CN108287021A - A kind of binary channels ultraphotic spectrum microscopic imaging device and obtain different phase spectral information method - Google Patents
A kind of binary channels ultraphotic spectrum microscopic imaging device and obtain different phase spectral information method Download PDFInfo
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Abstract
The present invention relates to a kind of binary channels ultraphotic spectrum microscopic imaging device and obtain different phase spectral information method.Device is controlled by imaging object, inverted light microscope, light colimated light system, Acousto-optic filtering system, beam splitter, spectral detection system, imaging system and computer and is formed with analysis system, imaging object, inverted light microscope, light colimated light system, binary channels Acousto-optic filtering system, beam splitter are sequentially connected, spectral detection system, imaging system are connected with beam splitter, computer control with analysis system respectively, and simultaneous computer control is also connected with binary channels Acousto-optic filtering system with analysis system.Enter light colimated light system after imaging object imaging and carries out shrink beam and collimation；Binary channels Acousto-optic filtering system carries out Acousto-optic filtering；Filtered beam is divided into two bundles light by beam splitter, and is sent to computer control and analysis system, completes analysis and storage, while extracting the spectrum at two different-wavebands of imaging object.
Description
Technical field
The present invention relates to a kind of optical microscopy imaging field, specifically a kind of binary channels ultraphotic spectrum microscopic imaging device and
Obtain different phase spectral information method.
Background technology
The visualization microscopy information that target object can be obtained using optical microscope imaging method, for medical treatment ＆ health, agriculture
Industry production, physics, chemistry and materialogy etc. field suffer from highly important meaning.Optical microscopy imaging means are a variety of
It is various, and all have the characteristics that respective and advantage.For example, the micro-imaging method that matches can be situated between using difference technology with transparent to light
Matter carries out high-contrast image, and a kind of new method is provided for the transparent substances micro-imaging such as cell.But it is micro- using matching
Imaging method has Halo effect and phasing back problem in imaging process, in addition, thickness of the micro-imaging method that matches to sample
Degree requires, and general no more than 5 microns, this strongly limits the application fields for the microscopy that matches.Two-photon fluorescence it is micro- at
As technology space high resolution, micron dimension can be reached, excitation light source is infrared band long wave laser, to the light of biological tissue
It damages small.But two-photon fluorescence micro-imaging technique, mechanism is complicated, high to the performance requirement of laser, and equipment is sufficiently expensive, and
And the depth of imaging is limited, generally in millimeter magnitude, therefore, actual application prospect is also uncertain.In addition, laser scanning is copolymerized
The resolving power of burnt micro-imaging technique, imaging is higher than common light microscope, and stray light and diffraction light can obtain in imaging
Inhibit to effective, good imaging quality.But in imaging process, it is easy to cause light injury to biological tissue, is not suitable for live body group
The imaging knitted.
Acousto-optic filtering technology is combined with optical microscopy imaging technology, may be implemented imaging object ultraphotic spectrum it is micro- at
Picture, this method can obtain the high-resolution image information and spectral information of imaging object simultaneously, and excitation light source is common wide
Light does not need laser as light source.Paper《The visible to the near infrared narrow band
acousto-optic tunable filter and the hyperspectralmicroscopic imaging on
biomedicine study》And patent（A kind of micro- ultra-optical spectrum imaging system）Propose the ultraphotic based on Acousto-optic filtering technology
It composes micro- at system.The technology, which overcomes traditional optical micro-imaging, can not obtain the deficiency of imaging object spectral information.Currently,
During ultraphotic spectrum micro-imaging based on Acousto-optic filtering, although the image and spectral information of imaging object can be obtained simultaneously,
But the spectrum obtained is single wave band, each width ultraphotic spectrum micro-image only includes the letter of a certain specific band of imaging object
Breath, which has limited ultraphotic spectral technologies to the withdrawing spectral information ability of imaging object, will certainly influence the structure to imaging object
Identification and its constituent analysis.
