CN210243498U - Inclined type layered light excitation microscopic imaging device and layered light excitation illuminator - Google Patents
Inclined type layered light excitation microscopic imaging device and layered light excitation illuminator Download PDFInfo
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- CN210243498U CN210243498U CN201920932013.0U CN201920932013U CN210243498U CN 210243498 U CN210243498 U CN 210243498U CN 201920932013 U CN201920932013 U CN 201920932013U CN 210243498 U CN210243498 U CN 210243498U
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Abstract
The utility model provides a micro-image device of tilting stratiform light excitation and stratiform light excitation illuminator belongs to micro-imaging technical field. The layered light exciting illuminator includes one illumination objective with one small aperture diaphragm in the back focal plane and with optical axis deviated 0-1 cm from that of the cylindrical lens, the scanning lens and the tube lens. The utility model provides a micro-imaging device is aroused to tilting stratiform light includes the light source, and stratiform light arouses the illuminator, and the meticulous translation platform of two dimension, sample translation platform receive objective and camera, and the device can realize the high flux fluorescence data acquisition under the stratiform light illumination, tracks the unimolecular process in the cell.
Description
Technical Field
The utility model relates to a microscopic imaging technique, concretely relates to inclined lamellar light excites microscopic imaging device and lamellar light excites illuminator.
Background
Fluorescence imaging methods are powerful weapons for dynamic observation of living cell single molecule signaling, and have been widely used in the study of intracellular single molecule behavior. However, for single molecule imaging deep in the cell, the fluorescence background is high due to the lack of background removal methods like total internal reflection illumination, which hinders accurate spatial localization of single molecules. In order to solve the problem of low signal-to-back ratio (SBR) of deep cell imaging in the wide-field epi-illumination mode, a large number of layered light technologies are gradually developed, and the imaging scheme for cells can generally reach an illumination thickness below 2 micrometers and an imaging field of view of about 10 micrometers. However, the prior art has more or less inconvenience in use and operation in the practical application of biological research, and the fluorescence imaging instrument also has the defects of complex operation, high cost and difficult maintenance, needs a specially trained person to perform shooting operation, and is difficult to realize high-throughput data acquisition.
SUMMERY OF THE UTILITY MODEL
To the problem that exists among the above prior art, the utility model provides a tilting stratiform light excites microscopic imaging device and stratiform light excites illuminator, is particularly suitable for the formation of image of high flux cell nuclear in-process.
The utility model provides a micro-imaging device of tilting stratiform light excitation includes: the device comprises a light source, a layered light excitation illuminator, a two-dimensional fine translation stage, a sample translation stage, a receiving objective and a camera;
the light source adopts a Gaussian beam with a certain width, the Gaussian beam can be expanded by a laser or collimated by an optical fiber emergent laser, and the light source generally comprises a plurality of collimated lasers with different wavelengths;
the layered light exciting illuminator structure one may include a cylindrical lens, an aperture stop, and an illumination objective lens. The space distance between the cylindrical mirror and the small aperture diaphragm is the focal length of the cylindrical mirror, the small aperture diaphragm is positioned at the back focal plane of the illumination objective lens, and the optical axis of the illumination objective lens deviates 0-1 cm relative to the optical axis of the cylindrical mirror;
the layered light excitation illuminator structure II can comprise a one-dimensional scanning galvanometer, a scanning lens, a tube lens, an aperture diaphragm and an illumination objective lens, wherein the space distance between the scanning galvanometer and the scanning lens is the focal length of the scanning lens, the space distance between the scanning lens and the tube lens is the sum of the focal lengths of the scanning lens and the tube lens, the aperture diaphragm is positioned at the back focal plane of the illumination objective lens, and the optical axis of the illumination objective lens deviates 0-1 cm relative to the optical axes of the scanning lens and the tube lens;
the layered light excitation illuminator is integrally positioned on the two-dimensional fine translation stage, and the included angle of the optical axis of the illumination objective relative to the sample translation stage is 0-90 degrees; the layered light exiting the illumination objective is focused to a depth on the sample, exciting the fluorescence of the sample at that depth, which is received by the receiving objective and transmitted to the camera to form an image.
The sample translation stage has the capability of fine adjustment along the optical axis direction of the receiving objective lens (the movement precision is higher than 10 nanometers), and is generally realized by adopting a single-axis piezoelectric ceramic displacement stage or a three-dimensional piezoelectric ceramic displacement stage (the position of a layered light excitation sample can be changed by controlling the sample translation stage to move along the optical axis of the receiving objective lens so as to realize three-dimensional scanning imaging of the sample, and a computer is used for reconstructing the acquired image so as to realize three-dimensional microscopic imaging).
