RU2712789C1 - Fiber-glass confocal scanning microscope - Google Patents

Abstract

FIELD: physics.

SUBSTANCE: invention relates to devices for recording radiation excited in local areas of the medium when focusing laser radiation. Fiber-optic confocal scanning microscope has a laser radiation source, a Y-circulator, a lens, a confocal diaphragm, a photodetector and spatial scanning devices of the analyzed region of the object. First unidirectional input of the optical waveguide of the Y-circulator is connected to the object illumination source. Optical beam of the output end of the first waveguide is directed to the end of the second waveguide at a certain angle in the axis of the second waveguide within the limits of the aperture angle of the optical fiber. Output end of the optical bidirectional second waveguide is an aperture which forms a light beam of illumination of the object through the lens and simultaneously is a confocal diaphragm filtering the radiation of the object medium response. Radiation of the medium response passes back through the lens and the second waveguide to the end of the third unidirectional waveguide. Third waveguide is located along the axis of the second waveguide. Output of the third waveguide is the output of the Y-circulator, connected to the input of the fiber-optic spectrometer. Radiating end of the second waveguide is connected to the mechanical system of displacement of this butt-end for scanning of the radiator scaled by the lens of the image of the aperture in the conjugated plane combined with the object.

EFFECT: technical result consists in expansion of functional capabilities of device and simplification of microscope design.

1 cl, 1 dwg

Description

The invention relates to devices for detecting radiation excited in local areas of the medium when focusing laser lighting, and the subsequent synthesis of two-dimensional and three-dimensional images based on the results of spatial scanning of an object with a light beam. The device can be used for spectral studies of various biological environments, including fluorescence diagnostics for solving applied problems of medicine. Confocal microscopy has several advantages compared to traditional optical microscopy, including adjustable depth of field, eliminating out-of-focus information that degrades the image, and the possibility of sequential analysis of optical sections of thick samples.

The well-known "Microscope Apparatus", which is the first description of a confocal microscope (MARVIN MlNSKY AM / 111 7- ATTORNEYS United States Patent office Patented Dec. 19, 1961 3,013,467 MICROSCOPY APPARATUS Marvin Minsky, 44 Bowdoin St., Cambridge, Mass. Filed Nov. 7 1957, Ser. No. 695,107 4 Claims. C1. 88-14). The device according to the invention (M. Minsky Patent) contains a radiation source 10, 12 with a point collimating diaphragm 16, a lens 11, a confocal aperture 26, a photodetector 28, a beam splitter plate 17, the reflecting surface of the plate 17 facing the lens 11 and the photodetector 28 s confocal diaphragm 26, and the transparent surface of the plate 17 faces the light source 10, 12 with a point collimating diaphragm 16, a device for spatial scanning of an object (Patent M. Minsky). From the description of the patent it follows that the radiation of the source passes through the dividing plate to the object, and returns in a different way: it is reflected from the dividing plate and sent through the confocal diaphragm to the photodetector. In this case, the dividing plate performs the function of a non-reciprocal device - three port circulator (Y-circulator). This technical solution eliminates out-of-focus rays delayed by the confocal aperture and, based on the data on the displacement of the sample by the spatial scanning device, constructs a two-dimensional or three-dimensional image with high contrast. The mechanical system for scanning the object of research is constructed using a resonator based on bending vibrations excited by electromagnetic devices synchronized with the scanning electron beam of the oscilloscope.

The main disadvantage of this device is the system of focusing the light beam of a point source 16 on the object and the position control system of the confocal diaphragm 26, should ensure that the image of the illuminated local area of the object is aligned with the point confocal diaphragm located in the conjugate plane of the lens. With a high resolution of the microscope, due to the "double focusing", high demands are placed on the optomechanics of the device. In addition, the formation of a point light source from an extended source using the diaphragm 16 does not allow to obtain a sufficiently high power density of the scanning beam of the illuminator at the object. The presented mechanical scanner system with a resonator, on which the object of research is fixed, cannot provide independent displacement along the coordinate axes and has limited possibilities for studying various media exposed to vibration.

