CN110554491B

CN110554491B - Miniature phase difference digital microscope for observing unstained cells of living body

Info

Publication number: CN110554491B
Application number: CN201811517647.6A
Authority: CN
Inventors: 王冬芳; 余宁梅; 汪丽
Original assignee: Xian University of Technology
Current assignee: Xian University of Technology
Priority date: 2018-12-12
Filing date: 2018-12-12
Publication date: 2022-04-22
Anticipated expiration: 2038-12-12
Also published as: CN110554491A

Abstract

The invention discloses a micro-phase difference digital microscope, which consists of a light source, an aperture diaphragm, a micro-current control chip, an objective lens group, a phase plate, an image sensor, a voice coil motor and control and display equipment. Compared with a common microscope, the condenser part of the common microscope is removed, the sample is directly irradiated by a parallel light source, and meanwhile, a micro-current control chip of the common phase difference microscope is placed behind an aperture diaphragm and a lens. Then a portion of the light emitted from the light source is irradiated toward the sample after passing through the circular aperture stop, and a portion of the light becomes diffracted light while another portion of the light is directed straight through. The two beams of coherent light then undergo interference enhancement at the image plane. The invention has simple structure, is convenient to carry, and can well observe cells.

Description

Miniature phase difference digital microscope for observing unstained cells of living body

Technical Field

The invention belongs to the field of optical microscopes, and particularly relates to a miniature phase difference digital microscope for observing unstained cells of a living body.

Background

Phase contrast microscopy was invented by Netherlands scientist Zermike in 1935 and thus received the Nobel physical prize in 1953 for the observation of unstained specimens. Because the refractive index and the thickness of the living cell parts in the unstained specimen are different, when light passes through, the wavelength and the amplitude do not change, only the phase changes slightly, and the light cannot be distinguished by human eyes. In general, the phase difference can be observed by changing the phase difference with a phase difference microscope, and changing the phase difference into an amplitude difference by utilizing diffraction and interference phenomena of light.

At present, in a channel of a microfluidic chip, aiming at living cells or unstained cells, because the refractive index and the thickness of the inside of the living cells in an unstained specimen are different, when light passes through, the wavelength and the amplitude are not changed, only the phase is slightly changed, and the light cannot be distinguished by human eyes. And if the method of direct imaging display is adopted, because the size of the common cell is equivalent to the pixel point, and the cell transparency is not obviously different from the surrounding medium, the problem that the cell boundary is unclear is encountered during imaging. Then the display effect and recognition rate of the cells are not satisfactory in the process of designing the cell collecting system.

Disclosure of Invention

The invention aims to provide a micro phase difference digital microscope for observing unstained cells of a living body, which can solve the problems that the size of the existing phase difference microscope is too large and the developing boundary of the cells in a microfluidic chip channel is not clear.

The technical scheme adopted by the invention is as follows: a miniature phase difference digital microscope for observing unstained cells in a living body comprises a light source, an aperture diaphragm, an objective lens group, a phase plate and a voice coil motor are sequentially arranged along the light emitting direction of the light source, a micro-current control chip is arranged between the aperture diaphragm and the objective lens group, a group of symmetrically arranged detectors are arranged at two ends in a channel of the micro-current control chip, the voice coil motor is connected with a control and display device, and a CMOS image sensor is arranged on the voice coil motor.

The present invention is also characterized in that,

the centers of the light source, the aperture diaphragm, the micro-current control chip, the objective lens group, the phase plate and the voice coil motor are on the same straight line.

The magnification of the objective lens group is 5-10 times.

When the magnification of the objective lens group is 5 times, the length of the microscope is 77-79 mm.

The phase plate is a circular phase plate, the thickness range of the phase plate is 9.90 mm-10.80 mm, and the step length is 0.02 mm.

The thickness of the phase plate is 10.20mm, and the distance between the phase plate and the light screen is 42.4 mm.

The aperture diaphragm adopts a circular aperture diaphragm, and the diameter of the aperture diaphragm is 1.0 mm.

The material of the microfluidic chip is common glass BK7 or PDMS.

The objective lens group is composed of two double cemented lenses.

The light source adopts a laser diode as the light source.

The invention has the beneficial effects that: put the slide in front of convex lens for convex lens can aggregate erection, form the battery of lens, like this when designing digital microscope behind, be convenient for integrate convex lens and voice coil motor and image sensor, have portable miniaturized advantage. The improved micro phase difference microscope can be used in a fixed display system of cells in a microfluidic channel, and can also be used as a universal micro phase difference microscope for displaying cells related to biological research and medical diagnosis.

