CN116183562A - Polarization modulation fluorescence microscopic imaging device and method - Google Patents
Polarization modulation fluorescence microscopic imaging device and method Download PDFInfo
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Abstract
The invention discloses a polarization modulation fluorescence microscopic imaging device and a method, wherein the device comprises an incident illumination modulation module, a microscopic system module, an emergent imaging modulation module and a control and image processing module, wherein the incident illumination modulation module and the emergent imaging modulation module are controlled by the control module, continuous polarization modulation illumination is carried out on a sample through the microscopic system module, and real-time image acquisition is carried out by utilizing an imaging sensing area array in the emergent imaging modulation module. Finally, obtaining a polarization parameter image of the sample through an image processing module. The device has the capability of rapidly and continuously carrying out polarization modulation and multi-parameter polarization image acquisition, and can be used for detecting the anisotropic arrangement of fluorescent molecules and the influence of fluorescent molecules with different orientations on a light field. The invention has extremely strong characterization capability on the boundary between fluorescence and non-fluorescence and biological cell boundary, and has great advantages in resolution and sensitivity.
Description
Technical Field
The invention belongs to the field of optical microscopic imaging, and particularly relates to a polarization modulation fluorescence microscopic imaging device and method.
Background
The fluorescence microscopic imaging technology realizes fluorescence imaging of a specific target by specifically labeling fluorescent protein, fluorescent dye and the like on a sample, is one of the most commonly used optical measurement methods at present, and is widely applied to researches of nanotechnology, life science and the like. The existing fluorescence microscopic imaging method mainly comprises a wide-field fluorescence microscopic imaging technology, a structured light obvious microscopic imaging technology, a laser scanning confocal technology, a two-photon fluorescence microscopic imaging technology and the like. However, the actual imaging result of the prior art is limited by the photo-bleaching and phototoxicity caused by excitation light and loss light intensity, the photon yield of fluorescent markers, the efficiency of a photoswitch and the like; at the same time, to achieve as high a resolution as possible, these techniques often require the use of high numerical aperture objectives, thus resulting in a relatively small imaging field of view and a relatively slow imaging speed.
Disclosure of Invention
The invention aims to provide a polarization modulation fluorescence microscopic imaging device.
The technical scheme for realizing the purpose of the invention is as follows: a polarization-modulated fluorescence microscopy imaging apparatus comprising: the system comprises an incident illumination modulation module, a microscope system module, an emergent imaging modulation module and a control and image processing module, wherein the control and image processing module is used for controlling the incident illumination modulation module to emit linearly polarized light with different polarization angles, controlling the emergent imaging modulation module to collect polarized light intensity images with different angles, and carrying out data processing on the collected polarized light intensity images with different angles to obtain polarized parameter images of a sample to be detected, the microscope system module comprises a dichroic mirror, an objective lens and an objective table, the objective table is provided with a sample, the linearly polarized light emitted by the incident illumination modulation module is incident on the sample through the dichroic mirror and the objective lens, and a light signal reflected by the sample enters the emergent imaging modulation module through the objective lens and the dichroic mirror.
Preferably, the incident illumination modulation module comprises a light source, a light source connecting piece, a linear polaroid and a microscope connecting piece, wherein the linear polaroid is arranged in the light source connecting piece, the front end of the light source connecting piece is connected with the light source, and the rear end of the light source connecting piece is connected with the microscope connecting piece.
Preferably, the emergent imaging modulation module comprises a quarter wave plate, a linear polaroid and an imaging sensing area array camera, the optical signals reflected by the sample reach the imaging sensing area array camera through the quarter wave plate and the linear polaroid, and the imaging sensing area array camera obtains the polarization parameter image.
Preferably, the fast axis of the 1/4 wave plate is in the XY plane and at a 45 degree angle to the X axis, and the fast axis of the linear polarizer is along the X axis.
Preferably, the incident illumination modulation module adopts a high-pressure mercury lamp fluorescence excitation light source and is provided with fluorescent modules with different wavelengths.
Preferably, the light intensity of the collected polarized light intensity image is specifically:
considering the above equation as a function of the light intensity I and the polarization angle θ only, the above equation can be changed to:
I i ＝a 0 +a 1 sin2θ i +a 2 cos2θ i
wherein:
angle of incidence polarization theta i Can be modulated from 0 to 180, the total number of modulation steps being n=180°/18 °, spread according to the fourier series, so a 0 、a 1 、a 2 Can be expressed as:
preferably, the polarization parameters in the obtained polarization parameter image include polarization intensity, phase delay, and azimuth angle.