Therefore, the spectral information for how extracting imaging object different-waveband simultaneously obtains in a width ultraphotic composes micro-image
The spectral information that imaging object is more rich is taken, is ultraphotic spectrum micro-imaging field concern.
Invention content
The purpose of the present invention is overcoming above-mentioned deficiency, in composing micro-imaging based on the ultraphotic of Acousto-optic filtering technology, use
Binary channels radio frequency source exports the radiofrequency signal of two different frequencies, while driving acousto-optic filter, while obtaining in difference
Two Acousto-optic filtering signals of cardiac wave length can obtain imaging object at two simultaneously on same width ultraphotic spectrum micro-image
The spectral information of different-waveband.
In order to achieve the above objectives, the technical scheme is that：Device is accurate by imaging object, inverted light microscope, light
Lineal system, binary channels Acousto-optic filtering system, beam splitter, spectral detection system, imaging system and computer control and analysis
System forms, and imaging object, inverted light microscope, light colimated light system, binary channels Acousto-optic filtering system, beam splitter connect successively
It connects, spectral detection system, imaging system are connected with beam splitter, computer control with analysis system respectively after being connected in parallel, together
When computer control be also connected with binary channels Acousto-optic filtering system with analysis system.
Imaging object is placed on the objective table of inverted light microscope, the broadband light warp from inverted light microscope light source
It after imaging object, is received by the object lens of inverted light microscope, shrink beam and collimation is carried out by light colimated light system after light-emitting window, it is accurate
Collimated optical beam carries out binary channels Acousto-optic filtering through Acousto-optic filtering system, and filtered beam is divided into light beam 1 and light beam 2 after beam splitter,
The beam intensity ratio of light beam 1 and light beam 2 is 1:19.Light beam 1 reaches spectral detection system and extracts its spectral information, and light beam 2 reaches imaging
System carries out ultraphotic and composes micro-imaging.Spectral information from spectral detection system and the ultraphotic from imaging system compose micrograph
As information is received and stored with analysis system by computer control；Computer is controlled controls sound with analysis system by control software
The parameter of light filtering system, spectral detection system and imaging system realizes that imaging Object Spectra information and ultraphotic spectrum are micro-
The best acquisition of image information forms complete light path-optical signal connection.
The imaging object is the non-staining slice of general biological tissue.
The inverted light microscope is general inverted light microscope, by wideband light source, objective table, micro- object
The compositions such as mirror and eyepiece.Transmitted light beam is exported through microscope light-emitting window after the imaged object of light beam that wideband light source is sent out, and is arrived
Shrink beam and collimation are carried out up to light colimated light system.
The light colimated light system is made of convex lens group and concavees lens group, and the light beam from microscope light-emitting window is through light standard
Direct line system converges and forms collimated light beam after collimating, and binary channels Acousto-optic filtering is carried out into Acousto-optic filtering system.
The Acousto-optic filtering system is located at the rear of light colimated light system, by acousto-optic filter and binary channels radio frequency source group
At.The collimated light beam that acousto-optic filter receives the output of light colimated light system simultaneously carries out it Acousto-optic filtering, and binary channels radio frequency source can be with
The radiofrequency signal of two different frequencies is exported simultaneously, and is loaded on acousto-optic filter simultaneously, and binary channels radio frequency source is filtered with acousto-optic
It is connected by radio frequency line between wave device.
The optical beam-splitter is K9 optical glass sheets, and the filtered beam from Acousto-optic filtering system is divided into light beam 1
With light beam 2, the beam intensity ratio of light beam 1 and light beam 2 is 1:19.Light beam 1 reaches spectral detection system and extracts its spectral information, light beam 2
It reaches imaging system and carries out ultraphotic spectrum micro-imaging.
The spectral detection system is fiber grating spectrograph, including fibre-optical probe, spectro-grating and high sensitivity
Photodiode array, the spectral resolution in visible-range are 0.2nm, and fibre-optical probe is received from optical beam-splitter
Light beam 1 is sent into spectro-grating and is divided, and filtered beam reaches photodiode array, two pole of photoelectricity after spectro-grating is divided
Pipe array will measure the intensity of different wave length signal light in filtered beam, and data are sent into computer control and analysis system.
