CN108982455B - Multi-focus light section fluorescence microscopic imaging method and device - Google Patents
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Abstract
The invention discloses a multi-focus light section fluorescence microscopic imaging method and a system, belonging to the technical field of optical imaging. The laser beam emitted by the laser is phase-modulated and then becomes a beam having a plurality of focal points. Rotating the galvanometer to scan the light beam along the X-axis direction; the light beam is moved back and forth along the Y-axis direction by changing the focal length of the electrically driven variable-focus concave lens. The movement of the multi-focal beam in both the X and Y directions can form a virtual light slice. Compared with a Gaussian light section in a traditional light section fluorescence microscope, the light section has weak diffusion along the direction of the illumination optical axis, has weak background light in a focusing range, and can image a fluorescence sample with lower background noise in a larger field range.
Description
Technical Field
The invention relates to the technical field of optical imaging, in particular to a multi-focus light section fluorescence microscopic imaging method and a device.
Background
The first optical microscope in the world emerges at the end of the sixteen century, which greatly expands the understanding of people on the microscopic world, and in the next centuries, the optical microscope is continuously improved, and the imaging speed, the resolution, the sensitivity and the like are greatly developed, thereby playing an increasingly important role in the field of biological imaging. With the rapid development of biotechnology, a deeper understanding of physiological processes is required, which also puts higher demands on 3D real-time imaging technology. However, the development of 3D real-time imaging techniques also faces many challenges — successful 3D real-time imaging techniques need to achieve high spatial resolution, high imaging speed, good photoslice capability, low photodamage, and photobleaching capability simultaneously.
There are many techniques for 3D real-time imaging such as wide-field microscopy, confocal microscopy, two-photon fluorescence microscopy, and light slice fluorescence microscopy. Among them, the fluorescence microscopic technique of optical section has been rapidly developed in these years due to its advantages of high speed, low photobleaching, and non-invasive imaging.
Because of the non-invasive property of the fluorescence microscopic technique of the optical section, the fluorescence microscopic technique has a very important position in the field of biological imaging. However, due to the diffusive nature of the illumination light slice, the field of view that the optical slice fluorescence microscopy can image is very limited, which limits its application to imaging large samples. In order to solve the problem, a field stitching technology and a light section fluorescence microscope are combined with each other, so that although the imaging range of the light section fluorescence microscope can be successfully expanded, an electric displacement platform needs to be added in a system, which increases the implementation cost and difficulty, and a sample needs to be stitched after being imaged for multiple times, which greatly reduces the imaging speed and cannot observe some fast physiological processes.
In comparison, the light beam is modulated and shaped, the shape of the light slice is changed, the diffusion of the light slice is slowed down, and the field of view can be expanded under the condition that the imaging speed is guaranteed. The application of the Bessel beam and the Airy beam in the fluorescence microscopy of the light section is the successful implementation of the scheme, but the light section formed by the two beams can generate more serious side lobes and cause serious background noise, and a related algorithm is required to be used for restoring the obtained image.
Disclosure of Invention
The invention aims to provide a multi-focus light section fluorescence microscopic imaging method, which can expand the field range of a light section fluorescence microscope without increasing background noise.
It is another object of the present invention to provide a light section fluorescence microscopy imaging device for implementing the above method, which can be used to implement the above method, wherein the incident light beam is phase modulated by a phase mask to generate a light beam having a plurality of focal points, and the distances between the focal points are substantially equal. Then, by galvanometer scanning, discrete optical slices are formed. Then, by adjusting the electrically driven variable focal concave lens, the focal point of the light beam is moved back and forth along the optical axis direction of the illumination objective lens, and by such movement, the light slices formed by the scanning of the galvanometer are connected with each other, forming a virtual light slice which is diffused more slowly along the direction of the illumination optical axis. The formed light slice has a larger field range, and the background light at the focused position is weaker, so that the background noise can be better eliminated when a sample is imaged.
In order to achieve the above object, the multi-focus optical section fluorescence microscopic imaging method provided by the invention comprises the following steps:
1) after phase modulation, laser forms a series of multi-focus beams with the same distance along the direction of an illumination optical axis;
2) scanning the light beam along the X-axis direction, and moving the light beam back and forth along the Y-axis direction to obtain a light slice with a certain imaging range along the Y direction;
3) and collecting fluorescence emitted by the fluorescence sample along the Z-axis direction to obtain a two-dimensional light intensity signal image of the sample at the axial position.
