CN113252605A - Phase contrast microscopy method and system based on multi-step phase shift - Google Patents
Phase contrast microscopy method and system based on multi-step phase shift Download PDFInfo
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- CN113252605A CN113252605A CN202110506858.5A CN202110506858A CN113252605A CN 113252605 A CN113252605 A CN 113252605A CN 202110506858 A CN202110506858 A CN 202110506858A CN 113252605 A CN113252605 A CN 113252605A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
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- G02B21/0052—Optical details of the image generation
- G02B21/0056—Optical details of the image generation based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
- G01N2021/4173—Phase distribution
- G01N2021/4186—Phase modulation imaging
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Abstract
The invention discloses a multi-step phase shift-based phase contrast microscopic measurement method and system. The method has no requirement on the coherence of the light source, has simple structure, can effectively avoid adverse effects caused by environmental vibration, and has important significance on quantitative phase measurement microscopy.
Description
Technical Field
The present invention relates to the field of optics and quantitative phase measurement.
Background
Phase contrast microscopy techniques are widely used for microscopic observation of living cells, causing non-contact and qualitatively responsive phase changes in the sample. However, the phase contrast microscopy can only be used for observing samples with small phase changes, and cannot quantitatively measure the phase of the samples. The advantages of digital holography, non-destructive, non-invasive, full-field measurement, etc., make it applicable to quantitative phase measurements. However, the digital holographic method requires complex and large amount of calculation, has high requirements for light source coherence, the whole system has complex structure and high requirements for measuring environmental vibration and the like, and cannot meet the requirements for measuring quantitative phase under any environment.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a multi-step phase shift-based phase contrast microscopy method and system, and the method is used for quantitatively measuring the phase from a phase contrast microscopy light path.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a phase contrast microscopy method based on multi-step phase shift is characterized by comprising the following steps:
s1, adjusting a light path, and collecting parallel light at one point on a phase type spatial light modulator after the parallel light passes through a lens without placing a sample to be detected, wherein the point is marked as an O point;
s2, placing a sample in front of the lens, and recording pha as the phase distribution of the sample to be detected;
s3, loading a constant phase tha1 on the O point of the phase type spatial modulator, and collecting an object beam intensity image modulated by the spatial light modulator by using image collecting equipment, wherein the image is marked as I1＝3+2cos(tha1-pha)-2cos(tha1)-2cos(pha)；
S4, loading a constant phase tha2 on the O point of the phase type spatial modulator, and collecting an object beam intensity image modulated by the spatial light modulator by using image collecting equipment, wherein the image is marked as I2＝3+2cos(tha2-pha)-2cos(tha2)-2cos(pha)；
S5, an equation set is constructed by utilizing the relation between I1 and tha1 and pha and the relation between I2 and tha2 and pha, and the phase distribution pha of the sample to be detected can be solved.
The phase type spatial light modulator in the step S1 may be a reflective phase type spatial light modulator or a transmissive phase type spatial light modulator.
The parallel light in step S1 may be natural light or a laser light source.
The sample to be measured in step S2 may be any transmissive and reflective phase-type object such as a living cell.
The range of the constant phase tha1 in step S3 is any real number that is not zero.
The value of the constant phase tha2 in step S4 is any real number that is not zero, is not equal to tha1, and has a difference of not 2 pi.
The technical scheme adopted by the invention for solving the technical problems is as follows: the device comprises a light source, a beam expanding and collimating device, a sample to be measured, a convex lens, a beam splitting prism, an imaging lens, a transmission type phase spatial light modulator, a reflection type phase spatial light modulator, a camera and a computer. The light emitted by the light source is collimated by the collimating device and then passes through the lens to be converged into one point on the transmission type spatial light modulator, the point is marked as an O point, the light emitted by the light source is collimated by the collimating device and then passes through a sample to be detected and then carries sample information to serve as an object beam, the object beam is modulated by the transmission type phase type spatial light modulator with the phase tha1 loaded at the O point, an object beam intensity image I1 is recorded by a CCD, the light emitted by the light source is collimated by the collimating device and then passes through the sample to be detected and then carries the sample information to serve as the object beam, the object beam is modulated by the transmission type phase type spatial light modulator with the phase tha2 loaded at the O point, the object beam intensity image I2 is recorded by the CCD, and an equation set is established by using the intensity images recorded twice, so that the phase distribution of the sample to be detected can be detected.
The method overcomes the limitation that the traditional multiplication microscopy only can measure a sample with tiny phase change, can calculate the real phase of the sample by establishing an equation set through multi-step phase shift, has no requirement on the coherence of a light source, has a simple structure, can effectively avoid adverse effects caused by environmental vibration, and has important significance for quantitative phase measurement microscopy.
Drawings
FIG. 1 is a schematic optical path for measuring phase information of a sample using multi-step phase-shift phase-contrast microscopy;
FIG. 2 is a sample phase information measurement system for a transmissive phase-type spatial light modulator using multi-step phase-shift phase-contrast microscopy;
FIG. 3 is a system for measuring phase information of a sample of a reflective phase-type spatial light modulator using multi-step phase-shift phase-contrast microscopy;
in fig. 1: 1-object beam, 2-convex lens, 3-phase type spatial light modulator, 4-imaging lens and 5-camera;
in fig. 2: 1-a light source, 2-a lens group, 3-a sample to be measured, 4-a convex lens, 5-a transmission type phase spatial light modulator, 6-an imaging lens and 7-a camera;
in fig. 3: the method comprises the following steps of 1-a light source, 2-a lens group, 3-a sample to be measured, 4-a convex lens, 5-a beam splitter prism, 6-a camera, 7-an imaging lens and 8-a reflective phase type spatial light modulator.
