CN105891116B - A kind of multifocal confocal scanning microscopic system and method based on volume holographic record - Google Patents
A kind of multifocal confocal scanning microscopic system and method based on volume holographic record Download PDFInfo
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- CN105891116B CN105891116B CN201610494544.7A CN201610494544A CN105891116B CN 105891116 B CN105891116 B CN 105891116B CN 201610494544 A CN201610494544 A CN 201610494544A CN 105891116 B CN105891116 B CN 105891116B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The invention discloses a kind of multifocal confocal scanning microscopic systems and method based on volume holographic record, wherein including scanning light beam recording device, emits excitation beam and records multiple groups pointolite array；Scanning light beam reduction apparatus restores the multiple groups pointolite array being recorded, and multifocal illumination is formed on sample；Second lens focus the multiple groups pointolite array being reduced, form focal beam spot on sample focal plane；Image pickup part carries out real time imagery to sample image；The third lens, the fluorescence signal that sample surfaces issue is focused on the photosensitive element of image pickup part, the Laser Scanning Confocal Microscope image taking speed that can solve the prior art using the present invention is slow, significant component of exciting light is blocked by the other parts other than pin hole in turntable to be caused to excite light utilization efficiency low, and luminous energy wastes serious technical problem.
Description
Technical field
The present invention relates to medical instrument more particularly to a kind of multifocal confocal scanning based on volume holographic record are aobvious
Micro-system and method.
Background technique
Confocal laser endomicroscopy can filter the stray light other than focus, effectively improve using conjugation pinhole filter
The resolution ratio and picture contrast of optical microscopy, and can be realized slice scanning and 3-D image reconstruct, it is therefore, extensive
Applied to fields such as biomedicines.Since Confocal laser endomicroscopy is a kind of technology of point-to-point detection, therefore, it is necessary to cooperate to sweep
It retouches technology and is just able to achieve two-dimensional imaging.Common confocal scanning mode has 2-D vibration mirror scanning and platform motion scan, single-point
The scanning mode of acquisition keeps image taking speed slow, it is difficult to meet dynamic observation needs.It is such as micro- using the copolymerization coke of turntable scan-type
Although mirror can be realized multifocal while acquire simultaneously fast imaging, but be limited to the size and arrangement of pinhole array, and suitable one
The exciting light divided is blocked by the other parts other than pin hole in turntable, and excitation light utilization efficiency is low, and luminous energy waste is serious.
Therefore, the existing technology needs to be improved and developed.
Summary of the invention
The purpose of the present invention is to provide a kind of multifocal confocal scanning microscopic system based on volume holographic record and sides
Method, slow with the Laser Scanning Confocal Microscope image taking speed for solving the prior art, significant component of exciting light is by other than pin hole in turntable
Other parts block cause excite light utilization efficiency it is low, luminous energy wastes serious technical problem.
Technical scheme is as follows:
A kind of multifocal confocal scanning microscopic system based on volume holographic record, wherein including
Scanning light beam recording device emits excitation beam and records multiple groups pointolite array；
Scanning light beam reduction apparatus restores the multiple groups pointolite array being recorded, and multifocal illumination is formed on sample；
Second lens focus the multiple groups pointolite array being reduced, form focal beam spot on sample focal plane；
Image pickup part carries out real time imagery to sample image；
The third lens focus on the fluorescence signal that sample surfaces issue on the photosensitive element of image pickup part.
The multifocal confocal scanning microscopic system based on volume holographic record, wherein the scanning light beam record
Device includes beam emissions portion, beam splitting component, reflecting mirror, pinhole array turntable, light beam output precision, the first lens and volume holographic
Turntable is recorded, the excitation beam of the beam emissions portion transmitting is partially transmitted part reflection, reflective portion by beam splitting component
Light beam is incident on the volume holographic record turntable after the reflecting mirror again secondary reflection as reference light, transmissive portion light beam
Pointolite array is formed by the pinhole array on the pinhole array turntable, pointolite array light beam penetrates light beam output precision
Afterwards by the first collimated, it is incident to the volume holographic record turntable and remembers with the reflective portion light beam in the volume holographic
Interfere and be recorded on record turntable, further includes control unit and the driving pinhole array turntable and volume holographic record turntable
The motor of synchronous rotation, pinhole array turntable and the volume holographic described in the output axis connection of the motor record turntable, described
Control unit controls the frequency of the motor rotation.