4) Moving the sample along the Z axis, repeating the step 3) to obtain a plurality of two-dimensional light intensity signal images, and performing three-dimensional reconstruction on the plurality of two-dimensional light intensity signal images to obtain three-dimensional imaging information of the fluorescent sample.
In the above technical solution, the X-axis direction is a direction perpendicular to the illumination optical axis and the detection optical axis, the Y-axis direction is a direction along the illumination optical axis, the Z-axis direction is a direction along the detection optical axis, and the three directions are perpendicular to each other two by two to form a three-dimensional rectangular coordinate system. In step 2), the light beam is scanned along the X-axis direction, so that the light beam forms discontinuous light slices. Through the steps 1) and 2), a virtual light slice is generated, compared with a light slice formed by a traditional Gaussian beam, the light slice is diffused more slowly along the direction of an illumination optical axis, and background light is weaker in a focusing range, so that an image with lower background noise can be obtained in a larger field range.
In step 1), in order to obtain a light beam with multiple focus points, it is necessary to convert laser light into radial polarized light, and then perform phase modulation on the radial polarized light.
Another specific scheme is that the phase modulation function used in step 1) is:
wherein, (r, theta) represents the polar coordinate of a certain point on the light beam, r is the normalized distance between the point and the optical axis, and theta is the included angle between the polar coordinate vector of the section of the light beam vertical to the optical axis and the laser emergent optical axis.
In another specific scheme, in the step 2), the light beam is moved back and forth, so that the focal point moves along the Y axis to form the light beam, and the moving range of the light beam is the distance between any two focal points of the multi-focal light beam.
In order to achieve the above another object, the present invention provides a multi-focus optical section fluorescence microscopic imaging apparatus, including an illumination system for forming an optical section, a sample stage for carrying a fluorescence sample, a detection system for detecting fluorescence emitted from the fluorescence sample, and a processor, wherein the illumination system includes, arranged in sequence along an optical path: a laser; a radial polarization converter for converting the laser beam into radial polarized light; the phase mask plate is used for modulating the phase of the radial polarized light and converting the radial polarized light into a multi-focus light beam; a uniaxial galvanometer for scanning the light beam irradiated on the fluorescent sample along the X-axis direction; and an electrically driven variable focal length concave lens that continuously changes a focal length; the processor is used for controlling the focal length of the electrically-driven variable-focus concave lens to continuously change, controlling the sample stage to move along the Z-axis direction in a fixed step length, and reconstructing a plurality of two-dimensional light intensity signal images collected by the detection system to obtain three-dimensional imaging information of the fluorescent sample.
In the above technical solution, after phase modulation, the laser beam forms a plurality of focusing points, and the focusing points scan along the X axis and move back and forth along the Y axis to form a virtual large-field optical slice. The fluorescence sample is excited by the light slice to generate fluorescence, and the fluorescence is collected by a detection system to obtain a two-dimensional image containing the information of the fluorescence sample on the XY plane. Then, the sample stage is controlled by the processor to move along the Z axis to obtain a plurality of two-dimensional images, and a three-dimensional imaging result of the fluorescent sample is obtained through reconstruction. Compared with the traditional light section fluorescence microscope, the light section generated by the device provided by the scheme is less prone to diffusion in the Y-axis direction, the background light in the focusing range is weaker, a larger view field can be generated, and the generated background noise is weaker.
Specifically, in order to obtain a light beam with a plurality of focuses, the modulation function on the phase mask plate used in the present invention is:
wherein, (r, theta) represents the polar coordinate of a certain point on the light beam, r is the normalized distance between the point and the optical axis, and theta is the included angle between the polar coordinate vector of the section of the light beam vertical to the optical axis and the laser emergent optical axis.
The other specific scheme is that the detection system comprises a detection objective lens, an optical filter, a tube lens and a CCD camera which are sequentially arranged along the Z-axis direction. The detection objective is used for collecting fluorescence excited by a fluorescence sample, the optical filter is used for filtering stray light, the tube lens is used for focusing the fluorescence on the CCD camera, and the CCD camera is used for recording a fluorescence signal on the light sensing surface and transmitting the fluorescence signal to the processor.
Another specific scheme is that a beam expanding lens for expanding laser beams emitted by the laser is arranged between the laser and the radial polarization converter; a first convex lens group used for adjusting the size of the laser beam is arranged between the phase mask plate and the single-shaft galvanometer; a second convex lens group and a lighting objective lens are sequentially arranged between the electrically-driven variable-focus concave lens and the sample stage.
