RU2527316C1 - Interference microscope - Google Patents

Abstract

FIELD: physics.

SUBSTANCE: interference microscope comprises a light field microscope for forming an enlarged image of an object in a back focal plane, a 4f optical system of two Fourier lenses, the front focal plane of which coincides with the back focal plane of the light field microscope. An output image recorder is placed in the back focal plane of the 4f optical system. A beam splitter is placed inside the 4f optical system before the common focal plane, said splitter forming two spaced-apart light beams which converge in the common focal plane of the 4f optical system, where two flat mirrors are placed perpendicular to the optical axis of each of the beams. Said mirrors reflect incident radiation in the opposite direction, and a pinhole, which transmits only non-scattered radiation and forms the reference beam, is placed in front of one of said mirrors. The beam splitter forms two parallel spaced-apart light beams with zero path difference, and reflecting surfaces of the flat mirrors lie in the same plane.

EFFECT: high measurement accuracy.

6 cl, 5 dwg

Description

The present invention relates to microscopy and can be used in biology, medicine, mechanical engineering, optical instrumentation.

Interference microscopes are known, see, for example, M. Franson “Phase-contrast and interference microscope”, Moscow: State Publishing House of Physics and Mathematics, 1960. They are designed to study phase objects.

Phase objects transparent to visible optical radiation are widespread both in industry and in biology and medicine. These include various polymer films, crystals, optical micro-parts, fiber optic products and, finally, biological objects - cells, etc. These objects are described by three-dimensional (3D) spatial distribution of the refractive index, which is associated with the density, temperature, concentration and other physical parameters of the object (Ch. West. Holographic interferometry. M: Mir, 1982).

When studying phase objects, the task arises of visualizing them, since they are usually transparent to probe radiation. To date, the following methods are widely known:

- the phase contrast method, also called the Zernike method, consisting in the fact that the phase shift in the light beam passing through the phase object is converted into a change in the brightness of the image (Bennett, A., Osterberg, H. Jupnik, H. and Richards, O. Phase Microscopy: Principles and Applications, John Wiley and Sons, Inc., New York, 1951);

- the method of interference contrast, consisting in the fact that the light beam is divided into two beams, one of which passes through the phase object, and the second passes it; the interference of the two beams can detect and measure the optical path difference included phase object (Hariharan P., Optical Interferometery 2 ^nd ed, Academic Press, 2003);

- the method of differential interference contrast (Nomarsky method, DIC), which consists in the fact that the polarized light beam is divided into two orthogonally polarized coherent beams that pass through the phase object by optical paths of different lengths, are reduced and interfere (Murphy, D., Differential interference contrast (DIC) microscopy and modulation contrast microscopy, in Fundamentals of Light Microscopy and Digital Imaging, Wiley-Liss, New York, 2001);

- the dark field method, which consists in the fact that to form an image of a phase object, only light beams scattered by this object are recorded (Roskin GI, Microscopic technology, M .: Publishing house "Soviet Science", 1946);

- polarization contrast method, consisting in the fact that an anisotropic phase object is placed between two polarization filters, the plane of polarization of one of which is rotated 90 ° relative to the other; only those light beams pass through the second filter, the plane of polarization of which turned out to be a rotated phase object (G. Roskin, Microscopic Technique, M.: Sovetskaya Nauka Publishing House, 1946).

Currently, research requires not only to observe and evaluate various geometric parameters (area, perimeter), but also to measure their local and integral characteristics.

Quantitative studies of the characteristics of phase objects can be carried out using interference microscopes based on the method of interference contrast, since only they allow measuring the optical path difference (ORX). Other methods allow you to either visualize or measure the derivative in the direction from ORX (differential interference contrast method). However, the widespread use of interference microscopes was hampered by the lack of automated methods for decoding interferograms, which hindered the introduction of these microscopes in the practice of laboratory research and routine measurements.

Previously, interference microscopes were constructed using two-beam schemes of Michelson and Mach-Zehnder interferometers (AN Zakharyevsky, AF Kuznetsova. Interference biological microscopes. Cytology, 1961).