Drawings

FIG. 1 is a schematic structural diagram of a miniature phase-difference digital microscope for observing unstained cells in a living body according to the present invention;

FIG. 2 is an Abel imaging schematic of a miniature phase-contrast digital microscope for observing unstained cells in vivo according to the present invention;

FIG. 3 is a schematic structural diagram of a microfluidic chip of a micro phase-difference digital microscope for observing unstained cells in a living body according to the invention;

FIG. 4 is a schematic diagram showing the results of the change in interference pattern with phase plate thickness for a micro phase contrast digital microscope of the present invention used to observe unstained cells in vivo;

FIG. 5 is a diagram showing the effect of the simulated interference pattern of the micro phase-contrast digital microscope for observing unstained cells in vivo according to the present invention.

In the figure, 1 is a light source, 2 is an aperture diaphragm, 3 is a micro-current control chip, 4 is an objective lens group, 5 is a phase plate, 6 is an image sensor, 7 is a voice coil motor, 8 is control and display equipment, and 9 is a detector.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

As shown in fig. 1, the structure of a micro phase difference digital microscope for observing unstained cells in a living body of the invention comprises a light source 1, an aperture diaphragm 2, an objective lens group 4, a phase plate 5 and a voice coil motor 7 are sequentially arranged along the light emitting direction of the light source 1, a micro-current control chip 3 is arranged between the aperture diaphragm 2 and the objective lens group 4, a pair of symmetrically arranged detectors 9 is arranged at two ends in a channel of the micro-current control chip 3, the voice coil motor 7 is connected with a control and display device 8, and a CMOS image sensor 6 is arranged on the voice coil motor 7.

The centers of the light source 1, the aperture diaphragm 2, the micro-current control chip 3, the objective lens group 4, the phase plate 5 and the voice coil motor 7 are on the same straight line.

The light source 1 uses a laser diode as a light source.

Wherein, the material of the microfluidic chip 3 is selected from a common glass material BK7 or PDMS.

The working principle of the miniature phase difference digital microscope for observing the unstained cells of the living body is as follows: the condenser in the ordinary microscope is partially removed, and the sample is directly irradiated by the parallel light source 1, so that the distance between the sample cell and the objective lens group 4 can be greatly shortened, meanwhile, the objective table of the ordinary phase difference microscope is changed into a micro-current control chip 3 which is arranged between the aperture diaphragm 2 and the objective lens group 4, and the direct light can be seen to be completely focused on the phase plate 5 after passing through the objective lens group 4, as shown in figure 2, according to the Abel imaging principle, after the parallel light beam passes through the cell sample, the direct light is focused on the phase plate 5 through the objective lens group 4, the diffracted light is focused on the image sensor 6, and then the two coherent light beams are interfered on the image plane, so that the position of the phase plate 5 is the right focus of the lens group.

As shown in FIG. 3, the optical path history of the digital microscope with micro phase difference for observing unstained cells in vivo according to the present invention is: the light emitted from the light source 1 passes through the aperture diaphragm 2, irradiates a cell sample through a detector 9 in a micro-current control chip 3 channel, at the cell boundary, one part of the light source is changed into diffracted light through the cell boundary, the phase of the diffracted light is correspondingly changed, the other part of the light passes through the direct light, the direct light is completely focused on the cylindrical phase plate 5 after passing through the objective lens group 4, the direct light is delayed by pi/2 through the phase plate 5, the diffracted light at the cell boundary is focused on the image sensor 6 (replaced by a light screen in simulation), and then two beams of coherent light are subjected to interference enhancement on an image plane. In order to eliminate chromatic aberration as much as possible and improve imaging quality, an achromatic objective lens is adopted as the objective lens and consists of two double-cemented lenses.

Under the influence of the structure of the existing phase difference microscope, when the microscope is simulated, the aperture diaphragm 2 and the phase plate 5 are designed to be circular, because the circular shape is easier to process than the annular shape in practice, the aperture diaphragm 2 is changed into a light-transmitting circular hole, the diameter is generally 1.0-2.0 mm, and the phase plate 5 is changed into a coated cylinder (the surface coated with a film opposite to the direct light from the sample). The invention adopts the circular aperture diaphragm 2 with the diameter of 1.0mm, which not only ensures the light field formed by the light passing through the aperture diaphragm 2, but also can meet the display of the field of view.

The choice of the objective 4 can influence the cell magnification and the optical path difference through which the light passes, thus influencing the interference effect. Generally, the magnification is selected to be 5-10 times, which is enough to image the image clearly on the light screen. In the invention, the cell magnification is 5 times, if the object distance is 5mm, the image distance is about 50mm through simulation, so that the position of the light screen 6 can be known.

