, полученное с использованием лазерного сканирующего микроскопа изображение обрабатывают в два этапа, на первом этапе обработку осуществляют вдоль медленной оси сканирования, на втором этапе обработку осуществляют вдоль быстрой оси сканирования.The technical result is achieved by the fact that in the known method of microscopic examination of biological samples labeled with phosphorescent probes in vitro, including multiplex marking of a biological sample with phosphorescent material, their visualization and determination of photoluminescence lifetime using laser scanning microscopy, nanoscale anti-stokes are used for multiplex marking of a biological sample

(NAF) having a size not exceeding 200 nm, which is excited by a continuous IR laser with a wavelength of 980 nm, the image obtained using a laser scanning microscope is processed in two stages, at the first stage, the processing is carried out along the slow axis of the scan, at the second stage, processing carried out along the fast axis of the scan.

The present invention meets the criteria of "novelty" and "inventive step", since the conducted patent information research did not identify sources of scientific and technical literature and patent documentation that would read the novelty of the proposed method, as well as technical solutions with essential features of the proposed method.

The technical result of the proposed method is:

- an increase in sensitivity in the visualization of phosphorescent nanoparticles deposited on biological samples;

- reducing the contribution of the noise component (increasing the signal-to-noise ratio);

- increasing the accuracy of determining the localization of discrete phosphorescent nanoparticles and their aggregates on biological samples;

- increasing the accuracy of determining the photoluminescence lifetime of discrete phosphorescent nanoparticles and their aggregates deposited on biological samples from the image.

The proposed method is illustrated in graphic material.

представлены результаты работы заявленного способа микроскопического исследования биологических образцов, основанного на определении времени жизни фотолюминесценции и локализации антистоксовых

по их изображению.In FIG. 1 on the example of a one-dimensional photoluminescence signal of a number of anti-Stokes

presents the results of the claimed method of microscopic examination of biological samples based on determining the lifetime of photoluminescence and localization of anti-Stokes

in their image.

In FIG. 2 presents a two-dimensional image of the simulated photoluminescence signal of a number of discrete phosphorescent nanoparticles (left panel) and the image restored using the proposed method (right panel). The insets show enlarged images of the areas circled in the image by white circles. To demonstrate the sensitivity of the reconstruction algorithm used to the image, random noise was added. It should be noted that as a result of deconvolution, the image size decreases significantly and a significant increase in the signal-to-noise ratio is observed. The latter effect is demonstrated by the absence of noise in the reconstructed image.

, подготовленных в фосфатно-солевом растворе.In FIG. 3 presents the results of the proposed method for the study of biological samples labeled with phosphorescent nanomaterials, using an example of a section of a liver (an example of a specific implementation). Images were obtained by scanning a laser beam at a speed of about 10 cm / s and a recording time of a signal from one point of about 10 μs. The sample under consideration was a thin section of liver tissue isolated from a laboratory mouse three hours after injection of biocompatible anti-Stokes nanocomplexes

prepared in phosphate-saline solution.

After isolation, liver tissue was fixed with 10% neutral buffered formalin for 48 hours. After that, the tissue was dehydrated in a series of alcohol solutions, placed in paraffin, and cut into sections with a thickness of about 5 μm.

(показаны зеленым цветом), локализовавшихся в печени после внутривенной инъекции. Масштабный отрезок - 50 мкм. На фиг. 3b, с показаны графики поверхности исходного (b) и восстановленного в результате деконволюции (с) изображения тех же фотолюминесцентных наночастиц на печени, что и на (а). Полученное время жизни фотолюминесценции наночастиц, накопившихся в печени лабораторной мыши, составляло 0,4-0,8 мс.In FIG. 3a shows an overlay of a sectional image of a liver obtained in transmitted light mode (shown in gray) and a photoluminescent image

(shown in green) localized in the liver after intravenous injection. The scale segment is 50 microns. In FIG. 3b, c shows surface graphs of the original (b) and reconstructed as a result of deconvolution (c) images of the same photoluminescent nanoparticles on the liver as on (a). The obtained lifetime of photoluminescence of nanoparticles accumulated in the liver of a laboratory mouse was 0.4-0.8 ms.