Preferably, depolarization intensity I dp The phase delay sin delta and the azimuth angle phi are respectively as follows:
the invention also provides a polarization modulation fluorescence microscopic imaging method, which comprises the following specific steps: step 1, inputting initial parameters of a stepping motor in a control and image processing module, wherein the initial parameters comprise motor rotation speed and rotation angle, acceleration time, dwell time and modulation mode, and setting camera exposure time and an image storage path.
Step 2, the control and image processing module sends out a control signal to control the stepping motor to drive the linear polaroid of the incident light modulation module to reach a set angle, and simultaneously controls the incident light modulation module to modulate the polarization state of the light beam and the emergent imaging modulation module to acquire images;
and step 3, the control and image processing module performs data processing on the collected multiple light intensity images to obtain a polarization parameter image of the sample to be detected.
Compared with the prior art, the invention has the remarkable advantages that: the invention does not need special designed complex conditions such as fluorescent protein, a scintillating fluorescent probe, structural light illumination and the like, and only utilizes the polarization characteristic of light to rapidly and continuously modulate the polarization state of light beams; the invention can be used for detecting the influence of the anisotropic arrangement of fluorescent molecules and fluorescent molecules with different orientations on the light field; the invention has extremely strong characterization capability on the boundary between fluorescence and non-fluorescence and biological cell boundary, and has great advantages in resolution and sensitivity.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims thereof as well as the appended drawings.
Drawings
The drawings are only for purposes of illustrating particular embodiments and are not to be construed as limiting the invention, like reference numerals being used to refer to like parts throughout the several views.
Fig. 1 is a diagram of an apparatus of the present invention, I is an incident light modulation module, II is a microsystem module, III is an exit imaging modulation module, and IV is a control and image processing module.
FIG. 2 is a simulated image of fluorescent dipole point pairs with a fluorescence polarization modulation at 50nm apart with a 90℃orientation difference.
Fig. 3 is an image of a fluorescence polarization modulated biological sample.
Detailed Description
It is easy to understand that various embodiments of the present invention can be envisioned by those of ordinary skill in the art without altering the true spirit of the present invention in light of the present teachings. Accordingly, the following detailed description and drawings are merely illustrative of the invention and are not intended to be exhaustive or to limit or restrict the invention. Rather, these embodiments are provided so that this disclosure will be thorough and complete by those skilled in the art. Preferred embodiments of the present invention are described in detail below with reference to the attached drawing figures, which form a part of the present application and are used in conjunction with embodiments of the present invention to illustrate the innovative concepts of the present invention.
The invention is conceived as shown in fig. 1, a polarization modulation fluorescence microscopic imaging device is mainly used for detecting the anisotropic arrangement of fluorescent molecules in a space scattering map and the influence of fluorescent molecules with different orientations on a light field, adopts excitation light with different polarization angles as an excitation light source of a fluorescence sample, and utilizes the anisotropic property of the fluorescence sample to obtain light intensity images with different polarization angles, and specifically comprises the following steps: the system comprises an incident illumination modulation module, a microscope system module, an emergent imaging modulation module and a control and image processing module, wherein the control and image processing module is used for controlling the incident illumination modulation module to emit linearly polarized light with a set polarization angle, controlling the emergent imaging modulation module to collect polarized light intensity images with different angles, and carrying out data processing on the collected polarized light intensity images with different angles to obtain polarized parameter images of a sample to be detected, the microscope system module comprises a dichroic mirror, an objective lens and an objective table, the objective table is provided with a sample, the linearly polarized light emitted by the incident illumination modulation module is incident on the sample through the dichroic mirror and the objective lens, and a light signal reflected by the sample enters the emergent imaging modulation module through the objective lens and the dichroic mirror.
In a further embodiment, the incident illumination modulation module includes a light source, a light source connector, a linear polarizer, and a microscope connector. The linear polaroid is arranged in the light source connecting piece, the front end of the light source connecting piece is connected with the light source, and the rear end of the light source connecting piece is connected with the microscope connecting piece.
In a further embodiment, the outgoing imaging modulation module comprises a quarter wave plate, a linear polarizer and an imaging sensing area array camera. The optical signal reflected by the sample reaches the imaging sensing area array camera through the quarter wave plate and the linear polaroid, and the imaging sensing area array camera obtains a polarization parameter image.