The closest in combination of features is the Dilor company (France), which is focused on carrying out measurements with high spectral resolution, in which the fluorescence images of objects are reconstructed only on the basis of the recorded spectra (A.V. Feofanov, “Spectral Laser Scanning Confocal Microscopy in Biological Research”. Advances in Biological Chemistry - vol. 47, 2007, p. 371-410, fig. 3 on p. 381). The device contains a laser source of radiation that excites fluorescence, a device for non-reciprocal switching of the radiation directed to the object and the radiation response of the medium in the form of a beam splitter, a lens, a confocal aperture, a photodetector with the function of decomposing the fluorescence radiation into the spectrum and recording it, a device for spatial scanning with an optical beam of the analyzed region of the object due to the movement of the lens and synchronous scanning with moving mirrors.

The disadvantage of this device is the presence of a beam splitter plate and, accordingly, the need for a high-precision synchronous focusing system - focusing radiation on the object and combining the image of the focal spot in the conjugate plane of the lens with a point confocal aperture. In this case, only one laser wavelength is used to excite fluorescence, and its change requires reconfiguration of the device. The scanning system, built on the synchronous movement of mirrors, is characterized by the complexity of mechanical components and has limitations on the dynamic characteristics of the scanner.

The technical result of the invention is to expand the functionality of the device and simplify the design of the microscope through the use of fiber optic Y-circulator as a non-reciprocal switching device directed to the object of radiation and radiation response medium (Fiber optic laser spectrum analyzer. Patent RU 2632993 from 04.04.2016. Published: 11.10 .2017, Bull. No. 29).

The advantage of this system is that it does not contain selective fission mirrors, optomechanical elements to perform optical alignment of combining the image of the focal spot of the scanning light beam with the confocal diaphragm in the conjugate plane of the lens. The proposed principle of switching optical beams solves the problem of their separation regardless of the spectral and mode composition of the radiation, as well as polarization. In the considered Y-circulator, nonreciprocity is due to the topology of spatial switching of optical beams.

The radiation beam of the output end of the first (unidirectional) optical waveguide coupled to a laser source that excites fluorescence is directed to the end of the second waveguide at a certain angle defined by the guiding system of substrates with grooves within the aperture angle of the optical fiber. The radiation from the output end of the second waveguide is focused by the lens on the surface or in the volume of the object. The reflected and fluorescence radiation of the illuminated local area of the object within the spatial angle of the numerical aperture is collected by the lens and introduced back into the output end of the second (bi-directional) waveguide. The radiation beam of the object from the second waveguide is directed to the end of the third (unidirectional) waveguide located along the axis of the second waveguide. The output of the third waveguide is the output of the Y-circulator and is connected to the input of a fiber optic spectrometer. To register the fluorescence spectrum in the optical waveguide of the laser source and the optical waveguide of the input of the spectrometer, transmission and blocking filters of laser radiation are additionally included. Thus, the output aperture of the second waveguide is both the output aperture of the laser source and the input aperture of the photodetector (confocal diaphragm), which blocks out-of-focus radiation rays of the object. High precision mechanics is excluded, which ensures that the image of a point source of fluorescent radiation excited by a focused laser beam is combined with a point confocal diaphragm, since the confocal diaphragm and the point source of laser radiation are combined in one aperture. There remains only one independent degree of freedom - focusing of the laser radiation exciting the fluorescence in the spatial coordinates of the object. The consequence of this is a new possibility of constructing a microscope scanning device, when spatial scanning of the image of the aperture of the end face of the second waveguide in a conjugate plane aligned with the object is carried out by mechanical scanning of the end face of the second waveguide (confocal diaphragm) in the corresponding displacement scale. This solution expands the possibilities of constructing scanners along with well-known technical solutions - scanning fluorescent signals with three-dimensional submicron resolution by moving mirrors, object displacement and lens displacement. The device further comprises transmitting and trapping optical filters at the output of the laser source and the input of the fiber optic spectrometer in accordance with the known technical solution, which provides the allocation of fluorescent radiation against the background of the exciting laser radiation.

This technical result is achieved by the fact that in a fiber optic confocal microscope containing a laser radiation source, a non-reciprocal switching device directed to the radiation object and the radiation response medium, a lens, a confocal aperture, a photodetector, a spatial scanning device of the analyzed region of the object according to the invention, as a non-reciprocal device a fiber optic Y-circulator is used, which forms the spatial separation of the switched optical beams, first whose unidirectional input of the optical waveguide is connected to the object light source, and the optical beam of the output end of this waveguide is directed to the end of the second waveguide at an angle to the axis of the second waveguide, defined by the guiding system of substrates with grooves within the aperture angle of the optical fiber, the output end of the optical bidirectional second the waveguide is an aperture that forms a light beam of illumination of the object through the lens and, at the same time, an aperture, which is a confocal diaphragm a mission filtering the radiation response of the medium of the object passing back through the same lens and the second waveguide to the end of the third unidirectional waveguide located along the axis of the second waveguide, the output of which is the output of the Y-circulator connected to the input of the fiber-optic spectrometer, and the radiating end of the second waveguide is connected to a mechanical displacement system of this end for scanning the image of the aperture of the emitter scaled by the lens in the conjugate plane aligned with the object.

