A four-dimensional detection system for staring confocal microscopy morphology and spectrum

Abstract

The present invention discloses a staring type confocal microscopic morphology spectrum four-dimensional detection system, comprising an excitation unit, a front optical unit, a microscopic imaging unit, a middle scanning unit, and a rear receiving unit; the excitation light of the sample to be tested is introduced from the excitation unit and incident on the middle scanning unit; the galvanometer system of the middle scanning unit performs two-dimensional scanning, and the emitted excitation light is incident on the microscope objective lens after being reflected by a beam splitter, and focuses on the sample to be tested; the reflected light forms a conjugate microscopic image after imaging; after the signal fluorescence or reflected light returns, one path is finally emitted to an external photodetector, and the other path of signal fluorescence or reflected light is incident on a hyperspectral spectroscopic module to form a fluorescence spectrum or a reflection spectrum. The system fuses three-dimensional microscopic morphology and fluorescence or reflection spectrum data into four-dimensional spectral morphology microscopic data, has extremely high spectral resolution and depth resolution, and has great application value in measurement fields such as in-situ microbial detection and industrial sample microstructure detection.

Description

Technical Field

[0001] The invention belongs to the field of optical technology, and in particular relates to a staring type confocal microscopic morphology spectrum four-dimensional detection system. Background Art

[0002] At present, hyperspectral imaging is based on multi-channel spectral technology, integrating optical imaging and spectral measurement, and can simultaneously obtain the image information and corresponding spectral information of the target. Hyperspectral imaging can analyze, measure and process the structure and composition of substances, and has the advantages of high analysis accuracy and wide measurement range. It is widely used in the fields of petroleum, materials, agronomy, geological exploration, biochemistry, medicine and health, environmental protection, safety testing, etc. However, traditional hyperspectral imaging technology can only obtain the spectral information of the object to be tested, but cannot obtain the three-dimensional morphological information of the object to be tested, and cannot restore the true four-dimensional information of objects with morphological and chromatic characteristics. Traditional three-dimensional reconstruction technology cannot collect the spectral information of objects. The microscope system can magnify and observe the detailed image information of the sample to be tested, and is an important tool for detecting the microscopic properties of the sample. How to realize the integrated collection of the microscopic three-dimensional morphology and spectral information of the sample to be tested is a difficulty in the current industry. In addition, when hyperspectral imaging technology is used for microscopic detection, most commercial hyperspectral imagers can only work in a single detection mode, which is difficult to use for multi-mode detection of samples with different characteristics. Summary of the invention

[0003] In order to overcome the problems in the prior art, the present invention discloses a staring-type confocal microscopic morphology spectrum four-dimensional detection system.

[0004] A staring confocal microscopic morphology spectrum four-dimensional detection system comprises an excitation unit, a front optical unit, a microscopic imaging unit, a middle scanning unit, and a rear receiving unit; the excitation unit comprises a reflector and an optical fiber collimator, the excitation light of the sample to be tested is introduced from the excitation unit, and is incident on the middle scanning unit after being adjusted by the reflector; the middle scanning unit comprises a telecentric lens, a scanning lens, a galvanometer system, a first beam splitter, and a filter in sequence, the scanning lens and the telecentric lens optimize the light beam and expand the beam, and the galvanometer system performs two-dimensional scanning; the front optical unit comprises a stage, a microscope objective lens, a displacement stage, and a beam splitter, the excitation light emitted from the middle scanning unit is incident on the microscope objective lens after being reflected by the beam splitter, and is focused on the sample to be tested on the stage, and the displacement stage is used to The distance to the sample to be tested is adjusted to achieve focusing, and the fluorescence signal or reflected light signal excited by the sample to be tested returns along the original optical path; the microscopic imaging unit includes a fourth focusing lens and a first camera, and the light reflected from the sample to be tested is imaged at the position of the first camera through the fourth focusing lens to form a conjugate microscopic image; the rear-end receiving unit includes a second beam splitter, a first focusing lens, a pinhole, a second focusing lens, and a hyperspectral spectroscopic module, and the signal fluorescence or reflected light passes through the middle scanning unit along the original optical path and is incident on the second beam splitter in the rear-end receiving unit, is focused at the pinhole position through the first focusing lens, and is emitted to the external photodetector, and another path of signal fluorescence or reflected light passes through the second focusing lens and is incident on the hyperspectral spectroscopic module to form a fluorescence spectrum or a reflection spectrum.

[0005] The hyperspectral spectrometer module includes a slit, a collimating lens, a prism-grating-prism, a third focusing lens, and a second camera in sequence. Light emitted from the middle scanning unit to the slit position is collimated into parallel light by the collimating lens. After passing through the prism-grating-prism, light of different wavelengths is focused on different positions of the photosensitive surface of the second camera by the third focusing lens, thereby forming a spectral image.

[0006] The image plane of the microscopic imaging unit is conjugated with the image plane of the fluorescence spectrum or the reflection spectrum.

[0007] The translation stage in the front optical unit is replaced with a liquid zoom lens to achieve rapid zooming and improve the scanning speed of the system.