The imaging system is made of the imaging lens and ICCD of focus adjustable.Light beam 2 from optical beam-splitter
Imaging lens through focus adjustable are converged and are imaged on the photosurface of ICCD, are carried out ultraphotic and are composed micro-imaging, imaging data is sent
Enter computer control and analysis system.
The described computer control is made of with analysis system PC machine, and by USB connecting lines respectively with binary channels radio frequency
Source, spectral detection system and imaging system connection.Binary channels radio frequency source control software, spectral detection system control built in PC machine
System software, imaging system control software and Data Analysis Software.PC machine using binary channels radio frequency source control with software,
Spectral detection system control software and imaging system control software, respectively to multi-channel radio frequency source, spectral detection system with
And imaging system carries out parameter adjustment and control；PC machine receives the spectroscopic data from spectral detection system and comes from imaging
The ultraphotic of system composes micro-image, and by Data Analysis Software to spectroscopic data and ultraphotic compose micro-image analyzed with
And storage, it completes binary channels ultraphotic and composes micro-imaging process.
Based on above-mentioned hardware device and control software, it is real in the following manner to obtain different phase spectral information method
It is existing：Inverted light microscope is micro- to imaging object progress broadband bright field, and the imaging beam of imaged object transmission is through being inverted light
It learns microscope light-emitting window and enters light colimated light system；Light colimated light system collects the imaging beam from inverted light microscope, to it
Carry out shrink beam and collimation；Binary channels Acousto-optic filtering system carries out bilateral to the collimated light beam through light colimated light system shrink beam and after collimating
Road Acousto-optic filtering, filtered beam include spectral information of the imaging object in two different-wavebands；Beam splitter, which receives, comes from bilateral
The filtered beam for saying light filtering system is classified as 2 liang of beams of light beam 1 and light beam, and the intensity ratio of light beam 1 and light beam 2 is 1:19；
Spectral detection system receives light beam 1, extracts its spectral information, send to computer control and analysis system；Imaging system receives light
Beam 2 carries out ultraphotic and composes micro-imaging, and ultraphotic spectrum micro-image data are sent to computer control and analysis system；Computer control
System receives spectrum and ultraphotic spectrum micro-image data from spectral detection system and imaging system with analysis system, completes
Analysis and storage；Computer controls and analysis system, by binary channels radio frequency source control software, controls binary channels radio frequency source
The frequency of two radiofrequency signals of output, changes the filtered beam spectrum of Acousto-optic filtering system output, while extracting imaging object
Spectrum at two different-wavebands carries out optimum detection to the spectral information of imaging object by spectral detection system, and utilizes
Imaging system obtains corresponding ultraphotic and composes micro-image, completes binary channels ultraphotic and composes micro-imaging process.
The present invention solves in traditional Acousto-optic filtering ultraphotic spectrum micro-imaging, can only obtain the single band spectrum of imaging object
The deficiency of information and its ultraphotic spectrum micro-image, the radiofrequency signal of two different frequencies is generated using binary channels radio frequency source simultaneously,
Imaging object is extracted simultaneously by acousto-optic filter in the spectrum of two different-wavebands, while obtaining the more rich light of imaging object
Spectrum and microscopic image information.The stability of the system is good, easy to control, according to the different characteristics of imaging object, can carry out
Two band center wavelength of filtered beam continuously adjusting or randomly selecting（101）.
Description of the drawings
Attached drawing 1 is binary channels ultraphotic spectrum microscopic imaging device figure.
Attached drawing 2 is binary channels ultraphotic spectrum micro-imaging flow chart.