The proposed method is as follows.

, имеющие размер, не превышающий 200 нм, наносят на исследуемый образец (срез ткани или культуру клеток) и возбуждают непрерывным ИК-лазером с длиной волны 980 нм. При этом наночастицы распределяются по образцу, связываясь с определенными участками. Взаимодействие наночастиц с окружающими молекулами сопровождается изменением времени жизни их фотолюминесценции.Biologically compatible anti-Stokes nanocomplexes

having a size not exceeding 200 nm, applied to the test sample (tissue section or cell culture) and excited by a continuous infrared laser with a wavelength of 980 nm. In this case, the nanoparticles are distributed over the sample, binding to certain areas. The interaction of nanoparticles with surrounding molecules is accompanied by a change in the lifetime of their photoluminescence.

The obtained biological samples are examined using a laser scanning microscope.

Images are obtained with the following microscope settings:

возбуждают непрерывным ИК-лазером с длиной волны 980 нм.Anti-stock

excited by a continuous infrared laser with a wavelength of 980 nm.

The photoluminescence signal of anti-Stokes nanophosphors is recorded using a non-scanning mode of detection (NDD mode).

Optical filters isolate the detection spectral range corresponding to the spectral range of photoluminescence emission used for marking anti-Stokes nanoparticles. When using nanoparticles doped with Tm ³⁺ _{, the} signal is recorded in the spectral ranges 445-485 nm and 790-810 nm, while doping with Er ³⁺ - 500-550 nm.

Moreover, to obtain images of individual nanoparticles and their aggregates distributed over the studied biological sample, a high-aperture immersion lens (for example, Apochromat 63x / 1.2 W) is used.

To obtain information about the morphology of the sample, a laser with a wavelength of 405 nm is used, which effectively excites endogenous fluorophores. The autofluorescence signal of the test biological sample is collected in a separate track to prevent overlapping autofluorescence and photoluminescence signals of nanoparticles.

The size of the resulting image is limited by a specified number of pixels (for example, 512 × 512 pixels).

Select the optimal scan speed (about 10 μs / pixel) at which the maximum signal to noise ratio is achieved. Scanning at such a speed is realized when excited by a laser beam with an intensity higher than the saturation energy density of anti-Stokes nanophosphors (> 0.1 J cm ^-2 ).

The resulting images are saved in an accessible format and exported for subsequent processing using a special algorithm implemented in the Matlab software package.

, в предлагаемом способе изображение представляют в виде массива, состоящего из последовательности строк, каждая из которых содержит одномерный профиль сигнала вдоль быстрой оси сканирования.Based on the fact that the scan time of one line is commensurate or much longer than the life time used for marking anti-Stokes

, in the proposed method, the image is presented in the form of an array consisting of a sequence of lines, each of which contains a one-dimensional signal profile along the fast scanning axis.

A sequential two-stage processing using the deconvolution algorithm is applied to the array thus obtained. In this case, the processing is carried out first along the slow (by varying the row number), then along the fast axis of the scan (by varying the column number).

Processing along the slow scan axis involves deconvolution using a Gaussian as an impulse function that describes the normal distribution of the signal along the slow scan axis. This procedure allows one to limit the lines containing the photoluminescence signal of nanoparticles and significantly reduce the contribution of the noise component.