The invention is illustrated by the circuit shown in FIG. 1, which shows a fiber optic confocal scanning microscope. Fiber optic Y-circulator (See Fig. 1), which forms a spatial separation of switched optical beams, includes optical fibers 1, 2, and 3 located on the substrate 4. The confocal aperture and simultaneously the aperture of the laser emitter 5 correspond to the output end of the optical waveguide 2. Laser source 6, coupled to the fiber 7 through the optical connector 8 is connected to the input of the collimator 9, which ensures the operation of the transmission filter 10. The optical output of the collimator 9 is connected to the fiber 1 of the Y-circulator. The lens 11 forms a scanning laser beam 12 at the object 13. The output of the fiber 3 is connected to the input of the collimator 14 containing the trap filter 15. The output of the collimator 14 through the optical connector 16 is connected to the input of the fiber-optic spectrometer 17. The emitting end of the fiber 2 is connected to a mechanical bias system of this end 18.

The operation of the optical fiber confocal scanning microscope (see Fig. 1) is as follows. The laser source 6, through the optical waveguide 7, the optical connector 8 transmits the radiation to the input of the collimator 9, forming a collimated radiation beam with an aperture necessary for the functioning of the filter 10, which transmits the main radiation line of the laser 6 and suppresses the rest of the radiation. The output radiation of the collimator 9 is fed to the input of the fiber 1 of the Y-circulator. The output optical beam of fiber 1 is directed to the input end of fiber 2 at a predetermined angle to the axis of fiber 2. The emitting end 5 of fiber 2 is located in the focal plane of the lens 11, which forms the scanning beam 12, focused in the conjugate plane of the lens 11 on the studied object 13. In the local volume focused laser radiation, the medium of the object 13 creates a fluorescence radiation. This radiation within the boundaries of the spatial angle 12 together with the reflected laser radiation by the lens 11 is focused on the end face 5 of the fiber 2, the aperture of which serves as a confocal diaphragm, since other rays, except the radiation from the focusing area, cannot be introduced into the fiber 2. The fluorescent radiation from fiber 2 in accordance with the direction of the optical beam is introduced into the fiber 3, then enters the collimator 14, passes through a blocking filter 15, which suppresses laser radiation and transmits fluorescence radiation. The output of the collimator 14 through the optical connector 16 is connected to the input of the fiber optic spectrometer 17, which registers the fluorescence spectrum of the scanned area of the object 13. The emitting end of the fiber 2 is connected to the mechanical system of the bias of this end 18. The offset of the confocal diaphragm 5, the aperture of which is also the emitter, leads to a shift of the focal spots in the conjugate plane of the lens 11 located on the object 13.

Claims (1)

A fiber optic confocal scanning microscope containing a laser radiation source, a device for non-reciprocal switching of radiation and radiation from the medium, a lens, a confocal aperture, a photodetector, a device for spatial scanning of the analyzed region of an object, characterized in that a fiber-optic Y-circulator is used as a device for non-reciprocal switching forming the spatial separation of switched optical beams, the first unidirectional input of an optical wave the novode of which is connected to the illumination source of the object, and the optical beam of the output end of this waveguide is directed to the end of the second waveguide at an angle to the axis of the second waveguide, defined by the guide system of substrates with grooves within the aperture angle of the optical fiber, the output end of the optical bidirectional second waveguide is an aperture, forming a light beam of illumination of the object through the lens and, at the same time, being a confocal diaphragm, filtering the radiation of the response of the medium of the object, passing back through the same lens and the second waveguide to the end face of the third unidirectional waveguide located along the axis of the second waveguide, the output of which is the output of the Y-circulator connected to the input of the optical fiber spectrometer, and the radiating end of the second waveguide is connected to the mechanical bias system of this end to scan the scaled the image lens of the aperture of the emitter in the conjugate plane, combined with the object.

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