Specifically, the fast axis of the 1/4 wave plate is in the XY plane and forms an included angle of 45 degrees with the X axis, and the fast axis of the linear polarizer is along the X axis. The 1/4 wave plate and the linear polaroid form a polarized parameter imaging light path. The XY plane is parallel to the ground and takes the fast axis of the linear polarizer as the X-axis direction. The Y-axis is perpendicular to the fast axis of the linear polarizer.
In a further embodiment, the incident illumination modulation module adopts a high-pressure mercury lamp fluorescence excitation light source and is provided with fluorescent modules with different wavelengths, so that fluorescence or non-fluorescence polarization microscopic imaging can be realized. In some embodiments, the light source used is a high pressure mercury lamp fluorescent excitation light source, with 365nm laser light as the excitation light source. A tele objective lens with a magnification of 100X was used, with a numerical aperture of 0.8.
In a further embodiment, the control and image processing module controls the rotation of the linear polarizer in the incident illumination modulation module, so that the incident light can be controlled to be incident in linear polarized light with different polarization angles, and the incident light can be rotated according to equidistant angles, wherein the maximum angle is 360 degrees.
In a further embodiment, the control and image processing module programs the control program using associated software. The linearly polarized light is incident at equal intervals, and corresponding images are acquired by the synchronous imaging sensing area array.
In a further embodiment, the control and image processing module performs data processing on the collected multiple light intensity images to obtain a polarization parameter map of the sample to be testedLike an image. The polarization parameter includes depolarization intensity I dp Phase delay sin delta, and azimuth angle phi. The specific process is as follows:
rotating the linear polarization field from 0 degree to 180 degrees, and collecting the light intensity I i The method comprises the following steps:
considering the above equation as a function of the light intensity I and the polarization angle θ only, the above equation can be changed to:
I i ＝a 0 +a 1 sin2θ i +a 2 cos2θ i
the three parameters in the above formula are:
angle of incidence polarization theta i Can be modulated from 0 to 180, the total number of modulation steps being n=180°/18 °, spread according to the fourier series, so a 0 、a 1 、a 2 Can be expressed as:
obtaining three polarization parameter images with different characteristics of the sample to be detected, which are respectively depolarization intensity I dp Phase delay sin delta, and azimuth angle Φ:
the working process of the invention is as follows: the sample is placed on a slide and placed on a microscope stage. The light source switch is turned on, the linear polarizer in the incident illumination modulation module is controlled by the computer to periodically rotate, so that incident light with different polarization angles passes through the dichroic mirror to excite a sample, the excited sample is focused on the surface of the sample through the objective lens, and the excited sample emits radiation light which passes through the objective lens, the dichroic mirror, the quarter wave plate and the polarization analyzer in turn. And corresponding light intensity images are acquired by using the imaging sensing area array. The imaging sensing area array in the emergent imaging modulation module acquires an image every time the linear polarizer rotates once in the incident illumination modulation module. The collected polarized light intensity image is processed by a control and image processing module to obtain a required polarized parameter image.
A polarization modulation fluorescence microscopic imaging method comprises the following specific steps:
step 1, inputting initial parameters of a stepping motor in a control and image processing module, wherein the initial parameters comprise motor rotation speed and rotation angle, acceleration time, dwell time and modulation mode, and setting camera exposure time and an image storage path.
Step 2, the control and image processing module sends out a control signal to control the stepping motor to drive the linear polaroid of the incident light modulation module to reach a set angle, and simultaneously controls the incident light modulation module to modulate the polarization state of the light beam and the emergent imaging modulation module to acquire images;
specifically, the incident light modulation module enables incident light modulation to be (i-1) 180/10 degrees with a Z axis in a YZ plane, i=1, 2,3 … and n, wherein the total modulation step number n=10, and the emergent imaging modulation module acquires images and synchronously acquires polarized light intensity pictures;
the collected light intensity I i The method comprises the following steps:
considering the above equation as a function of the light intensity I and the polarization angle θ only, the above equation can be changed to:
I i ＝a 0 +a 1 sin2θ i +a 2 cos2θ i
wherein:
angle of incidence polarization theta i Can be modulated from 0 to 180, the total number of modulation steps being n=180°/18 °, spread according to the fourier series, so a 0 、a 1 、a 2 Can be expressed as:
and step 3, the control and image processing module performs data processing on the collected multiple light intensity images to obtain a polarization parameter image of the sample to be detected.