Specific implementation mode
The invention will be further described with reference to the accompanying drawings and embodiments
In Fig. 1,101 be imaging object, and 102 be inverted light microscope, and 103 be light colimated light system, and 104 be the filter of binary channels acousto-optic
Wave system is united, and 105 be beam splitter, and 106 be spectral detection system, and 107 be imaging system, and 108 be that computer control is with analysis
System.Wherein imaging object（101）, inverted light microscope（102）, light colimated light system（103）, binary channels Acousto-optic filtering system,
Beam splitter（105）It is sequentially connected, spectral detection system, imaging system（107）After being connected in parallel respectively with beam splitter
（105）, computer control and analysis system（108）It is connected, simultaneous computer control and analysis system（108）Also said with bilateral
Light filtering system（104）It is connected.
In order to describe in more detail this system, specific binary channels ultraphotic spectrum micro-imaging process is made in conjunction with Fig. 2 as follows
Explanation：
Step 201：System is opened, i.e. the information initializing of whole system, includes mainly inverted light microscope, the filter of binary channels acousto-optic
Wave system is united（104）Middle binary channels radio frequency source, spectral detection system（106）And imaging system（107）Default parameters be arranged with
And computer controls and analysis system（108）The unlatching of control software.
Step 202：According to imaging object（101）Imaging demand, to inverted light microscope, light colimated light system
（103）And binary channels Acousto-optic filtering system（104）Carry out the setting of parameter.
Step 203：It will be through binary channels Acousto-optic filtering system（104）The filtered beam of outgoing is split, and is divided into intensity ratio
It is 1:19 light beam 1 and light beam 2, light beam 1 enter spectral detection system（106）, light beam 2 enter imaging system（107）.
Step 204：Adjust spectral detection system（106）Parameter, to light beam 1 carry out spectral information extraction；It is adjusted to
As system（107）Parameter utilize light beam 2 carry out ultraphotic compose micro-imaging.
Step 205：Utilize computer analysis and control system（108）Analyzing processing is carried out to imaging results, according to image
Position, imaging definition and filtering signal spectral information etc., parameter adjustment is carried out to front end optical system, is obtained more preferable
Imaging definition；To binary channels Acousto-optic filtering system（104）The frequency of two radiofrequency signals of middle radio frequency source output carries out only
Vertical adjustment, while extracting imaging object（101）In the spectral information of two different-wavebands；Parameter tune is carried out to spectrum investigating system
Section, ensures the optimum detection of spectral information；To imaging system（107）Parameter regulation is carried out, is clearly surpassed with obtaining target object
Spectrum micro-image.
Step 206：The binary channels ultraphotic spectrum micro-imaging result of acquisition is stored, binary channels ultraphotic composes micro-imaging
Process terminates.
Claims (9)
1. a kind of binary channels ultraphotic spectrum microscopic imaging device and acquisition different phase spectral information method, it is characterised in that：
1）Device is by imaging object, inverted light microscope, light colimated light system, Acousto-optic filtering system, beam splitter, spectral detection
System, imaging system and computer control with analysis system form, imaging object, inverted light microscope, light colimated light system,
Binary channels Acousto-optic filtering system, beam splitter are sequentially connected, and spectral detection system, imaging system are divided with light respectively after being connected in parallel
Beam device, computer control is connected with analysis system, simultaneous computer control and analysis system also with binary channels Acousto-optic filtering system
It is connected；
2）Obtain different phase spectral information specific method：Inverted light microscope is micro- to imaging object progress broadband bright field,
The imaging beam of imaged object transmission enters light colimated light system through inverted light microscope light-emitting window；Light colimated light system, which is collected, to be come
From the imaging beam of inverted light microscope, shrink beam and collimation are carried out to it；Binary channels Acousto-optic filtering system through light to collimating system
Collimated light beam after shrink beam of uniting and collimation carries out binary channels Acousto-optic filtering, and filtered beam includes imaging object in two different-wavebands
Spectral information；Beam splitter receives the filtered beam from binary channels Acousto-optic filtering system and is classified as 2 liang of light beam 1 and light beam
The intensity ratio of beam, light beam 1 and light beam 2 is 1:19；Spectral detection system receives light beam 1, extracts its spectral information, send to computer
Control and analysis system；Imaging system receive light beam 2, carry out ultraphotic compose micro-imaging, by ultraphotic spectrum micro-image data send to
Computer controls and analysis system；Computer is controlled receives the light from spectral detection system and imaging system with analysis system
Spectrum and ultraphotic compose micro-image data, complete analysis and storage；Computer controls and analysis system, passes through binary channels radio frequency
Source control software, the frequency of two radiofrequency signals of control binary channels radio frequency source output, changes Acousto-optic filtering system output
Filtered beam spectrum, while the spectrum at two different-wavebands of imaging object is extracted, by spectral detection system to imaging object
Spectral information carry out optimum detection, and obtain corresponding ultraphotic using imaging system and compose micro-image, complete binary channels ultraphotic
Compose micro-imaging process.