The technical effects of the utility model:
the utility model provides a be equipped with illumination objective in two kinds of stratiform light excitation illuminator structures, behind the focus of gauss light beam formation stratiform light, because illumination objective's optical axis is skew 0-1 cm for the optical axis of cylindrical mirror or scanning lens and tube mirror, the direction of propagation that realizes the stratiform light is certain contained angle with the receipt objective optical axis to the stratiform light excitation micro-imaging device of tilting has been obtained. The inclined lamellar light excitation microscopic imaging device can excite the visual field to be between 5 and 16 micrometers, so that the signal background ratio is improved by orders of magnitude, and the inclined lamellar light excitation microscopic imaging device is compatible with the receiving objective lens with the highest fluorescence collection efficiency at present. The utility model allows the user to conveniently and rapidly find and shoot the single molecule dynamic process in the single cell depth, and realizes the high flux fluorescence data acquisition under the laminar light illumination; in addition to this, the present invention enables tracking of single molecule processes in cells at specific locations in a tissue or cell mass, with the benefit of the long working distance of the illumination objective.
Drawings
Fig. 1 is a schematic diagram of an optical path of a layered light formed by the focus of a gaussian beam according to a first embodiment of the present invention; wherein (a) is a main view plane; (b) is a top view;
fig. 2 is a schematic view of a tilted layered light-excited microscopic imaging device according to a first embodiment of the present invention;
fig. 3 is a schematic view of the gaussian beam focusing to form a layered light according to the second embodiment of the present invention; wherein (a) is a main view plane; (b) is a top view;
fig. 4 is a schematic view of a tilted layered light-excited microscopic imaging device according to a second embodiment of the present invention;
wherein, 1 is a small hole diaphragm; 2-an illumination objective lens; 3-a two-dimensional fine translation stage; 4-a sample translation stage; 5-receiving objective lens; 6-a camera; 7-cylindrical mirror; 8, a tube mirror; 9-a scanning lens; 10-scanning galvanometer.
Detailed Description
The present invention will be further explained with reference to the accompanying drawings.
Example one
As shown in fig. 1, the layered light excitation illuminator of the present invention includes a cylindrical mirror, an aperture stop, and an illumination objective. The space distance between the cylindrical mirror and the small aperture diaphragm is the focal length of the cylindrical mirror, and the small aperture diaphragm is positioned at the back focal plane of the illumination objective lens. The optical axis of the illumination objective is offset by a certain distance (in an embodiment it may be 5 mm) with respect to the optical axis of the cylindrical mirror.
As shown in fig. 2, the tilted layered light-excited microscopic imaging apparatus of the present embodiment includes: the device comprises a light source, a layered light excitation illuminator, a two-dimensional fine translation stage, a sample translation stage, a receiving objective and a camera.
Wherein the light source employs a gaussian beam having a width, and in the exemplary system a gaussian beam having a diameter of 1.0 cm is employed. The Gaussian beam can be expanded by a laser or collimated by emergent laser of an optical fiber. The light source typically comprises a plurality of collimated laser beams of different wavelengths.
The sample translation stage has the ability of fine adjustment along the optical axis direction of the receiving objective lens (the movement precision is higher than 10 nanometers), and is generally realized by adopting a single-axis piezoelectric ceramic displacement stage or a three-dimensional piezoelectric ceramic displacement stage (the position of a layered light excitation sample can be changed by controlling the sample translation stage to move along the optical axis of the receiving objective lens, so that the three-dimensional scanning imaging of the sample is realized, and the acquired image is reconstructed by using a computer, so that the three-dimensional microscopic imaging is realized).
Wherein, the receiving objective adopts a high numerical aperture objective, and the embodiment adopts a Nikon 100 times 1.49 numerical aperture objective.
Wherein, the camera adopts electron multiplication CCD or sCMOS to increase the collection sensitivity, and the embodiment adopts DU-897EMCCD of Andor company.
The included angle between the receiving objective and the illuminating objective is not 0 or 90 degrees; adjusting the propagation direction of the layered light to deviate from the distance between the optical axes of the illumination objective lenses by moving the illumination objective lenses, adjusting the inclination between the propagation direction of the layered light emitted from the illumination objective lenses and the optical axes of the illumination objective lenses so that the plane in which the layered light is located is perpendicular to the optical axes of the reception objective lenses, thereby generating fluorescence along the optical axes of the reception objective lenses; controlling the one-dimensional sample translation stage to move along the optical axis of the receiving objective lens, so that the depth of the layered light incident into the sample is changed, and the depth of the sample is scanned; the imaging unit processes the image, thereby realizing three-dimensional microscopic imaging.
Example two
The layered light excitation illuminator of the utility model is shown in figure 3, comprising a one-dimensional scanning galvanometer, a scanning lens, a tube lens, a small-hole diaphragm and an illumination objective. The space distance between the scanning galvanometer and the scanning lens is the focal length of the scanning lens, the space distance between the scanning lens and the tube lens is the sum of the focal lengths of the scanning galvanometer and the scanning galvanometer, and the aperture diaphragm is positioned on the back focal plane of the illumination objective lens. The optical axis of the illumination objective is offset by a certain distance (in an embodiment 5 mm) with respect to the optical axis of the scanning lens and the tube lens.