In another specific embodiment, the focal length of the electrically driven variable focal length concave lens varies over the distance between any two focal points of the multi-focal beam. Through the change of the focal length, the light beam moves back and forth in the Y-axis direction, and a light slice with a wider field range is obtained. The electrically driven variable focus concave lens is controlled by a processor to achieve continuous variation of focal length.
The principle of the invention is as follows:
in the traditional light slice fluorescence microscopic technology, a common Gaussian beam is used to form a light slice through the focusing of a cylindrical mirror or the scanning of a vibrating mirror, the light slice formed in the mode can diffuse quickly in the Y-axis direction, only a small part of the focused light slice can effectively excite the fluorescence of a sample, and the field range is very limited.
In the invention, a multi-focus light beam is formed by modulating the phase mask plate, then the light beam moves back and forth in the Y-axis direction by electrically driving the focal length change of the focal length variable concave lens, and in the process of quick movement, a plurality of focal points are connected with each other to form a light slice with a larger usable range in the Y-axis direction. The light section formed by the method can be well focused in a long distance, the field range of the light section fluorescence microscope is greatly expanded, and the well focused light section can well inhibit background noise.
Compared with the prior art, the invention has the beneficial effects that:
the invention greatly improves the field range of the fluorescence microscopic technology of the light section and reduces the background noise of imaging.
Drawings
FIG. 1 is a schematic structural diagram of a light section fluorescence microscopic imaging device according to an embodiment of the present invention;
fig. 2 is a schematic diagram of phase distribution of a phase mask according to an embodiment of the present invention;
fig. 3 is a light intensity distribution of a multi-focus light beam formed after a laser beam passes through a phase mask plate on a YZ plane according to an embodiment of the present invention;
FIG. 4(a) is a diagram of the distribution of light intensity of the light slices on the YZ plane, which is finally obtained by the embodiment of the present invention; (b) the light intensity distribution diagram of the traditional Gaussian light slice on the YZ plane is shown;
FIG. 5 is a graph comparing the point spread function of the system with the Y-axis normalized intensity distribution curve of the conventional light section fluorescence microscopy technique according to the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described with reference to the following embodiments and accompanying drawings.
Device embodiment
Referring to fig. 1, the multi-focus light-slice fluorescence microscopic imaging device of the present embodiment includes a laser 1, a beam expanding lens 2, a radial polarization converter 3, a phase mask 4, a first convex lens group (convex lens 5, convex lens 6), a uniaxial galvanometer 7, an electrically-driven variable-focus concave lens 8, a second convex lens group (convex lens 9, convex lens 10), an illumination objective lens 11, a sample stage 12, a detection objective lens 13, an optical filter 14, a tube lens 15, a CCD camera 16, and a computer 17.
The device embodiment of the invention can be divided into four parts: an illumination system for producing an illuminated light slice, a sample stage 12 for carrying a sample, a detection system for detecting fluorescence emitted by the sample, and a processor, which in this embodiment is a computer 17.
Wherein, lighting system includes that follow the light path and arrange in proper order: the device comprises a laser 1, a beam expanding lens 2, a radial polarization converter 3, a phase mask plate 4, a convex lens 5, a convex lens 6, a single-axis vibrating mirror 7, an electrically-driven variable focus concave lens 8, a convex lens 9, a convex lens 10 and an illumination objective lens 11.
The laser 1 emits laser light; the beam expanding lens 2 expands the beam and collimates the laser; the radial polarization converter 3 converts the incident light beam into a radially polarized light beam; the phase mask 4 phase-modulates the radially polarized light beam to form a light beam with a plurality of focal points, the phase distribution on the phase mask 4 is shown in fig. 2, and its modulation function is:
wherein, (r, theta) represents the polar coordinate of a certain point on the light beam, r is the normalized distance between the point and the optical axis, and theta is the included angle between the polar coordinate vector of the section of the light beam vertical to the optical axis and the laser emergent optical axis.
The convex lens 5 and the convex lens 6 adjust the size of the light beam; the uniaxial galvanometer 7 reflects the light beam, and the light beam can be scanned along the X-axis direction at the sample through rotation; the electrically driven variable focal length concave lens 8 is controlled by the computer 17, and the focal length is changed, so that the light beam irradiated on the sample moves back and forth along the Y-axis direction; the illumination objective 11 projects an excitation beam onto the fluorescent sample.
The sample stage 12 carrying the sample can be moved by the computer 17 in fixed steps along the Z-axis direction.
The detection system comprises the following components arranged in sequence along the Z-axis direction: a detection objective 13, a filter 14, a tube lens 15 and a CCD camera 16.