1. The Michelson beam splitting scheme is used in the academician V.P. microinterferometer Linnik (1933). Linnik's microscope allows you to work with micro-lenses of large numerical apertures. To study phase microobjects, they should be placed on a mirror located in the subject channel. In this case, the radiation passes through them twice (reflection scheme) (VP Linnik. A device for interference research of reflecting objects under a microscope (“microinterferometer”), DAN USSR, 1933). For large or dense objects, a double pass of radiation leads to strong refraction at the edges of the object and the inability to reconstruct the phase in these places. The need to use a glass slide with mirror coating is also a disadvantage of this scheme. For the first time, an automated version of the Linnik MII-4 interference microscope was developed by Professor V.P. Tychinsky (1989) and named “Airiskan” (V. P. Tychinsky. Computer phase microscope. M: Knowledge, 1989).

2. The Mach-Zehnder beam splitting scheme is used in a Horn microscope (Horn, 1950). In its composition, it has two identical branches in which the studied drug and the comparison drug are located. Radiation passes through the studied object once (light transmission scheme) (G.A. Dunn. Transmitted-light interference microscopy: a technique bom before its time. Proceedings of the Royal Microscopical Society, 1998). An automated version of the Horn microscope is also known (Zicha and Dunn, 1995) (G.A. Dunn. Transmitted-light interference microscopy: a technique bom before its time. Proceedings of the Royal Microscopical Society, 1998). This microscope uses the phase step method (K. Creath. Phase-shifting speckle interferometry. Appl. Opt. Vol.24, No. 18, 1985). The discrete phase shift is provided by the transverse movement of the compensation wedge controlled from the stepper motor. This method is called DRIMAPS (Digitally Recorded Interference Microscopy with Automatic Phase Shifting - Digital Interference Microscopy with Automatic Phase Shift). The design of the Horn microscope is difficult to operate and configure, since it requires two identical channels with the same preparations: the study drug and the comparison drug.

The main disadvantage of two-beam interference microscope schemes is the need to use coherent radiation. This leads to large noise in the interferograms and, as a result, to an increase in the error in the reconstruction of the phase from interferograms. The separation in space of the object and supporting branches also leads to the fact that the rays going into them react differently to vibration and temperature fluctuations, which greatly impairs the vibration resistance of the interference microscope.

The current trend in interference microscopy is the creation of an interference attachment (module) to a commercial microscope that uses light from a conventional halogen lamp or LED with a short coherence length. This allows one to significantly reduce the noise characteristic of coherent radiation (speckle noise, etc.). The use of radiation sources with a short coherence length leads to the necessity of using interferometer circuits with combined object and reference branches. Such interferometers are usually compensated for by white light, i.e. the optical path difference in the center of the field of view is zero.

Known interference microscope described in the work of B. Bhaduri et.al. “Diffraction phase microscopy with white light.” Optics Letters, Vol.37, No.6, PP.1094-1096, 2012. This interference microscope contains a commercial inverted light field microscope with a light source in the form of a halogen lamp to form an enlarged image of the object in the output plane of this microscope, 4f optical system from two Fourier lenses, the front focal plane of which coincides with the rear focal plane of the bright field microscope, and the output image recorder is located in the rear focal plane 4f of the optical system. A diffraction grating is located in the front focal plane 4f of the optical system, which produces many diffraction orders containing all information about the object. Inside the 4f optical system in the common focal plane (Fourier plane) there is an amplitude spatial filter consisting of a point diaphragm combined with an optical axis (zero diffraction order) forming a reference beam and a rectangular diaphragm that completely transmits radiation in the first diffraction order. In the rear focal plane 4f of the optical system, two plane beams converge at a small angle in the plane of the recorder and form an object interferogram in bands of finite width. This scheme, in fact, is a Mach-Zehnder double-beam interferometer in which the reference beam is formed from radiation transmitted through a point diaphragm, and the second object beam from radiation in the first diffraction order. The disadvantage of this interference microscope is that the object and reference beams are formed at the entrance to the 4f system and therefore they travel different paths in free air space. Therefore, they will be subject to air fluctuations in different ways, which leads to instability of the interference pattern. Another drawback is that only the Fourier algorithm can be used to decipher the interference picture. A more accurate method - the phase shift method - requires changing the optical path length of one of the arms of the interferometer, which is difficult to do in this scheme.

