CN108287021B - Dual-channel hyperspectral microscopic imaging device and method for acquiring spectral information in different stages - Google Patents
Dual-channel hyperspectral microscopic imaging device and method for acquiring spectral information in different stages Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
- G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
- G01J3/28—Investigating the spectrum
- G01J3/2823—Imaging spectrometer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
- G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
- G01J3/02—Details
- G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
- G01J3/0208—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using focussing or collimating elements, e.g. lenses or mirrors; performing aberration correction
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0064—Optical details of the image generation multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G—PHYSICS
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- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/008—Details of detection or image processing, including general computer control
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- G—PHYSICS
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- G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
- G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
- G01J3/28—Investigating the spectrum
- G01J2003/283—Investigating the spectrum computer-interfaced
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
Abstract
The invention relates to a double-channel hyperspectral microscopic imaging device and a method for acquiring spectral information in different stages. The device comprises an imaging object, an inverted optical microscope, an optical collimation system, an acousto-optic filtering system, an optical beam splitter, a spectrum detection system, an imaging system and a computer control and analysis system, wherein the imaging object, the inverted optical microscope, the optical collimation system, the double-channel acousto-optic filtering system and the optical beam splitter are sequentially connected, the spectrum detection system and the imaging system are respectively connected with the optical beam splitter and the computer control and analysis system, and meanwhile the computer control and analysis system is also connected with the double-channel acousto-optic filtering system. Imaging an imaging object, and then entering a light collimation system to perform beam shrinking and collimation; the two-channel acousto-optic filtering system performs acousto-optic filtering; the optical beam splitter divides the filtered light beam into two light beams and sends the two light beams to the computer control and analysis system to complete analysis and storage, and simultaneously, the optical spectrums at two different wave bands of the imaging object are extracted.
Description
Technical Field
The invention relates to the field of optical microscopic imaging, in particular to a dual-channel hyperspectral microscopic imaging device and a method for acquiring spectral information at different stages.
Background
The visual microscopic information of the target object can be obtained by utilizing the optical microscopic imaging method, and the method has very important significance for the fields of medical health, agricultural production, physics, chemistry, materialics and the like. Optical microscopy imaging means are diverse and have their own features and advantages. For example, the phase contrast microscopic imaging method can be used for carrying out high-contrast imaging on the transparent medium by using the phase contrast technology, and provides a new method for microscopic imaging of transparent objects such as cells. However, the phase contrast microscopy has the problems of halo effect and phase inversion during the imaging process, and in addition, the phase contrast microscopy requires a sample thickness of generally not more than 5 micrometers, which greatly limits the application field of phase contrast microscopy. The two-photon fluorescence microscopic imaging technology has high spatial resolution, can reach micron level, adopts infrared band wavelength laser as an excitation light source, and has small light damage to biological tissues. However, the two-photon fluorescence microscopic imaging technology has the advantages of complex mechanism, high performance requirement on a laser, very expensive equipment, limited imaging depth, and general millimeter level, so that the practical application prospect is not clear. In addition, the resolution of the imaging is higher than that of a common optical microscope by using a laser scanning confocal microscopic imaging technology, stray light and diffracted light in imaging can be effectively inhibited, and the imaging quality is good. However, in the imaging process, photodamage is easily caused to biological tissues, and the imaging method is not suitable for imaging living tissues.
The method can simultaneously acquire high-resolution image information and spectrum information of the imaging object, and the excitation light source is common broadband light without laser serving as a light source. Papers The visible to the near infrared narrow band acousto-optic tunable filter and the hyperspectralmicroscopic imaging on biomedicine study and patents (a microscopic hyperspectral imaging system) propose hyperspectral microscopic systems based on acousto-optic filtering techniques. The technology overcomes the defect that the traditional optical microscopic imaging can not acquire the spectral information of an imaging object. At present, in the hyperspectral microscopic imaging process based on acousto-optic filtering, although images and spectrum information of an imaging object can be obtained at the same time, the obtained spectrum is in a single wave band, and each hyperspectral microscopic image only contains information of a certain specific wave band of the imaging object, so that the extraction capability of a hyperspectral technology on the spectrum information of the imaging object is limited, and the structural identification and the component analysis of the imaging object are influenced.