Image processing along the fast scanning axis is carried out in selected rows using the following algorithm:

1) perform a search for local maxima among the elements of the row. Moreover, to search for the exact location of the nanoparticles, a threshold is set that represents the minimum peak intensity (MRI, Min Peak Height);

2) limit (find the beginning and end) each of the detected peaks;

аппроксимируется функцией, представленной в общем виде как:3) approximate each of the peaks using the point spread function (PSF), which describes the growth and subsequent decay of the photoluminescent signal. In this case, the photoluminescence signal

approximated by a function, represented in general terms as:

,

,

where G is the intensity distribution of the exciting laser beam and describes the propagation of a traveling wave in the positive direction of the x axis with velocity v. This intensity distribution is described by a Gaussian function with a beam constriction w, which is equal to the diameter of the focused beam:

;

;

P (x) is the studied profile of the photoluminescence signal; G (t) is the dependence of the photoluminescence of nanoparticles on time, described by a function of the form [Hehlen, M.P .; Kuditcher, A .; Lenef, A.L .; Ni, H .; Shu, Q .; Rand, S. C; Rai, J .; Rai, S. Physical Review In 2000, 61, 1116]:

G (t) = - A exp (-t / τ _R ) + В exp (-t / τ _ph ),

.where τ _R - characteristic time apkonversionnogo energy transfer; τ _ph is the anti-Stokes photoluminescence lifetime

.

Thus, the detected signal S (t) is an overlay of the surface profile of the nanoparticle signal and the function of the time decay of the photoluminescence intensity G (t):

,

,

where erƒ is the error function.

Function S is a more accurate impulse function compared to that used in the known method, which is used for further deconvolution.

4) using the parameters (t _R and τ _ph ) obtained as a result of approximating the signal profile, deconvolve the profile using the Lisi-Richardson algorithm implemented in the Matlab environment;

5) save the parameters in a table with the results and go to the next line. To prevent the loss of signal of large particles, as well as the contribution of long-term noise, the presence of correlation between adjacent lines is monitored.

An example of a specific performance is given in the form of an extract from the experimental protocol.

, имеющие размер, не превышающий 200 нм, наносят на исследуемый образец (печень белой мыши) и возбуждают непрерывным ИК-лазером с длиной волны 980 нм. При этом наночастицы распределяются по образцу, связываясь с определенными участками. Взаимодействие наночастиц с окружающими молекулами сопровождается изменением времени жизни их фотолюминесценции.Biocompatible anti-Stokes nanocomplexes

having a size not exceeding 200 nm is applied to the test sample (white mouse liver) and excited by a continuous infrared laser with a wavelength of 980 nm. In this case, the nanoparticles are distributed over the sample, binding to certain areas. The interaction of nanoparticles with surrounding molecules is accompanied by a change in the lifetime of their photoluminescence.

The obtained biological samples are examined using a laser scanning microscope.

Images are obtained with the following microscope settings:

возбуждают непрерывным ИК-лазером с длиной волны 980 нм.Anti-stock

excited by a continuous infrared laser with a wavelength of 980 nm.

The photoluminescence signal of anti-Stokes nanophosphors is recorded using a non-scanning mode of detection (NDD mode).

Optical filters isolate the detection spectral range corresponding to the spectral range of photoluminescence emission used for marking anti-Stokes nanoparticles. When using nanoparticles doped with Tm ³ _{, the} signal is recorded in the spectral ranges 445-485 nm and 790-810 nm, while doping with Er ³⁺ - 500-550 nm.

Moreover, to obtain images of individual nanoparticles and their aggregates distributed over the studied biological sample, a high-aperture immersion lens (for example, Apochromat 63x / 1.2 W) is used.

To obtain information about the morphology of the sample, a laser with a wavelength of 405 nm is used, which effectively excites endogenous fluorophores. The autofluorescence signal of the test biological sample is collected in a separate track to prevent overlapping autofluorescence and photoluminescence signals of nanoparticles.

The size of the resulting image is limited by a specified number of pixels (for example, 512 × 512 pixels).

(>0.1 Дж × см^-2).Select the optimal scan speed (about 10 μs / pixel) at which the maximum signal to noise ratio is achieved. Scanning at this speed is realized when excited by a laser beam with an intensity higher than the density of saturation energy of anti-Stokes

(> 0.1 J × cm ^-2 ).