The multifocal confocal scanning microscopic system based on volume holographic record, wherein the scanning light beam record
Device further includes one for limiting the diaphragm of record area area, and the diaphragm is set to the light beam of the volume holographic record turntable
Incidence end.
The multifocal confocal scanning microscopic system based on volume holographic record, wherein the scanning light beam reduction
Device includes beam emissions portion, volume holographic record turntable, the first lens, light beam output precision and pinhole array turntable, the light
The light beam of beam emission part transmitting is irradiated in the volume holographic record turntable, and the volume holographic record turntable restores after being irradiated by light beam
The pointolite array, pointolite array light beam focus after first lens, light beam output precision and by the needles
Pin hole on hole array turntable, pointolite array light beam are formed focal beam spot after second lens focus on sample, institute
Scanning light beam reduction apparatus is stated to further include control unit and drive the pinhole array turntable and volume holographic record turntable same
The motor of rotation is walked, pinhole array turntable described in the output axis connection of the motor and the volume holographic record turntable, the control
Unit processed controls the frequency of the motor rotation.
The multifocal confocal scanning microscopic system based on volume holographic record, wherein the light beam output precision
For dichroscope, the exciting light that the beam emissions portion issues, the fluorescence that reflected sample surface is excited are transmitted.
The multifocal confocal scanning microscopic system based on volume holographic record, wherein the light beam output precision
For beam splitter.
The multifocal confocal scanning microscopic system based on volume holographic record, wherein the pinhole array turntable
Including the several groups pinhole array uniformly distributed along pinhole array disk peripheral, the pinhole array includes several evenly distributed needles
The correspondence pin hole distance in hole, two groups of adjacent pinhole array of sequence is half pinhole size.
The multifocal confocal scanning microscopic system based on volume holographic record, wherein the image pickup part is taken the photograph for CCD
As head.
The microscopic method of the described multifocal confocal scanning microscopic system based on volume holographic record, wherein including with
Lower step:
Step A00: writing scan light beam starts beam emissions portion, and the excitation beam of beam emissions portion transmitting passes through beam splitting
Component is partially transmitted part reflection, and reflective portion light beam, which is incident to after the reflecting mirror again secondary reflection as reference light, to be turned
On dynamic volume holographic record turntable, the pinhole array on pinhole array turntable of the transmissive portion light beam Jing Guo the rotation forms point
Array of source, pointolite array light beam penetrate light beam output precision after by the first collimated, be incident to the volume holographic of rotation
Record turntable is simultaneously interfered and is recorded on volume holographic record turntable with reflective portion light beam；
Step B00: scanning photo-reduction, the light beam of beam emissions portion transmitting are irradiated in the volume holographic record turntable of rotation, body
Holographic recording turntable restores the pointolite array after being irradiated by light beam, pointolite array light beam is defeated by the first lens, light beam
The pin hole on the pinhole array turntable is focused and passed through after component out, pointolite array light beam is by after second lens focus
Focal beam spot is formed on sample；
Step C00: sample light is collected and imaging, and pointolite array excitation beam excites sample surfaces to issue fluorescence, swashed
The fluorescence of hair is collected by the second lens, is reflected after focusing on pinhole array turntable by light beam output precision, the fluorescence light reflected
Beam focuses on the image pickup part through the third lens.
The microscopic method of the multifocal confocal scanning microscopic system based on volume holographic record, wherein in step
In A00, start motor after starting beam emissions portion, motor drives volume holographic record turntable and pinhole array turntable to rotate synchronously.
Beneficial effects of the present invention: the present invention is aobvious by providing a kind of multifocal confocal scanning recorded based on volume holographic
Micro-system and microscopic method can record multiple groups point light source battle array in the different positions that volume holographic records turntable by carousel mode
Column can restore corresponding pointolite array, be formed on sample when being reversely incident on volume holographic record turntable using reference light
Multifocal illumination, and the burnt multi-point scanning of copolymerization is realized by way of rotation, the present invention can be realized the imaging of rapid copolymerization coke
Meanwhile increasing substantially the utilization rate of exciting light.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of scanning light beam recording device in the present invention.