Another interference microscope is known as described by Z. Wang et al. “Spatial light interference microscopy (SLIM)”, Optics Express, Vol.19, No.2, PP.1016-1028, 2011. It, like the previous interference microscope, consists of a commercial inverted microscope, but with phase contrast (Zernike) to form an enlarged image of the object in the output plane of this microscope. Next, an optical module in the form of a 4f optical system of two Fourier lenses is added to the microscope, the front focal plane of which coincides with the rear focal plane of the bright field microscope, and the output image recorder is located in the rear focal plane 4f of the optical system. Inside the 4f optical system in the common focal plane (Fourier plane) is not an amplitude, but a phase spatial filter that operates on reflection. The filter is made in the form of a purely phase spatio-temporal light modulator controlled by a computer, which shifts the phase of the unscattered radiation focused near the optical axis, i.e. It works as a Zernike variable phase filter. When using an annular diaphragm in a condenser, the phase filter also has the form of a ring. The light reflected from this part of the modulator forms a reference beam. The light that has undergone diffraction on the object is reflected from the rest of the modulator and forms an object beam. As a result, in the rear focal plane 4f of the optical system, in the plane of the recorder, two plane beams converge at zero angle and form an interferogram similar to that obtained when the interferometer is tuned to an infinitely wide (zero) band. Since one of the light beams is formed from radiation scattered on the object, such an interferogram can be considered as a Gabor hologram. To decrypt the interferogram, four frames with a phase shift of π / 2 are used. The main disadvantage of this interference microscope is the use of an expensive phase space-time digital light modulator. Another drawback is that a more complex mathematical procedure is used to obtain quantitative information about a phase object than in the conventional phase shift method. In particular, additional recording of the amplitude image formed from the scattered radiation, normalized to the amplitude of the unscattered radiation, is required.

Known interference microscope described in the work of N.T. Shaked “Quantitative phase microscopy of biological samples using a portable interferometer”, Optics Letters, Vol.37, No.11, PP.2016-2018, 2012, closest to the proposed interference microscope.

This interference microscope contains a bright field microscope to form an enlarged image of the object in the rear focal plane of the microscope, 4f an optical system of two Fourier lenses, the front focal plane of which coincides with the rear focal plane of the light field microscope, and a recorder is located in the rear focal plane 4f of the optical system of the output image, inside the 4f optical system to the common focal plane there is a beam splitter forming two spatially separated light beams, each of which converges in the common focal plane 4f of the optical system, where two flat mirrors are placed perpendicular to the optical axis of each of the beams, and which reflect radiation incident on them in the opposite direction, and a point aperture is located in front of one of the mirrors, combined with optical axis, which transmits only non-scattered radiation and forms a reference beam.

The disadvantage of this microscope is that the beam splitter face of the beam splitter is located at an angle of 45 degrees to the optical axis of the microscope, because the beam splitter is made in the form of a cube (gluing from two prisms with angles of 90, 45, 45 degrees). As a result, the beam splitter forms two spatially separated light beams, the optical axes of which are located at 90 degrees. Therefore, two flat mirrors, reflecting the incident radiation on them in the opposite direction, are located at an angle of 90 degrees to each other. It is impossible to install these mirrors exactly at the same distance from the side faces of the beam splitting cube, this distance will always be different, and therefore the optical path difference between the beams will always exist. For radiation with a short coherence length of the order of 27 μm, which is used in this microscope, the optical path difference must be reduced to a value of about 1 μm, much smaller than the coherence length, in order to achieve good band contrast. This is a rather difficult task of fine-tuning the interferometer.

The technical problem solved by the present invention is to reduce the requirements for alignment of the interferometer, increase its stability and, ultimately, to improve the accuracy of measurements.

The solution of this problem is achieved by the fact that in an interference microscope containing a bright field microscope to form an enlarged image of the object in the rear focal plane of this microscope, 4f an optical system of two Fourier lenses, the front focal plane of which coincides with the rear focal plane of the light field microscope, and in the rear focal plane 4f of the optical system there is an output image recorder, inside 4f of the optical system to the common focal plane p there is a beam splitter forming two spatially separated light beams, each of which converges in the common focal plane 4f of the optical system, where two flat mirrors are placed, perpendicular to the optical axis of each of the beams, which reflect radiation incident on them in the opposite direction, and in front of one of the mirrors a point diaphragm is located, combined with the optical axis, which transmits only unscattered radiation and forms a reference beam, the beam splitter forms two parallel spatial for separating a light beam with a zero path difference, and the reflective surfaces of flat mirrors lie in the same plane.