[0008] Beneficial effects of the present invention:

[0009] The confocal probe principle is used to detect the three-dimensional morphological information and fluorescence or reflection spectrum information of the sample to be tested. The three-dimensional morphological data and fluorescence or reflection spectrum data of the sample to be tested are fused into a four-dimensional data cube. The system has the characteristics of high depth resolution, high spectral resolution, and excellent data fusion accuracy. The new spectral morphological data body greatly improves the accuracy of microscopic detection and analysis of materials.

[0010] The present invention takes into account the lack of three-dimensional morphology restoration and the single detection mode of hyperspectral imaging under microscopic detection, and uses the confocal probe principle to collect three-dimensional morphology data and fluorescence or reflection spectrum data of the sample to be tested. The three-dimensional morphology data and fluorescence or reflection spectrum data of the sample to be tested are fused into a four-dimensional spectral morphology data set, which increases the means and methods of the system for object detection, greatly improving the availability and richness of the system. This is of great significance for analyzing the microscopic properties of the sample to be tested, and has great application value in measurement fields such as in-situ microbial detection and industrial sample microstructure detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a structural schematic diagram of a staring confocal microscopic morphology spectrum four-dimensional detection system;

[0012] In the figure, the stage 1, the microscope objective 2, the translation stage 3, the beam splitter 4, the telecentric lens 5, the scanning lens 6, the galvanometer system 7, the first beam splitter 8, the filter 9, the second beam splitter 10, the first focusing lens 11, the pinhole 12, the second focusing lens 13, the hyperspectral spectroscopic module 14, the slit 15, the collimating lens 16, the prism-grating-prism 17, the third focusing lens 18, the reflector 19, the fiber collimator 20, the fourth focusing lens 21, the first camera 22, and the second camera 23. DETAILED DESCRIPTION

[0013] The present invention will be further described below in conjunction with the accompanying drawings.

[0014] A staring confocal microscopic morphology spectrum four-dimensional detection system includes an excitation unit, a front optical unit, a microscopic imaging unit, a middle scanning unit and a rear receiving unit. Based on the confocal probe principle, the system can simultaneously collect three-dimensional morphology data and fluorescence or reflection spectrum data of the sample to be tested. The three-dimensional morphology data and fluorescence or reflection spectrum data of the sample to be tested are fused into a four-dimensional spectral morphology data set. The excitation light is introduced from the excitation unit to the middle scanning unit, and the galvanometer system realizes scanning by changing the angle of incidence to the scanning lens. The displacement stage adjusts the distance between the microscope objective and the sample to be tested so that the sample is placed on the focal plane of the microscope objective. The point on the sample to be tested is excited to fluoresce or the point generates a reflected light signal. The excitation light can be focused at different depths of the sample to be tested, and only the fluorescence signal on the focal plane of the microscope objective in the front optical unit can be collected by the rear receiving unit, and the fluorescence signal of the non-focal plane will be blocked by the confocal pinhole in the rear receiving unit. The sample to be tested can realize fluorescence spectrum imaging at different depths through tomographic scanning.

[0015] Confocal microscopic morphology detection is to continuously change the distance between the displacement stage of the front optical unit and the sample to be tested, and locate the axial distance between the sample to be tested and the microscope objective lens by collecting the maximum value of the focused light intensity signal in the rear receiving unit, so as to accurately obtain the precise three-dimensional position data of the surface point of the sample to be tested. Then, the fluorescence or reflection spectrum data is collected through the hyperspectral spectrometer module of the rear receiving unit; finally, the two-dimensional points of the sample to be tested are scanned by the galvanometer system to obtain the three-dimensional morphology of the sample to be tested and the hyperspectral fluorescence or reflection spectrum of each spatial point.

[0016] like Figure 1 As shown, the excitation unit includes a reflector 19 and a fiber collimator 20. The excitation light of the sample to be tested is introduced from the excitation unit and incident on the middle scanning unit after being adjusted by the reflector 19. The system can scan the three-dimensional morphology of the sample to be tested through a confocal probe. The optical properties of the sample to be tested can be characterized by collecting fluorescence data or reflection spectrum data.

[0017] The middle scanning unit includes a telecentric lens 5, a scanning lens 6, a galvanometer system 7, a first beam splitter 8, and a filter 9 in sequence. The scanning lens 6 and the telecentric lens 5 optimize the light beam and expand the beam, and the galvanometer system 7 performs two-dimensional scanning. The galvanometer system 7 realizes scanning by changing the angle of incidence to the scanning lens 6. The scanning lens 6 and the telecentric lens 5 optimize the scanning image plane and increase the spot diameter, so that the scanning beams of different angles are always converged on the entrance pupil of the micro-objective lens through the reflector (beam splitter) of the front optical unit. The laser is emitted from the middle scanning unit to the front optical unit.

