RU2442140C1 - Microfluometer for studying the fluorescence of single cells - Google Patents

Abstract

FIELD: cell microfluorometry.

SUBSTANCE: microfluorometer used to study the fluorescence of single cells comprises a fluorescent microscope with a camera/a microphotometer, a monitor and a light source for fluorescence incitement. The light source is a set of extra bright light-emitting semiconductor diodes with fixed light waves length. The diodes are interconnected with the diode switch and the signal monitor.

EFFECT: measuring sensitivity increase through unwanted emission reduction; measurement accuracy increase.

3 dwg

Description

The invention relates to microfluorimetric studies of single cells and can be used in biophysics, biology, physiology and medicine to study the effect of biologically active substances, including drugs, on single cells.

The closest analogue of this invention is a RatioMaster ™ microspectrofluorimeter from Photon Technology International, Inc., USA (http://www.pti-nj.com/RatioMaster/RatioMaster.htmt), designed to measure and visualize the concentration of various ions within cells by level fluorescence of pre-loaded into these cells fluorescent probes (dyes), the emission intensity of which is proportional to the concentration of the corresponding ions, and consisting of a fluorescence microscope equipped with a camera / microphotometer, a recorder and a radiation source excitation of fluorescence performed on an ultrahigh pressure xenon lamp and controlled by a scanning monochromator. Xenon lamp provides continuous illumination of the input of the monochromator in a wide spectral range; the excitation radiation of the wavelength set by the recorder from the output of the monochromator controlled by the recorder is transmitted by a fluorescence microscope to the cells previously loaded with a fluorescent probe. Emission from a fluorescent probe is collected by a fluorescence microscope and received by a camera / microphotometer, which transmits a cell emission signal for recording and analysis to a recorder, whose role is played by a computer with specialized software for fluorescence analysis. The known device along with the complexity and high cost has the following significant disadvantages:

1) the use of a monochromator to excite fluorescence when working on single cells does not provide sufficient sensitivity for measuring the fluorescence of a single cell due to the low signal-to-noise energy ratio due to the high (comparable with the emission signal) level of spurious radiation of the monochromator in the emission spectral region (due to light scattering on the gratings and also the presence of grating diffraction maxima above the first order), as a result of which the measured emission signal arriving at the recorder is I am fuzzy, which does not allow for normal measurement accuracy;

2) a high spread of the emission signal of the fluorescent probe recorded by such a device due to the unstable brightness and inconstancy of the discharge size in the lamp of an ultrahigh pressure of the illuminator, which is essentially impossible to reduce by filtering, since the energy time spectra of the noise of the lamp and emission signals partially overlap;

3) the possibilities of increasing the energy signal-to-noise ratio by increasing the excitation radiation power when using this device are strongly limited by the lamp power, the low (~ 1: 100) output of the monochromator, the final values of the quantum yield and absorption capacity of the fluorescent probes loaded into the cell, and also fast burnout of probes with continuous exposure to radiation on the cell, which leads to a decrease in sensitivity and measurement accuracy.

The objective of the invention is to increase the sensitivity of the measurement by reducing the level of spurious radiation in the spectral region of the emission and improving the accuracy of the measurement by reducing the spread of the recorded emission signal during microfluorimetric studies of single cells.

This goal is achieved by the fact that in a microfluorimeter for studying the fluorescence of a single cell containing a fluorescence microscope equipped with a camera / microphotometer, a recorder and a fluorescence excitation radiation source, according to the invention, the latter is made in the form of superbright emitting semiconductor diodes with a fixed radiation wavelength, interconnected a device for switching diodes and associated with the registrar, through which the synchronization of the inclusion of diodes with the register radio emission signal.

Figure 1 presents the implementation diagram of the proposed device.

A microfluorimeter for studying the fluorescence of a single cell contains an optical channel 1, an ultra-bright emitting semiconductor diode 2, a condenser 3, a filter 4, a light guide 5, a photometer 6, a camera 7, a fluorescence microscope 8, a preparation 9, a micro lens 10, a dichroic mirror 11, a lens 12, a device switching diodes 13, a current source 14, a recorder 15, which, as in the analogue, is a computer with specialized software for fluorescence analysis.

The fluorescence excitation radiation source contains five identical optical channels 1, each of which includes an ultra-bright emitting semiconductor diode 2 (wavelengths of maximum spectral density / radiation power of each diode = 340/20, 380/50, 440/70, 480/70, 530/70 nm / mW, respectively) and a capacitor 3 for matching the numerical aperture of the input end of the fiber 5 and the radiation pattern of diode 2. Elimination of the second harmonic of radiation and narrowing of the spectral band of the emitting diode is achieved by an optical filter 4. The optical fibers of all optical channels are converged into a single optical fiber with regular laying of fibers, the output branch of which is installed in the epifluorescence port of the microscope 8. Lens 12 projects the end face of the output branch of the optical fiber 5 through the dichroic mirror 11 into the plane of the exit pupil of the micro-lens 10, which ensures uniform illumination of the drug 9. Radiating a diode 2 in each optical channel is controlled by a current source 14 from the switching device of the diodes 13. The registrar 15 synchronizes the inclusion of emitting diodes with istratsiey emission signal obtained from the camera 7 and / or 6 photometer.