Reducing the requirements for alignment of the interferometer is achieved by the fact that the manufacture of the beam splitter is performed with a high degree of accuracy (Yu.G. Kozhevnikov. "Optical prisms", M .: Mashinostroenie, 1984).

The alignment of the reflecting surfaces of the mirrors in one plane is achieved by the fact that they are installed on a common plate.

Improving the stability of the interferometer is achieved by the fact that the beam splitter and mirrors are rigidly fixed on a common base, as a result of which the vibration has the same effect on the nodes of the interferometer, which does not affect the measurement results.

The combination of the above features significantly simplifies the alignment of the interferometer, increases its stability and accuracy of measurements.

The first variant of the interference microscope is possible, characterized in that the beam splitter is made in the form of a Kester prism.

A second variant of the interference microscope is possible, characterized in that the beam splitter is made in the form of gluing from two Dove prisms.

A third variant of the interference microscope is possible, characterized in that the beam splitter is made in the form of a cube, the beam splitting face of which is parallel to the optical axis of the incident beam.

Further, the invention is illustrated by specific examples of its implementation and the accompanying drawings.

Figure 1 illustrates a circuit diagram consisting of two parts: a commercial light field microscope I and an interference prefix II. Microscope I consists of a partially coherent light source 1, for example, an LED; a collecting optical element 2, for example a collector lens; an element restricting the cross section of the light beams 3, for example a field diaphragm; an element forming a point light source 4, for example a point diaphragm; an optical element 5 forming a parallel beam of radiation, for example a collimation lens; phase object 6; an optical element 7, for example a micro lens; reflective element 8, for example a flat mirror; an optical element 9, for example, an ocular lens, forming an enlarged image in the rear focal plane 10 of the optical element 9. The interference device II includes a 4f optical system of optical elements 11 and 12, for example, Fourier lenses, the front focal plane of which is aligned with the rear focal plane 10 of the microscope and in the rear focal plane 13 of which an image recorder 14 is installed, for example, a CCD; a beam splitter 15, for example, Kester's prism, Dove biprism or a cube located to the common focal plane 4f of the system; two retro-reflective elements 16 and 17, for example two flat mirrors, the reflective surfaces of which are mounted in the common focal plane 4f of the optical system using the support plate 18; spatial frequency filter 19, for example a pinhole, which is mounted on the retro-reflective element 17; a movable element 20, for example a piezoelectric motor, on which a retro-reflective element 17 is fixed.

Figure 2 illustrates the path of the rays in the Kester prism and the displacement of one of the reflective elements according to one of the embodiments of the present invention.

Another embodiment of the invention is based on the use of a Dove prism, namely: gluing of two Dove prisms is used as a beam splitter. Figure 3 illustrates the path of rays in the Dove prism and the displacement of one of the retro-reflective elements.

Another possible embodiment of the invention is based on the fact that the beam splitter is made in the form of a cube, the beam splitting face of which is parallel to the optical axis of the incident light beam. Figure 4 illustrates the path of the rays in a beam splitter cube and the displacement of one of the reflective elements.

Figure 5 shows the design of the base plate of the retro-reflective elements 16 and 17.

In Fig. 1, a parallel beam of partially-coherent radiation from a source 1 is formed using optical elements 2 and 5, between which there is a field diaphragm 3, which limits the cross section of the light beam, and a point diaphragm 4, acting as a point source.

A parallel beam of light passes through a phase object 6, which is located in the front focal plane of the optical element 7, the rear focal plane of which is aligned with the front focal plane of the optical element 9. An enlarged image of the phase object 6 is constructed in the rear focal plane 10 of the optical element 9. Reflective element 8 is auxiliary to reduce the construction of the microscope.

The back focal plane of the microscope is aligned with the front focal plane 4f of the optical system. In it, the radiation passes through the optical element 11, then through the beam splitter 15, after which two spatial frequencies of the same intensity are formed in the common focal plane.