The detection objective lens 13 is used for collecting fluorescence emitted by the fluorescence sample excited by the laser; the optical filter 14 is used for filtering the collected stray light; the tube lens 15 is used for collecting the collected fluorescence onto the CCD camera 16; the CCD camera 16 is used to record the fluorescence signal and transmit the signal to the computer 17.
The computer 17 reconstructs a plurality of two-dimensional fluorescence signals recorded by the CCD camera 16 on one hand, and reconstructs an imaging result of a three-dimensional fluorescence sample; on the other hand, the sample stage 12 carrying the fluorescent sample is controlled to move along the Z axis in a fixed step length, and on the other hand, the electrically-driven variable-focus concave lens 8 is controlled to continuously change the focal length of the sample stage, wherein the change range is the distance between two focuses in the obtained multi-focus light beam.
The process of three-dimensional imaging of the fluorescent sample by using the device is as follows:
laser beams emitted from the laser 1 are expanded and collimated by the beam expanding lens 2, then converted into radial polarized light by the radial polarization converter 3, and then phase-modulated by the phase mask 4, wherein the phase distribution on the phase mask 4 is as shown in fig. 2, and the beams are changed into multi-focus focused beams after the phase modulation.
The multifocal light beam is adjusted in size by the convex lens 5 and the convex lens 6, then irradiates the uniaxial galvanometer 7, and is reflected by the uniaxial galvanometer 7. The reflected light beam is scattered by the electrically driven variable-focus concave lens 8, then is focused and expanded by the convex lens 9 and the convex lens 10, and is projected onto a fluorescent sample (placed on a sample stage 12 for bearing the fluorescent sample) by an illumination objective lens 11.
The light intensity distribution of the multifocal excitation beam on the YZ section is shown in fig. 3, and the multifocal excitation beam is scanned on the X axis and the Y axis respectively through the rotation of the uniaxial galvanometer 7 and the focal length conversion of the electrically-driven variable-focus concave lens 8, so as to obtain a virtual large-field light slice, and the light intensity distribution of the light slice on the YZ section is shown in fig. 4 (a).
The fluorescence sample is excited by the light section to emit fluorescence, is collected by a detection objective lens 13, is filtered by an optical filter 14, is focused on a CCD camera 16 by a tube lens 15, and the CCD camera 16 transmits the recorded two-dimensional fluorescence signal to a computer 17.
The computer 17 controls the sample stage 12 carrying the fluorescent sample to move in fixed steps along the Z-axis direction, and an image with two-dimensional signals of the fluorescent sample is obtained at each axial position. And reconstructing the plurality of images to obtain a three-dimensional imaging result of the sample.
To verify the enlargement of the field of view of the fluorescence microscopy of light section by the method used in this example, the light intensity distributions on YZ cross-section of the light section obtained in this example were compared with those of the conventional gaussian light section, as shown in fig. 4, wherein (a) is the light section obtained by the present invention, and (b) is the conventional gaussian light section, and the light section obtained by the present invention was far slower in diffusion in the Y-axis direction by comparison.
By comparing the curves of the normalized light intensity distribution in the Y-axis direction in fig. 5, it can be seen that the method used in the present invention greatly expands the light slice fluorescence microscopy method, and the present invention expands the field of view of the light slice fluorescence microscopy technique eight times by full width at half maximum (FWHM) calculation in the curves.
Method embodiment
The multi-focus light section fluorescence microscopic imaging method of the embodiment is realized based on the light section fluorescence microscopic imaging device in the device embodiment, and comprises the following steps:
1) after phase modulation, laser forms a series of multi-focus beams with the same distance along the direction of an illumination optical axis;
2) scanning the light beam along the X-axis direction, and moving the light beam back and forth along the Y-axis direction to obtain a light slice with a certain imaging range along the Y direction;
3) and collecting fluorescence emitted by the fluorescence sample along the Z-axis direction to obtain a two-dimensional light intensity signal image of the sample at the axial position.
4) Moving the sample along the Z axis, repeating the step 3) to obtain a plurality of two-dimensional light intensity signal images, and performing three-dimensional reconstruction on the plurality of two-dimensional light intensity signal images to obtain three-dimensional imaging information of the fluorescent sample.
In the present embodiment, the X-axis direction is a direction perpendicular to the illumination optical axis and the detection optical axis, the Y-axis direction is a direction along the illumination optical axis, and the Z-axis direction is a direction along the detection optical axis. The three directions are mutually vertical pairwise to form a three-dimensional rectangular coordinate system.