The resulting images are saved in an accessible format and exported for subsequent processing using a special algorithm implemented in the Matlab software package.

, в предлагаемом способе изображение представляют в виде массива, состоящего из последовательности строк, каждая из которых содержит одномерный профиль сигнала вдоль быстрой оси сканирования.Based on the fact that the scan time of one line is commensurate or much longer than the life time used for marking anti-Stokes

, in the proposed method, the image is presented in the form of an array consisting of a sequence of lines, each of which contains a one-dimensional signal profile along the fast scanning axis.

A sequential two-stage processing using the deconvolution algorithm is applied to the array thus obtained. In this case, the processing is carried out first along the slow (by varying the row number), then along the fast axis of the scan (by varying the column number).

Processing along the slow scan axis involves deconvolution using a Gaussian as an impulse function that describes the normal distribution of the signal along the slow scan axis. This procedure allows one to limit the lines containing the photoluminescence signal of nanoparticles and significantly reduce the contribution of the noise component.

Image processing along the fast scanning axis is carried out in selected rows using the following algorithm:

1) perform a search for local maxima among the elements of the row. Moreover, to search for the exact location of the nanoparticles, a threshold is set that represents the minimum peak intensity (MRI, Min Peak Height);

2) limit (find the beginning and end) each of the detected peaks;

аппроксимируется функцией, представленной в общем виде как:3) approximate each of the peaks using the point spread function (PSF), which describes the growth and subsequent decay of the photoluminescent signal. In this case, the photoluminescence signal

approximated by a function, represented in general terms as:

,

,

where G is the intensity distribution of the exciting laser beam and describes the propagation of a traveling wave in the positive direction of the x axis with velocity v. This intensity distribution is described by a Gaussian function with a beam constriction w, which is equal to the diameter of the focused beam:

;

;

; Г(t) -зависимость фотолюминесценции наночастиц от времени, описываемая функцией вида [Hehlen, М.P.; Kuditcher, A.; Lenef, A.L.; Ni, Н.; Shu, Q.; Rand, S.С; Rai, J.; Rai, S. Physical Review В 2000, 61, 1116]:P (x) - studied profile of the photoluminescence signal

; G (t) -dependence of the photoluminescence of nanoparticles on time, described by a function of the form [Hehlen, M.P .; Kuditcher, A .; Lenef, AL; Ni, H .; Shu, Q .; Rand, S. C; Rai, J .; Rai, S. Physical Review In 2000, 61, 1116]:

Г (t) = - А exp (-t / τ _R ) + В exp (-t / τ _ph ),

.where τ _R - characteristic time apkonversionnogo energy transfer; τ _ph is the anti-Stokes photoluminescence lifetime

.

Thus, the detected signal S (t) is an overlay of the surface profile of the nanoparticle signal and the function of the time decay of the photoluminescence intensity G (t):

,

,

where erƒ is the error function.

Function S is a more accurate impulse function compared to that used in the known method, which is used for further deconvolution.

4) using the parameters (τ _R and τ _ph ) obtained as a result of approximating the signal profile, deconvolve the profile using the Lisi-Richardson algorithm implemented in the Matlab environment;

5) save the parameters in a table with the results and go to the next line. To prevent the loss of signal of large particles, as well as the contribution of long-term noise, the presence of correlation between adjacent lines is monitored.

Claims (1)

A method for microscopic examination of biological samples marked with in vitro phosphorescent probes, including multiplex marking of a biological sample with phosphorescent material, their visualization and determination of photoluminescence lifetime using laser scanning microscopy, characterized in that nanoscale anti-stokes phosphors (NAF) are used for multiplex marking of a biological sample, having a size not exceeding 200 nm, which are excited by a continuous infrared laser with a length of lny 980 nm, obtained using the laser scanning microscope image is treated in two stages, the first stage treatment is performed along the slow scan axis, the second stage treatment is carried out along the fast scan axis.

Images