Detailed Description
The invention will now be further described with reference to the following examples and drawings:
example 1: the invention designs a multi-step phase-shift phase-contrast microscopic transmission type phase spatial light modulator sample phase information measuring system as shown in figure 2, and the working flow is as follows:
light emitted by a light source 1 is collimated and expanded by a lens group 2, then passes through a convex lens 4, and then is converged at one point on a transmission phase type spatial light modulator 5 and is marked as an O point, a sample 3 is placed in front of the convex lens 4, and is marked as phase distribution of the sample 3 to be detected, and a constant phase is loaded on the O point of the transmission phase type spatial light modulator 5
And a camera 7 is used for collecting the intensity image of the object beam modulated by the transmission type phase-type spatial light modulator 5 and recording the image as
A constant phase is applied to the point O of the transmissive phase type spatial light modulator 5
And a camera 7 is used for collecting the intensity image of the object beam modulated by the transmission type phase-type spatial light modulator 5 and recording the image as
Simultaneous system of equations
Can obtain the product
The phase of the sample to be measuredpha＝angle[6-I1-I2+i·(I1+I2)]。
Example 2: the invention designs a multi-step phase-shift phase-contrast microscopic reflection type phase spatial light modulator sample phase information measuring system as shown in figure 2, and the working flow is as follows:
light emitted by a light source 1 is collimated and expanded by a lens group 2 and then passes through a convex lens 4, and then is converged at one point on a reflective phase type spatial light modulator 8 and is marked as an O point, a sample 3 is placed in front of the convex lens 4, and is marked as phase distribution of the sample 3 to be detected, and a constant phase is loaded on the O point of the reflective phase type spatial light modulator 8
The object beam is modulated by a reflective phase-type spatial light modulator 8, and the intensity image of the object beam reflected by the beam splitter prism is collected by a camera 6 and recorded as
A constant phase is applied to the point O of the transmissive phase type spatial light modulator 5
The object beam is modulated by a reflective phase-type spatial light modulator 8, and the intensity image of the object beam reflected by the beam splitter prism is collected by a camera 6 and recorded as
Simultaneous system of equations
Can obtain the product
Then the phase (pha) of the sample to be tested is equal to angle [6-I1-I2+i·(I1+I2)]。
Claims (8)
1. A phase contrast microscopic measurement method based on multi-step phase shift is characterized by comprising the following steps:
s1, adjusting a light path, and collecting parallel light at one point on a phase type spatial light modulator after the parallel light passes through a lens without placing a sample to be detected, wherein the point is marked as an O point;
s2, placing a sample in front of the lens, and recording pha as the phase distribution of the sample to be detected;
s3, loading a constant phase tha1 on the O point of the phase type spatial modulator, and collecting an object beam intensity image modulated by the spatial light modulator by using image collecting equipment, wherein the image is marked as I1＝3+2cos(tha1-pha)-2cos(tha1)-2cos(pha)；
S4, loading a constant phase tha2 on the O point of the phase type spatial modulator, and collecting an object beam intensity image modulated by the spatial light modulator by using image collecting equipment, wherein the image is marked as I2＝3+2cos(tha2-pha)-2cos(tha2)-2cos(pha)；
S5, establishing an equation set by utilizing the relation between I1 and tha1 and pha and the relation between I2 and tha2 and pha
The phase distribution pha of the sample to be measured can be solved.
2. A multi-step phase shift-based phase contrast phase microscopic measurement system comprises a light source, a beam expanding and collimating device, a sample to be measured, a convex lens, a beam splitting prism, an imaging lens, a transmission type phase type spatial light modulator, a reflection type phase type spatial light modulator, a camera and a computer. The light emitted by the light source is collimated by the collimating device and then passes through the lens to be converged into one point on the transmission type spatial light modulator, the point is marked as an O point, the light emitted by the light source is collimated by the collimating device and then passes through a sample to be detected and then carries sample information to serve as an object beam, the object beam is modulated by the transmission type phase type spatial light modulator with the phase tha1 loaded at the O point, an object beam intensity image I1 is recorded by a CCD, the light emitted by the light source is collimated by the collimating device and then passes through the sample to be detected and then carries the sample information to serve as the object beam, the object beam is modulated by the transmission type phase type spatial light modulator with the phase tha2 loaded at the O point, the object beam intensity image I2 is recorded by the CCD, and an equation set is established by using the intensity images recorded twice, so that the phase distribution of the sample to be detected can be detected.
3. The phase type spatial light modulator according to claim 1 may be a reflective phase type spatial light modulator or a transmissive phase type spatial light modulator.
4. The parallel light according to claim 1 may be natural light or laser light.
5. The sample to be measured according to claim 1 may be any of transmission-type and reflection-type phase-type objects such as living cells.
6. The range of constant phase tha1 of claim 1 is any real number that is not zero.
7. The constant phase tha2 of claim 1 takes on any real number that is not zero, is not equal to tha1, and differs by not 2 pi.
8. Pha according to claim 1, when
When the alpha is calculated, the alpha is alpha [6-I ]1-I2+i·(I1+I2)] 。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117631246A (en) * | 2023-12-25 | 2024-03-01 | 山东理工大学 | Quantitative phase imaging method and device |
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