[0018] The front optical unit includes a stage 1, a microscope objective lens 2, a displacement stage 3, and a beam splitter 4. The excitation light emitted from the middle scanning unit is incident on the microscope objective lens 2 through reflection from the beam splitter 4, and is focused on the sample to be tested on the stage 1. The displacement stage 3 is used to adjust the distance of the sample to be tested to achieve focusing, and the fluorescent signal or reflected light signal excited by the sample to be tested returns along the original light path. The displacement stage 3 in the front optical unit can be replaced by a liquid zoom lens to achieve fast zooming and improve the scanning speed of the system.

[0019] The microscopic imaging unit includes a fourth focusing lens 21 and a first camera 22. The light reflected from the sample to be tested is imaged at the position of the first camera 22 through the fourth focusing lens 21 to form a conjugate microscopic image. The image plane of the microscopic imaging unit is conjugate with the image plane of the fluorescence spectrum or the reflection spectrum. A color microscopic image can be observed in the microscopic imaging unit. The color microscopic image can be used to quickly focus and locate the position of the sample to be tested in the field of view. The system fuses the three-dimensional microscopic morphology and confocal fluorescence or reflection spectrum data into a four-dimensional spectral morphology microscopic data set.

[0020] The rear-end receiving unit includes a second beam splitter 10, a first focusing lens 11, a pinhole 12, a second focusing lens 13, and a hyperspectral spectroscopic module 14. The signal fluorescence or reflected light is incident on the second beam splitter 10 in the rear-end receiving unit through the middle scanning unit along the original optical path, is focused at the position of the pinhole 12 through the first focusing lens 11, and is emitted to the external photodetector. Another signal fluorescence or reflected light is incident on the hyperspectral spectroscopic module through the second focusing lens 13 to form a fluorescence spectrum or a reflection spectrum. The external photodetector can be a single photodetector or a dual-channel photodetector. If the single photodetector is replaced with a dual-channel photodetector, differential confocal detection is implemented, which can further improve the accuracy of the system's three-dimensional reconstruction to a certain extent.

[0021] The hyperspectral spectroscopic module includes a slit 15, a collimating lens 16, a prism-grating-prism 17, a third focusing lens 18, and a second camera 23 in sequence. Light is emitted from the middle scanning unit to the slit 15 and is collimated into parallel light by the collimating lens 16. After passing through the prism-grating-prism 17, light of different wavelengths is focused on different positions of the photosensitive surface of the second camera 23 by the third focusing lens 18, thereby forming a spectral image. The system scans the position of the point to be detected on the surface of the sample to be tested to achieve fluorescence or reflection spectrum scanning imaging in the staring state.

[0022] The embodiments described above can be further combined or replaced, and the embodiments are only descriptions of preferred embodiments of the present invention, and do not limit the concept and scope of the present invention. Without departing from the design concept of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention belong to the protection scope of the present invention. The protection scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A staring confocal microscopic morphology spectrum four-dimensional detection system, characterized in that: It includes an excitation unit, a front optical unit, a microscopic imaging unit, a middle scanning unit, and a rear receiving unit; The excitation unit includes a reflector and a fiber collimator. The excitation light of the sample to be tested is introduced from the excitation unit and incident on the middle scanning unit after being adjusted by the reflector. The middle scanning unit includes a telecentric lens, a scanning lens, a galvanometer system, a first beam splitter, and a filter in sequence. The scanning lens and the telecentric lens optimize the light beam and expand the beam, and the galvanometer system performs two-dimensional scanning. The front optical unit includes a stage, a microscope objective lens, a displacement stage, and a beam splitter. The excitation light emitted from the central scanning unit is reflected by the beam splitter and incident on the microscope objective lens, and is focused on the sample to be tested on the stage. The displacement stage is used to adjust the distance of the sample to be tested to achieve focusing. The fluorescence signal or reflected light signal excited by the sample to be tested returns along the original light path. The microscopic imaging unit comprises a fourth focusing lens and a first camera, and the light reflected from the sample to be tested is imaged at the position of the first camera through the fourth focusing lens to form a conjugate microscopic image; The rear-end receiving unit includes a second beam splitter, a first focusing lens, a pinhole, a second focusing lens, and a hyperspectral spectroscopic module. The signal fluorescence or reflected light passes through the middle scanning unit along the original optical path and is incident on the second beam splitter in the rear-end receiving unit. It is focused at the pinhole position through the first focusing lens and emitted to the external photodetector. Another path of signal fluorescence or reflected light passes through the second focusing lens and is incident on the hyperspectral spectroscopic module to form a fluorescence spectrum or a reflection spectrum. The hyperspectral spectrometer module includes a slit, a collimating lens, a prism-grating-prism, a third focusing lens, and a second camera in sequence. Light emitted from the middle scanning unit to the slit position is collimated into parallel light by the collimating lens. After passing through the prism-grating-prism, light of different wavelengths is focused on different positions of the photosensitive surface of the second camera by the third focusing lens, thereby forming a spectral image.

2. The system according to claim 1, characterized in that The image plane of the microscopic imaging unit is conjugated with the image plane of the fluorescence spectrum or the reflection spectrum.

3. The system according to claim 1, characterized in that The translation stage in the front optical unit is replaced with a liquid zoom lens to achieve rapid zooming and improve the scanning speed of the system.

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