The proposed device operates as follows. Preparation 9 — live single cells, previously loaded with a fluorescent probe selected for measuring the ion of interest, are located under the micro-lens 10 on a fluorescence microscope 8. Then, the excitation wavelength (or several wavelengths with multi-wave excitation mode) is set on the recorder 15, which corresponds to the maximum spectral absorption capacity selected fluorescence probe. In the process of measuring cell emission, the recorder provides periodic inclusion of a diode with a wavelength of maximum spectral radiation density equal to the set excitation wavelength (s). The excitation radiation from the optical unit through the optical fiber 5 is transmitted to a fluorescence microscope 8, is reflected by a dichroic mirror 11 into a micro lens 10, which focuses this radiation on the cells of the preparation 9 located below it. At the same time, the emission radiation is collected by a micro lens 10 to the camera 7 and / or photometer 6, the signal from which synchronously with the inclusion of the diode 2 is taken by the registrar 15 for storage and analysis. When various biologically active substances are applied to the cell, the concentration of the ion of interest inside it changes. This change is estimated by the registrar by the difference in the signals of cell emission at rest and when the applied substance acts on it.

The results of comparing the prototype and the proposed device in our experiments on the registration of calcium emission signals in the cytoplasm of single cells are shown in Figure 2. Single cells of the COS-1 line, previously loaded with a Fluo-4 calcium probe (Molecular Probes, USA), were placed on a glass using an Axiovert-100 microscope (Zeiss, Germany) with an Achroplan-40 microscope, NA = 0.8 (Zeiss, Germany) both in the prototype device and in the claimed microfluorimeter. Fluorescence was excited at a wavelength of 480 nm using a monochromator, which is part of the RatioMaster ™ prototype (Photon Technology International, Inc., USA), and using ultra-bright emitting semiconductor diodes of the claimed device. The emission signal from the IC-camera IC-200 (Photon Technology International, Inc., USA) and / or the M-40 microscopic photometer (Photon Technology International, Inc., USA) mounted on a microscope, both in the prototype and in the claimed device entered the registrar. In both cases, a personal computer with specialized software was used as a registrar. ATP application caused a short-term increase in the calcium ion Ca ²⁺ in the cytoplasm of a single COS-1 cell loaded with a Fluo-4 calcium probe in both cases. Ca ²⁺ signals were recorded from the same cell using the prototype (Fig.2A, C) and using the claimed device (Fig.2B, D). The lines above the fluorescence curves in the graphs of both experiments correspond to ATP applications, and the numbers correspond to the ATP concentration in nanomoles.

When COS-1 cells were stimulated with a purinoreceptor agonist, ATP, the recorded relative change in the emission intensity (ΔF / F) in the case of an analog was lower (Fig. 2A, ΔF / F = 2.0 ± 0.3, n = 3) than in the case of the proposed device ( Fig.2B, ΔF / F = 2.5 ± 0.2, n = 3). The energy signal-to-noise ratio was 15 for the proposed device and 7 for the monochromator, respectively. This is due to the presence of spurious radiation at the output of the monochromator in the spectral region of emission due to the non-ideality of its gratings and the resulting light scattering, as well as the presence of diffraction maxima above the first order in gratings. The instability of the brightness and the inconstancy of the size of the discharge body in the ultrahigh-pressure lamp of an analog illuminator led to a deviation in the fluorescence excitation radiation intensity and a higher dispersion of the recorded emission signal (Fig. 2A), compared with the claimed device (Fig. 2B). Achieved in the claimed device a reduction in spurious radiation and a decrease in the scatter of the recorded signal allowed us to increase the sensitivity and improve the accuracy of measuring the emission of a single cell with the application of the minimum (submicromole) concentrations of ATP, which was not achieved using the prototype. The response of the cell to 100 nM ATP is threshold in the case of registration by the prototype (Fig. 2B), and in the case of using the claimed device, such a response is confidently recorded (Fig. 2G), while the claimed device is also capable of detecting weaker fluorescence signals. The synchronization of the inclusion of light-emitting diodes with a camera / microphotometer in the proposed device can significantly reduce the burnout of the probe, and the fast switching of the diodes allows for multi-wave excitation at two or more wavelengths.

Achieved with the proposed device high sensitivity and accuracy of registration of the emission signal allow for the initial selection and testing of various biologically active substances and new drugs on single cells in biology, medicine and pharmacology. The characteristic service life of light emitting diodes is approximately 30-50 thousand hours versus 1-2 thousand hours for xenon lamps, which is an additional advantage of the claimed device according to the invention.

Claims (1)

A microfluorimeter for studying the fluorescence of single cells, containing a fluorescence microscope with a camera / microphotometer, a registrar and a fluorescence excitation radiation source, characterized in that super bright emitting semiconductor diodes with fixed radiation wavelengths interconnected by a diode switching device are used as a fluorescence excitation radiation source associated with the registrar, through which synchronization of the inclusion of diodes from the registrar emission signal.

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