In a further embodiment, deriving the polarization parameter using the correlation software comprises: depolarization intensity I dp Phase delay sin delta, and azimuth angle phi:
the method is mainly used for detecting the influence of the anisotropic arrangement of fluorescent molecules in the space scattering spectrum and the fluorescent molecules with different orientations on the light field. The device has the capability of rapidly and continuously carrying out polarization modulation and multi-parameter image acquisition, has extremely strong characterization capability on fluorescence and non-fluorescence junctions and biological cell boundaries, and has great advantages in resolution and sensitivity.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.
It should be appreciated that in the above description of exemplary embodiments of the invention, various features of the invention are sometimes described in the context of a single embodiment or with reference to a single figure in order to streamline the invention and aid those skilled in the art in understanding the various aspects of the invention. The present invention should not, however, be construed as including features that are essential to the patent claims in the exemplary embodiments.
It should be understood that modules, units, components, etc. included in the apparatus of one embodiment of the present invention may be adaptively changed to arrange them in an apparatus different from the embodiment. The different modules, units or components comprised by the apparatus of the embodiments may be combined into one module, unit or component or they may be divided into a plurality of sub-modules, sub-units or sub-components.
Claims (9)
1. A polarization-modulated fluorescence microscopy imaging apparatus, comprising: the system comprises an incident illumination modulation module, a microscope system module, an emergent imaging modulation module and a control and image processing module, wherein the control and image processing module is used for controlling the incident illumination modulation module to emit linearly polarized light with different polarization angles, controlling the emergent imaging modulation module to collect polarized light intensity images with different angles, and carrying out data processing on the collected polarized light intensity images with different angles to obtain polarized parameter images of a sample to be detected, the microscope system module comprises a dichroic mirror, an objective lens and an objective table, the objective table is provided with a sample, the linearly polarized light emitted by the incident illumination modulation module is incident on the sample through the dichroic mirror and the objective lens, and a light signal reflected by the sample enters the emergent imaging modulation module through the objective lens and the dichroic mirror.
2. The fluorescence microscopic imaging device according to claim 1, wherein the incident illumination modulation module includes a light source, a light source connector, a linear polarizer, and a microscope connector, wherein the linear polarizer is disposed in the light source connector, and a front end of the light source connector is connected to the light source, and a rear end of the light source connector is connected to the microscope connector.
3. The polarization-modulating fluorescence microscopic imaging device according to claim 1, wherein the outgoing imaging modulation module comprises a quarter wave plate, a linear polarizer and an imaging sensing area array camera, and the optical signal reflected by the sample reaches the imaging sensing area array camera through the quarter wave plate and the linear polarizer, and the imaging sensing area array camera obtains a polarization parameter image.
4. The polarization-modulating fluorescence microscopy imaging device of claim 3, wherein the fast axis of the 1/4 wave plate is in the XY plane and at a 45 degree angle to the X axis, and the fast axis of the linear polarizer is along the X axis.
5. The polarization-modulated fluorescence microscopy imaging device of claim 1, wherein the incident illumination modulation module employs a high-pressure mercury lamp fluorescence excitation light source and is configured with fluorescent modules of different wavelengths.
6. The polarization-modulated fluorescence microscopy imaging device of claim 1, wherein the light intensity of the collected polarized light intensity image is specifically:
the above equation is considered as a function between the light intensity I and the polarization angle θ, and is rewritten as:
I i ＝a 0 +a 1 sin2θ i +a 2 cos2θ i
wherein:
angle of incidence polarization theta i From 0 ° to 180 °, the total modulation step number is n=180°/18 °, and the modulation is spread according to the fourier series, so a 0 、a 1 、a 2 Expressed as:
7. the polarization-modulated fluorescence microscopy imaging device of claim 1, wherein the polarization parameters in the obtained polarization parameter image comprise polarization intensity, phase delay, and azimuth angle.
8. The polarization-modulated fluorescence microscopy imaging device of claim 7, wherein depolarization intensity I dp The phase delay sin delta and the azimuth angle phi are respectively as follows:
9. a method based on the system according to any one of claims 1 to 7, characterized by the specific steps of: step 1, inputting initial parameters of a stepping motor in a control and image processing module, wherein the initial parameters comprise motor rotation speed and rotation angle, acceleration time, dwell time and modulation mode, and setting camera exposure time and an image storage path.
Step 2, the control and image processing module sends out a control signal to control the stepping motor to drive the linear polaroid of the incident light modulation module to reach a set angle, and simultaneously controls the incident light modulation module to modulate the polarization state of the light beam and the emergent imaging modulation module to acquire images;
and step 3, the control and image processing module performs data processing on the collected multiple light intensity images to obtain a polarization parameter image of the sample to be detected.
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