Therefore, how to extract the spectrum information of different wave bands of the imaging object at the same time and acquire the richer spectrum information of the imaging object in one hyperspectral microscopic image is a concern in the hyperspectral microscopic imaging field.
Disclosure of Invention
The invention aims to overcome the defects, in hyperspectral microscopic imaging based on an acousto-optic filtering technology, a double-channel radio frequency source is adopted to output radio frequency signals with two different frequencies, an acousto-optic filter is driven simultaneously, two acousto-optic filtering signals with different center wavelengths are obtained simultaneously, and spectral information of an imaging object in two different wave bands can be obtained simultaneously on the same hyperspectral microscopic image.
In order to achieve the above purpose, the technical scheme of the invention is as follows: the device comprises an imaging object, an inverted optical microscope, an optical collimation system, a double-channel acousto-optic filtering system, an optical beam splitter, a spectrum detection system, an imaging system and a computer control and analysis system, wherein the imaging object, the inverted optical microscope, the optical collimation system, the double-channel acousto-optic filtering system and the optical beam splitter are sequentially connected, the spectrum detection system and the imaging system are respectively connected with the optical beam splitter and the computer control and analysis system after being connected in parallel, and meanwhile, the computer control and analysis system is also connected with the double-channel acousto-optic filtering system.
The imaging object is arranged on the object stage of the inverted optical microscope, broadband light from the light source of the inverted optical microscope is received by the objective lens of the inverted optical microscope after passing through the imaging object, the beam is condensed and collimated by the light collimating system after passing through the light outlet, the collimated light beam is subjected to double-channel acousto-optic filtering by the acousto-optic filtering system, the filtered light beam is divided into a light beam 1 and a light beam 2 after passing through the light beam splitter, and the light intensity ratio of the light beam 1 to the light beam 2 is 1:19. The light beam 1 reaches the spectrum detection system to extract the spectrum information thereof, and the light beam 2 reaches the imaging system to perform hyperspectral microscopic imaging. The spectrum information from the spectrum detection system and the hyperspectral microscopic image information from the imaging system are received and stored by the computer control and analysis system; the computer control and analysis system controls parameters of the acousto-optic filtering system, the spectrum detection system and the imaging system through control software, so that optimal acquisition of spectrum information and hyperspectral microscopic image information of an imaging object is realized, and complete light path-light signal connection is formed.
The imaging object is in general is a non-stained section of biological tissue.
The inverted optical microscope is a general inverted optical microscope and consists of a broadband light source, an objective table, a microscope objective lens, an ocular lens and the like. The light beam emitted by the broadband light source is transmitted through the imaging object and then is output through the light outlet of the microscope, and the light beam reaches the light collimation system to be condensed and collimated.
The light collimation system consists of a convex lens group and a concave lens group, light beams from a light outlet of the microscope are converged and collimated by the light collimation system to form parallel light beams, and the parallel light beams enter the acousto-optic filtering system to carry out double-channel acousto-optic filtering.
The acousto-optic filtering system is positioned behind the light collimating system and consists of an acousto-optic filter and a double-channel radio frequency source. The acousto-optic filter receives the parallel light beams output by the light collimation system and performs acousto-optic filtering on the parallel light beams, the two-channel radio frequency source can output radio frequency signals with two different frequencies at the same time and load the radio frequency signals onto the acousto-optic filter at the same time, and the two-channel radio frequency source is connected with the acousto-optic filter through a radio frequency line.
The optical beam splitter is a K9 optical glass sheet, a filtering light beam from the acousto-optic filtering system is divided into a light beam 1 and a light beam 2, and the light intensity ratio of the light beam 1 to the light beam 2 is 1:19. The light beam 1 reaches the spectrum detection system to extract the spectrum information thereof, and the light beam 2 reaches the imaging system to perform hyperspectral microscopic imaging.