In the common focal plane 4f of the system, two retroreflective elements 16 and 17 are located lying in the same plane. The last condition is fulfilled using the support plate 18, on which the mirrors are mounted. The reflective element 17 is mounted on the movable element 20, which biases the reflective element by a known value of Δ, which allows the method of interferometry of the phase steps. The radiation is freely reflected from the retroreflective element 16 and propagates in the opposite direction. Thus, an object bundle is formed. A spatial frequency filter is installed in front of the retro-reflective element 17, transmitting only unscattered radiation, which also, when reflected, propagates in the opposite direction. Thus, a reference beam is formed. In the reverse stroke, the reflected rays are combined in space by a beam splitter and pass through an optical element 12, which builds in the rear focal plane 13 an interference image of a phase object 6, fixed by the recorder 14.

The method of phase steps is implemented by creating a difference in the path of the interfering reference and object beams by shifting the reflective element 17 mounted on the movable element 20 by a known value Δ along the optical axis of the system.

Figure 2 shows in more detail the course of the rays in the Kester prism and the offset circuit of the retro-reflective element 17 by Δ using the movable element 20.

Figure 3 shows the path of the rays in the beam splitter, made in the form of gluing two prisms Dove. The shift implementation scheme is the same.

Figure 4 shows the path of the rays in the third embodiment of the beam splitting prism - cube.

Figure 5 shows a flat support plate 18 that implements the installation of reflective elements 16 and 17 when assembling in one plane, which ensures the operation of the interferometer in the mode of bands of infinite width.

The device operates in two stages as follows.

The first step is to calibrate the interference microscope. The optical system is imperfect, as a result of which various kinds of aberrations arise, distorting the image of the object under study. To eliminate such negative effects, at the first stage, the image formed by the optical system is recorded without the investigated phase object.

The second stage is the measurement process itself. The investigated phase object 6 is placed in the front focal plane of the microscope. In a beam splitting system, the optical path of the reference light beam is changed so as to realize a phase shift π / 2 (Δ = λ / 4) relative to the object beam. The sequential introduction of the known path difference Δ and registration of the interference image gives five images that cover the full period corresponding to the central wavelength of the LED. Pictures are corrected using the image obtained at the calibration stage, and then reconstruction of the initial image of the phase object is performed.

In addition to the above embodiments, embodiments are possible where, instead of two flat mirrors, one larger flat mirror is used. It is also possible that flat mirrors are supported by reflecting faces on one base plane. In addition, to implement the invention, it is possible to use a circuit when one of the planar mirrors is mounted on a piezoelectric element for implementing the phase shift method.

Thus, the claimed invention provides a solution to the above technical problems due to the following essential features of the device used: the beam splitter generates two parallel spatially separated light beams with zero travel difference, and the working surfaces of the reflective elements lie in the same plane, which simplifies alignment; the installation of a beam splitter and reflective elements eliminates the influence of vibrations.

Although the device claimed as an invention is described by the example of a number of its specific embodiments, it will be clear to those skilled in the art that numerous modifications of this device will be possible without going beyond the idea and scope of the legal protection of the invention as defined by the attached claims.

Claims (6)

1. An interference microscope containing a bright field microscope to form an enlarged image of the object in the rear focal plane of this microscope, 4f an optical system of two Fourier lenses, the front focal plane of which coincides with the rear focal plane of the light field microscope, and the optical focal plane 4f in the 4f optical plane of the system, an output image recorder is located; inside the 4f optical system, a beam splitter is located up to a common focal plane, forming two spatially separated light beams, each of which converges in the common focal plane 4f of the optical system, where two flat mirrors are placed perpendicular to the optical axis of each of the beams, which reflect the incident radiation on them in the opposite direction, and in front of one of the mirrors there is a point aperture aligned with the optical an axis that transmits only unscattered radiation and forms a reference beam, characterized in that the beam splitter forms two parallel spatially separated light beams with zero times stroke, and the reflecting surfaces of flat mirrors lie in the same plane. 2. The interference microscope according to claim 1, characterized in that the beam splitter is made in the form of a Kester prism. 3. The interference microscope according to claim 1, characterized in that the beam splitter is made in the form of gluing from two Dove prisms. 4. The interference microscope according to claim 1, characterized in that the beam splitter is made in the form of a cube, the beam splitting face of which is parallel to the optical axis of the incident light beam. 5. The interference microscope according to claim 1, characterized in that the flat mirrors with reflecting faces rest on one base plane. 6. The interference microscope according to claim 5, characterized in that one of the planar mirrors is mounted on a piezoelectric element to implement the phase shift method.

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