2. a kind of binary channels ultraphotic spectrum microscopic imaging device according to claim 1 and acquisition different phase spectral information side
Method, it is characterised in that the imaging object is the non-staining slice of general biological tissue.
3. a kind of binary channels ultraphotic spectrum microscopic imaging device according to claim 1 and acquisition different phase spectral information side
Method, it is characterised in that the inverted light microscope is general inverted light microscope, by wideband light source, objective table, is shown
The compositions such as speck mirror and eyepiece.
4. a kind of binary channels ultraphotic spectrum microscopic imaging device according to claim 1 and acquisition different phase spectral information side
Method, it is characterised in that the light colimated light system is made of convex lens group and concavees lens group, the light beam from microscope light-emitting window
Collimated light beam is formed after light colimated light system converges and collimates, binary channels Acousto-optic filtering is carried out into Acousto-optic filtering system.
5. a kind of binary channels ultraphotic spectrum microscopic imaging device according to claim 1 and acquisition different phase spectral information side
Method, it is characterised in that the Acousto-optic filtering system is located at the rear of light colimated light system, by acousto-optic filter and binary channels radio frequency
Source forms, and is connected by radio frequency line between binary channels radio frequency source and acousto-optic filter.
6. a kind of binary channels ultraphotic spectrum microscopic imaging device according to claim 1 and acquisition different phase spectral information side
Method, it is characterised in that the optical beam-splitter is K9 optical glass sheets, and the filtered beam from Acousto-optic filtering system is divided into
The beam intensity ratio of light beam 1 and light beam 2, light beam 1 and light beam 2 is 1:19, light beam 1 reaches spectral detection system and extracts its spectral information,
Light beam 2 reaches imaging system and carries out ultraphotic spectrum micro-imaging.
7. a kind of binary channels ultraphotic spectrum microscopic imaging device according to claim 1 and acquisition different phase spectral information side
Method, it is characterised in that the spectral detection system is fiber grating spectrograph, including fibre-optical probe, spectro-grating and Gao Ling
Sensitivity photodiode array, the spectral resolution in visible-range are 0.2nm, and fibre-optical probe, which receives, comes from optical beam splitting
The light beam 1 of device is sent into spectro-grating and is divided, and filtered beam reaches photodiode array, photoelectricity after spectro-grating is divided
Diode array will measure the intensity of different wave length signal light in filtered beam, and data are sent into computer control and are with analysis
System.
8. a kind of binary channels ultraphotic spectrum microscopic imaging device according to claim 1 and acquisition different phase spectral information side
Method, it is characterised in that the imaging system is made of the imaging lens and ICCD of focus adjustable, from optical beam-splitter
Imaging lens of the light beam 2 through focus adjustable are converged and are imaged on the photosurface of ICCD, are carried out ultraphotic and are composed micro-imaging, are imaged number
According to the control of feeding computer and analysis system.
9. a kind of binary channels ultraphotic spectrum microscopic imaging device according to claim 1 and acquisition different phase spectral information side
Method, it is characterised in that the described computer control is made of with analysis system PC machine, and by USB connecting lines respectively with binary channels
Radio frequency source, spectral detection system and imaging system connection；Binary channels radio frequency source control software, spectral detection system built in PC machine
System control software, imaging system control software and Data Analysis Software.
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