The spectrum detection system is a fiber bragg grating spectrometer and comprises a fiber optic probe, a beam splitting grating and a high-sensitivity photodiode array, the spectrum resolution in the visible light range is 0.2nm, the fiber optic probe receives a light beam 1 from an optical beam splitter and sends the light beam 1 into the beam splitting grating for splitting, a filtered light beam reaches the photodiode array after being split by the beam splitting grating, and the photodiode array measures the intensity of signal light with different wavelengths in the filtered light beam and sends data into a computer control and analysis system.
The imaging system consists of an imaging lens with adjustable focal length and an ICCD. The light beam 2 from the optical beam splitter is converged and imaged on the photosensitive surface of the ICCD through an imaging lens with adjustable focal length, hyperspectral microscopic imaging is carried out, and imaging data are sent to a computer control and analysis system.
The computer control and analysis system is composed of a PC and is respectively connected with the dual-channel radio frequency source, the spectrum detection system and the imaging system through USB connecting wires. The PC is internally provided with two-channel radio frequency source control software, spectrum detection system control software, imaging system control software and data analysis software. The PC machine respectively carries out parameter adjustment and control on the multichannel radio frequency source, the spectrum detection system and the imaging system by using the software for controlling the double-channel radio frequency source, the software for controlling the spectrum detection system and the software for controlling the imaging system; and the PC receives the spectrum data from the spectrum detection system and the hyperspectral microscopic image from the imaging system, analyzes and stores the spectrum data and the hyperspectral microscopic image through data analysis software, and completes the double-channel hyperspectral microscopic imaging process.
Based on the hardware equipment and the control software, the method for acquiring the spectrum information of different stages is realized by the following modes: the inverted optical microscope carries out broadband bright field microscopy on the imaging object, and imaging light beams transmitted by the imaging object enter the light collimation system through a light outlet of the inverted optical microscope; the light collimation system collects imaging light beams from the inverted optical microscope, and performs beam shrinking and collimation on the imaging light beams; the double-channel acousto-optic filtering system performs double-channel acousto-optic filtering on the parallel light beams condensed and collimated by the light collimating system, and the filtered light beams contain spectral information of an imaging object in two different wave bands; the optical beam splitter receives the filtered light beam from the double-channel acousto-optic filtering system and divides the filtered light beam into two beams, namely a light beam 1 and a light beam 2, wherein the intensity ratio of the light beam 1 to the light beam 2 is 1:19; the spectrum detection system receives the light beam 1, extracts spectrum information of the light beam and sends the spectrum information to the computer control and analysis system; the imaging system receives the light beam 2, performs hyperspectral microscopic imaging, and sends hyperspectral microscopic image data to the computer control and analysis system; the computer control and analysis system receives the spectrum and hyperspectral microscopic image data from the spectrum detection system and the imaging system, and analysis and storage are completed; the computer control and analysis system controls the frequencies of two radio frequency signals output by the two-channel radio frequency source through the software for controlling the two-channel radio frequency source, changes the spectrum of the filtering light beam output by the acousto-optic filtering system, extracts the spectrum of the imaging object at two different wave bands, optimally detects the spectrum information of the imaging object through the spectrum detection system, acquires corresponding hyperspectral microscopic images through the imaging system, and completes the two-channel hyperspectral microscopic imaging process.
The invention solves the defect that only single-band spectrum information and hyperspectral microscopic image of an imaging object can be obtained in the traditional acousto-optic filtering hyperspectral microscopic imaging, simultaneously generates two radio frequency signals with different frequencies by utilizing a double-channel radio frequency source, simultaneously extracts the spectrums of the imaging object in two different bands by using an acousto-optic filter, and simultaneously obtains richer spectrums and microscopic image information of the imaging object. The system has good stability and convenient control, and can continuously adjust or randomly select the central wavelengths of two wave bands of the filtering light beam according to different characteristics of an imaging object (101).