The layered light excitation illuminator is integrally positioned on the two-dimensional fine translation stage, the optical axis of the illumination objective lens has a certain included angle relative to the sample translation stage, the angle is determined by the type of the illumination objective lens, in the embodiment, a water immersion objective lens with the Nikon 0.8 numerical aperture working distance of 3.5 mm is adopted, and the included angle is 45 degrees.
Wherein the moving precision of the two-dimensional fine translation stage is higher than 10 microns. And adjusting the plane excited by the layered light to coincide with the focal plane of the receiving objective lens by adjusting the two-dimensional fine translation stage, and adjusting the area excited by the layered light to be in the central area of the imaging field of the receiving objective lens.
As shown in fig. 4, in the whole system, the gaussian beam enters the central point of the scanning galvanometer, and is reflected by the scanning galvanometer, the reflected light enters along the optical axes of the scanning lens and the tube mirror, and the scanning galvanometer scans the gaussian beam to form a light plane. Subsequently, through aperture screening of the aperture stop, light beams within the aperture range enter the illumination objective lens. Because the optical axis of the illumination objective lens is translated for a certain distance relative to the optical axis of the cylindrical lens, the light beam emitted from the illumination objective lens deflects for a certain angle relative to the illumination objective lens, and the light beam passes through the illumination objective lens to form layered light illumination near the front focal length of the light beam.
Finally, it is noted that the disclosed embodiments are intended to aid in further understanding of the invention, but will be understood by those skilled in the art that: various substitutions and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the present invention should not be limited to the embodiments disclosed, and the scope of the present invention is defined by the appended claims.
Claims (8)
1. A layered light excitation illuminator comprises a cylindrical lens, and is characterized in that the layered light excitation illuminator is provided with an illumination objective lens, an aperture diaphragm is arranged at the back focal plane of the illumination objective lens, the space distance between the cylindrical lens and the aperture diaphragm is the focal length of the cylindrical lens, and the optical axis of the illumination objective lens deviates 0-1 cm from the optical axis of the cylindrical lens.
2. A layered light excitation illuminator comprises a one-dimensional scanning galvanometer, a scanning lens and a tube lens, wherein the space distance between the scanning galvanometer and the scanning lens is the focal length of the scanning lens, and the space distance between the scanning lens and the tube lens is the sum of the focal lengths of the scanning lens and the tube lens.
3. An inclined lamellar light excitation microimaging device, which is characterized by comprising a light source, a lamellar light excitation illuminator according to claim 1 or 2, a two-dimensional fine translation stage, a sample translation stage, a receiving objective and a camera, wherein the light source is a Gaussian beam with a certain width, the lamellar light excitation illuminator is integrally positioned on the two-dimensional fine translation stage, the included angle of the optical axis of the illuminating objective relative to the sample translation stage is 0-90 degrees, the plane excited by the lamellar light is adjusted to be coincident with the focal plane of the receiving objective by adjusting the two-dimensional fine translation stage, the area excited by the lamellar light is adjusted in the central area of the imaging visual field of the receiving objective, the lamellar light emitted from the illuminating objective is focused to a depth of a sample, the fluorescence of the sample at the depth is excited, and the fluorescence is received by the receiving objective and then transmitted to the camera to form an.
4. The oblique layered light-excited microscopic imaging device according to claim 3, wherein the light source comprises a plurality of collimated laser beams with different wavelengths, and the Gaussian beam is realized by expanding the laser beam or collimating the emergent laser beam of the optical fiber.
5. The oblique layered light-excited microscopic imaging device according to claim 3, wherein the sample translation stage is a uniaxial piezoelectric ceramic displacement stage or a three-dimensional piezoelectric ceramic displacement stage.
6. The tilted layered light-excited microscopic imaging apparatus as claimed in claim 3, wherein the receiving objective lens is a high numerical aperture objective lens.
7. The oblique layered light-excited microscopic imaging apparatus as claimed in claim 3, wherein the camera employs an electron-multiplying CCD or sCMOS to increase the collection sensitivity.
8. The oblique layered light-excited microscopic imaging apparatus as claimed in claim 3, wherein the precision of the movement of the two-dimensional fine translation stage is higher than 10 μm.
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| CN201920932013.0U CN210243498U (en) | 2019-06-20 | 2019-06-20 | Inclined type layered light excitation microscopic imaging device and layered light excitation illuminator |
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| CN201920932013.0U CN210243498U (en) | 2019-06-20 | 2019-06-20 | Inclined type layered light excitation microscopic imaging device and layered light excitation illuminator |
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