The modulation function of the phase modulation in step 1) is as follows,
and (r, theta) represents the polar coordinate of a certain point on the light beam, r is the normalized distance between the point and the optical axis, and theta is the included angle between the polar coordinate vector of the section of the light beam vertical to the optical axis and the laser emergent optical axis.
Claims (9)
1. A multi-focus light section fluorescence microscopic imaging method is characterized by comprising the following steps:
1) after phase modulation, laser forms a series of multi-focus beams with the same distance along the direction of an illumination optical axis;
2) scanning the light beam along the X-axis direction, and moving the light beam back and forth along the Y-axis direction to obtain a light slice with a certain imaging range along the Y direction;
3) and collecting fluorescence emitted by the fluorescence sample along the Z-axis direction to obtain a two-dimensional light intensity signal image of the sample at the axial position.
4) Moving the sample along the Z axis, repeating the step 3) to obtain a plurality of two-dimensional light intensity signal images, and performing three-dimensional reconstruction on the plurality of two-dimensional light intensity signal images to obtain three-dimensional imaging information of the fluorescent sample; the X-axis direction is a direction perpendicular to the illumination optical axis and the detection optical axis, the Y-axis direction is a direction along the illumination optical axis, and the Z-axis direction is a direction along the detection optical axis.
2. The multi-focal light-slice fluorescence microscopy imaging method of claim 1, characterized in that:
in the step 1), before phase modulation is performed on the laser, the laser is converted into radial polarized light, and then phase modulation is performed on the radial polarized light.
3. The multi-focal light-slice fluorescence microscopy imaging method of claim 1, characterized in that:
the modulation function of the phase modulation in step 1) is:
wherein, (r, theta) represents the polar coordinate of a certain point on the light beam, r is the normalized distance between the point and the optical axis, and theta is the included angle between the polar coordinate vector of the section of the light beam vertical to the optical axis and the laser emergent optical axis.
4. The multi-focal light-slice fluorescence microscopy imaging method of claim 1, characterized in that:
in the step 2), the light beam is moved back and forth, so that the focal point moves along the Y axis to form light rays, and the moving range of the light beam is the distance between any two focal points of the multi-focus light beam.
5. The utility model provides a many focuses light section fluorescence microscopic imaging device, includes the lighting system that forms the light section, bears the sample platform of fluorescence sample, detects fluorescence sample and sends fluorescence's detecting system and a treater, its characterized in that:
the illumination system comprises, arranged in sequence along an optical path: a laser; a radial polarization converter for converting the laser beam into radial polarized light; the phase mask plate is used for modulating the phase of the radial polarized light and converting the radial polarized light into a multi-focus light beam; a uniaxial galvanometer for scanning the light beam irradiated on the fluorescent sample along the X-axis direction; and an electrically driven variable focal length concave lens that continuously changes a focal length; after phase modulation, laser forms a series of multi-focus beams with the same distance along the direction of an illumination optical axis;
the processor is used for controlling the focal length of the electrically-driven variable-focus concave lens to continuously change, controlling the sample stage to move along the Z-axis direction in a fixed step length, and reconstructing a plurality of two-dimensional light intensity signal images collected by the detection system to obtain three-dimensional imaging information of the fluorescent sample.
6. The multi-focal light-slice fluorescence microscopy imaging device of claim 5, wherein:
the modulation function of the phase mask plate for modulating the phase of the radial polarized light is as follows:
wherein, (r, theta) represents the polar coordinate of a certain point on the light beam, r is the normalized distance between the point and the optical axis, and theta is the included angle between the polar coordinate vector of the section of the light beam vertical to the optical axis and the laser emergent optical axis.
7. The multi-focal light-slice fluorescence microscopy imaging device of claim 5, wherein:
the detection system comprises a detection objective lens, an optical filter, a tube lens and a CCD camera which are sequentially arranged along the Z-axis direction.
8. The multi-focal light-slice fluorescence microscopy imaging device of claim 5, wherein:
a beam expanding lens for expanding the laser beam emitted by the laser is arranged between the laser and the radial polarization converter; a first convex lens group used for adjusting the size of the laser beam is arranged between the phase mask plate and the single-shaft galvanometer;
and a second convex lens group and a lighting objective lens for focusing and expanding the laser beams are sequentially arranged between the electrically-driven variable-focus concave lens and the sample stage.
9. The multi-focal light-slice fluorescence microscopy imaging device of claim 5, wherein:
the focal length variation range of the electrically-driven variable focus concave lens is the distance between any two focuses of the multi-focus light beam.
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