Drawings
FIG. 1 is a diagram of a dual channel hyperspectral microimaging apparatus.
FIG. 2 is a flow chart of dual channel hyperspectral microscopy imaging.
Detailed Description
The invention will be further described with reference to the accompanying drawings and examples
In fig. 1, 101 is an imaging object, 102 is an inverted optical microscope, 103 is a light collimation system, 104 is a two-channel acousto-optic filtering system, 105 is an optical beam splitter, 106 is a spectrum detection system, 107 is an imaging system, and 108 is a computer control and analysis system. The imaging object (101), the inverted optical microscope (102), the light collimation system (103), the double-channel acousto-optic filtering system and the optical beam splitter (105) are sequentially connected, the spectrum detection system and the imaging system (107) are respectively connected with the optical beam splitter (105) and the computer control and analysis system (108) after being connected in parallel, and meanwhile the computer control and analysis system (108) is also connected with the double-channel acousto-optic filtering system (104).
To describe the present system in more detail, a specific two-channel hyperspectral microscopy imaging procedure is described below in conjunction with fig. 2:
step 201: the system is started, namely the information of the whole system is initialized, and mainly comprises an inverted optical microscope, a double-channel radio frequency source in a double-channel acousto-optic filtering system (104), default parameter setting of a spectrum detection system (106) and an imaging system (107) and starting of software for controlling a computer control and analysis system (108).
Step 202: the inverted optical microscope, the light collimation system (103) and the two-channel acousto-optic filtering system (104) are subjected to parameter setting according to the imaging requirement of an imaging object (101).
Step 203: the filtered light beam emitted by the double-channel acousto-optic filtering system (104) is split into a light beam 1 and a light beam 2 with the intensity ratio of 1:19, the light beam 1 enters the spectrum detection system (106), and the light beam 2 enters the imaging system (107).
Step 204: adjusting parameters of a spectrum detection system (106) to extract spectrum information of the light beam 1; adjusting parameters of the imaging system (107) uses the light beam 2 for hyperspectral microscopy imaging.
Step 205: analyzing and processing the imaging result by using a computer analysis and control system (108), and adjusting parameters of a front-end optical system according to the position of an image, the imaging definition, spectral information of a filtering signal and the like to obtain better imaging definition; the frequencies of two radio frequency signals output by a radio frequency source in the double-channel acousto-optic filtering system (104) are independently adjusted, and meanwhile, the spectrum information of an imaging object (101) in two different wave bands is extracted; parameter adjustment is carried out on the spectrum detection system, so that optimal detection of spectrum information is ensured; the imaging system (107) is parametrically adjusted to obtain a clear hyperspectral microscopy image of the target object.
Step 206: and storing the obtained two-channel hyperspectral microscopic imaging result, and ending the two-channel hyperspectral microscopic imaging process.