In order to obtain the best interference enhancement effect, the invention also simulates the thickness of the phase plate 5: the phase plate 5 is used for changing the phase and illumination intensity of light passing through a specific area, and two methods are used for enabling diffraction light and direct light to generate interference phenomenon. The first is to adjust the direct light to be in phase with the diffracted light, which is to generate interference enhancement between the diffracted light and the direct light, so that the contrast between the sample cell and the environment (or other impurities such as air bubbles) is more obvious, and the cell is brighter. The second is that the phase difference between the diffracted light and the direct light is odd times of pi, the interference between the diffracted light and the direct light is weakened, and the cell is darker than the environment. In the invention, light emitted by the light source passes through the aperture diaphragm 2 with a small size and can be used as a parallel light source to reach the microfluidic chip 3, so that the circular phase plate 5 can only coat a direct light transmission area, and only interference enhancement can be carried out, namely, cells are brighter than the surrounding environment. The thickness of the phase plate 5 of the present invention is 9.90-10.80mm, the step size is 0.02mm, as shown in fig. 4, which is a schematic diagram of the interference pattern of the microscope of the present invention as a result of the change in the thickness of the phase plate, as can be seen in fig. 5: when the thickness of the phase plate 5 is 10.20mm, the interference effect is best when the distance between the coil motor 7 and the phase plate 5 is 42.4 mm.

The aperture diaphragm adopts a tiny circular aperture diaphragm, so that the parallelism of a laser diode light field is better; the objective lens is separated from the lens group, and the structure of the objective lens is different from that of a common large-size phase difference microscope, so that the objective lens is more convenient to integrate into a digital microscope; design miniaturization, whole structure designs out system length at present and only 77~79mm, carries and integrates very easily.

Example (b):

a beam of light emitted by a parallel light source passes through an aperture diaphragm 2 with the diameter of 1.0mm, directly irradiates the lower wall of a channel in a cylindrical microfluidic chip, passes through a pair of detectors 9 and irradiates on sample cells, a part of light is diffracted, direct light and diffracted light pass through the cylindrical upper wall, and are converged by an objective lens group 4, the direct light is focused on a circular phase plate 5, and the diffracted light passes through a side area. When the parallel direct light passes through the sample cell and its surrounding area, a part of the parallel direct light is diffracted due to the obstacle, and the diffracted light and the direct light are focused at different positions after passing through the objective lens group 4. It can be seen that the position of focusing the direct light is closer to the objective 4, the position of the phase plate 5 is near the focal plane of the objective, the position of focusing the diffracted light is the image plane, and the focusing positions of the two are different, so that it is necessary to design a cylindrical phase plate 5 for the direct light to pass through the coating region, and the purpose of the coating region is to change the direct light to delay its phase by pi, and then 2 is in phase with the diffracted light passing directly through the non-coating region, so we can observe the interference pattern on the light screen 6, as shown in fig. 5, the interference enhancement is generated at the cell edge, and there is obvious brightness and darkness with the environment.

Claims

1. The micro phase difference digital microscope for observing unstained cells in a living body is characterized by comprising a light source (1) for emitting parallel light, an aperture diaphragm (2), an objective lens group (4), a phase plate (5) and a voice coil motor (7) are sequentially arranged along the light emitting direction of the light source (1), a microfluidic chip (3) is arranged between the aperture diaphragm (2) and the objective lens group (4), a group of symmetrically-arranged detectors (9) are arranged at two ends in a channel of the microfluidic chip (3), the phase plate (5) is a circular phase plate, the aperture diaphragm (2) adopts the circular aperture diaphragm, a voice coil motor (7) is connected with a control and display device (8), a CMOS image sensor (6) is arranged on the voice coil motor (7), the CMOS image sensor (6) is replaced by an optical screen during simulation, and the thickness value range of the phase plate (5) is 9.90 mm-10.80 mm, the step length is 0.02mm, and the diameter of the aperture diaphragm (2) is 1.0 mm.

2. The digital microscope according to claim 1, wherein the light source (1), the aperture stop (2), the microfluidic chip (3), the objective lens group (4), the phase plate (5) and the voice coil motor (7) are aligned with each other.

3. The digital microscope according to claim 1, wherein the objective lens group (4) has a magnification of 5 to 10 times.

4. The digital microscope according to claim 3, wherein the length of the microscope is 77 to 79mm when the objective lens group (4) is magnified at 5 times.

5. The digital microscope according to claim 1, wherein the thickness of the phase plate (5) is 10.20mm and the distance between the phase plate (5) and the light screen is 42.4 mm.

6. The digital microscope with micro phase difference for observing unstained cells in living bodies as claimed in claim 1, wherein the material of the microfluidic chip (3) is selected from common glass BK7 or PDMS.

7. -the micro phase-contrast digital microscope for observing unstained cells of a living body according to claim 1, characterized in that the objective lens group (4) is composed of two cemented doublets.

8. The digital microscope according to claim 1, wherein the light source (1) is a laser diode as a light source.