Claims (9)
1. A dual-channel hyperspectral microscopic imaging device, which is characterized in that:
1) The device consists of an imaging object, an inverted optical microscope, an optical collimation system, an acousto-optic filtering system, an optical beam splitter, a spectrum detection system, an imaging system and a computer control and analysis system, wherein the imaging object, the inverted optical microscope, the optical collimation system, the double-channel acousto-optic filtering system and the optical beam splitter are sequentially connected, the spectrum detection system and the imaging system are respectively connected with the optical beam splitter and the computer control and analysis system after being connected in parallel, and meanwhile, the computer control and analysis system is also connected with the double-channel acousto-optic filtering system;
2) The specific method for acquiring the spectrum information of different stages comprises the following steps: the inverted optical microscope carries out broadband bright field microscopy on the imaging object, and imaging light beams transmitted by the imaging object enter the light collimation system through a light outlet of the inverted optical microscope; the light collimation system collects imaging light beams from the inverted optical microscope, and performs beam shrinking and collimation on the imaging light beams; the double-channel acousto-optic filtering system performs double-channel acousto-optic filtering on the parallel light beams condensed and collimated by the light collimating system, and the filtered light beams contain spectral information of an imaging object in two different wave bands; the optical beam splitter receives the filtered light beam from the double-channel acousto-optic filtering system and divides the filtered light beam into two beams, namely a light beam 1 and a light beam 2, wherein the intensity ratio of the light beam 1 to the light beam 2 is 1:19; the spectrum detection system receives the light beam 1, extracts spectrum information of the light beam and sends the spectrum information to the computer control and analysis system; the imaging system receives the light beam 2, performs hyperspectral microscopic imaging, and sends hyperspectral microscopic image data to the computer control and analysis system; the computer control and analysis system receives the spectrum and hyperspectral microscopic image data from the spectrum detection system and the imaging system, and analysis and storage are completed; the computer control and analysis system controls the frequencies of two radio frequency signals output by the two-channel radio frequency source through the software for controlling the two-channel radio frequency source, changes the spectrum of the filtering light beam output by the acousto-optic filtering system, extracts the spectrum of the imaging object at two different wave bands, optimally detects the spectrum information of the imaging object through the spectrum detection system, acquires corresponding hyperspectral microscopic images through the imaging system, and completes the two-channel hyperspectral microscopic imaging process.
2. The dual-channel hyperspectral microscopic imaging device of claim 1, wherein the imaging object is a general non-stained section of biological tissue.
3. The dual-channel hyperspectral microscopic imaging device of claim 1, wherein the inverted optical microscope is a general inverted optical microscope, and comprises a broadband light source, an objective table, a microscope objective lens, an ocular lens and the like.
4. The dual-channel hyperspectral microscopic imaging device as claimed in claim 1, wherein the light collimating system comprises a convex lens group and a concave lens group, and the light beams from the light outlet of the microscope are converged and collimated by the light collimating system to form parallel light beams, and enter the acousto-optic filtering system to perform double-channel acousto-optic filtering.
5. The dual-channel hyperspectral microscopic imaging device of claim 1, wherein the acousto-optic filtering system is positioned behind the light collimating system and consists of an acousto-optic filter and a dual-channel radio frequency source, and the dual-channel radio frequency source is connected with the acousto-optic filter through a radio frequency line.
6. The dual-channel hyperspectral microscopic imaging device as claimed in claim 1, wherein the optical beam splitter is a K9 optical glass sheet, the filtered light beam from the acousto-optic filtering system is split into a light beam 1 and a light beam 2, the light intensity ratio of the light beam 1 to the light beam 2 is 1:19, the light beam 1 reaches the spectrum detection system to extract the spectrum information thereof, and the light beam 2 reaches the imaging system to perform hyperspectral microscopic imaging.
7. The dual-channel hyperspectral microscopic imaging device of claim 1, wherein the spectrum detection system is a fiber bragg grating spectrometer, and comprises a fiber optic probe, a beam splitting grating and a high-sensitivity photodiode array, wherein the spectrum resolution in the visible light range is 0.2nm, the fiber optic probe receives a light beam 1 from a light beam splitter, the light beam is sent to the beam splitting grating for splitting, the filtered light beam is sent to the photodiode array after being split by the beam splitting grating, and the photodiode array measures the intensity of signal lights with different wavelengths in the filtered light beam and sends data to the computer control and analysis system.
8. The dual-channel hyperspectral microscopic imaging device as claimed in claim 1, wherein the imaging system is composed of an imaging lens with adjustable focal length and an ICCD, the light beam 2 from the optical beam splitter is converged by the imaging lens with adjustable focal length and imaged on a photosurface of the ICCD for hyperspectral microscopic imaging, and imaging data are sent to the computer control and analysis system.
9. The dual-channel hyperspectral microscopic imaging device as claimed in claim 1, wherein the computer control and analysis system is composed of a PC and is respectively connected with the dual-channel radio frequency source, the spectrum detection system and the imaging system through USB connecting wires; the PC is internally provided with two-channel radio frequency source control software, spectrum detection system control software, imaging system control software and data analysis software.
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