Fig. 2 scanning light beam of the present invention restores schematic diagram.
Fig. 3 is pin hole distribution schematic diagram in the present invention.
Fig. 4 is the structural schematic diagram of pinhole array turntable 16 in the present invention.
Fig. 5 is the structural schematic diagram of pinhole array in the present invention.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer and more explicit, right as follows in conjunction with drawings and embodiments
The present invention is further described.
Refering to fig. 1 and Fig. 2, the present invention provide a kind of multifocal confocal scanning microscopic system based on volume holographic record,
Wherein, including
Scanning light beam recording device 1 emits excitation beam and records multiple groups pointolite array；
Scanning light beam reduction apparatus 2 restores the multiple groups pointolite array being recorded, and multifocal illumination is formed on sample；
Second lens 3 focus the multiple groups pointolite array being reduced, form focal beam spot on sample focal plane；
Image pickup part 5 carries out real time imagery to sample image；
The third lens 4 focus on the fluorescence signal that sample surfaces issue on the photosensitive element of image pickup part.
Further, scanning light beam recording device 1 includes beam emissions portion 11, beam splitting component 12, reflecting mirror 13, pin hole battle array
Column turntable 16, light beam output precision 17, the first lens 18 and volume holographic record turntable 14, and what the beam emissions portion 11 emitted swashs
The beam that shines is partially transmitted part by beam splitting component 12 and reflects, and reflective portion light beam is after the reflecting mirror 13 again secondary reflection
It is incident on the volume holographic record turntable 14 as reference light, transmissive portion light beam is by the pinhole array turntable 16
Pinhole array forms pointolite array, and pointolite array light beam collimates after penetrating light beam output precision 17 by the first lens 18,
It is incident to the volume holographic record turntable 14 and interferes simultaneously on volume holographic record turntable 14 with the reflective portion light beam
It is recorded, the pinhole array turntable 16 of rotation is set, the rotation of turntable 14 is recorded by pinhole array turntable 16 and volume holographic, is changed
Become the scan position of scanning light beam, turntable different location can be recorded in volume holographic and record different pointolite arrays, realized altogether
The multifocal spot scan of microscopic system is focused, the time required to reducing imaging.
Scanning light beam recording device 1 further includes control unit 10 and the driving pinhole array turntable 16 and the volume holographic
The motor 19 that turntable 14 rotates synchronously is recorded, pinhole array turntable 16 described in the output axis connection of the motor 19 and the body are complete
Breath record turntable 14, described control unit 10 control the frequency that the motor 19 rotates to adapt to camera shooting demand, scanning light beam note
Recording device 1 further includes one for limiting the diaphragm 15 of record area area, and the diaphragm 15 is set to the volume holographic record and turns
The light beam incidence end of disk 14, reference light and object light through diaphragm 15 are interfered and are recorded on volume holographic record turntable 14.
Further, scanning light beam reduction apparatus 2 includes beam emissions portion 11, volume holographic record turntable 14, the first lens
18, light beam output precision 17 and pinhole array turntable 16, the light beam that the beam emissions portion 11 emits are irradiated in the volume holographic
Turntable 16 is recorded, the Lighting direction in Lighting direction and scanning light beam recording device 1 is on the contrary, volume holographic records turntable
16 irradiated by light beam after restore the pointolite array, pointolite array light beam passes through first lens 18, light beam output group
It is focused after part 17 and by the pin hole on the pinhole array turntable 16, the pointolite array light beam being reduced is with point after being focused
The state of light beam of light source is by the pin hole on pinhole array turntable 16, and exciting light is no longer by the other parts other than pin hole in turntable
It blocks, reduces the waste of luminous energy.
Pointolite array light beam forms focal beam spot, scanning light beam reduction on sample after being focused by second lens 3
Device 2 further includes control unit 10 and the pinhole array turntable 16 and volume holographic record turntable 14 is driven to rotate synchronously
Motor 19, pinhole array turntable 16 and the volume holographic described in the output axis connection of the motor 19 record turntable 14, the control
Unit 10 processed controls the frequency that the motor 19 rotates, and in the present embodiment, control unit 10 is a computer.
Preferably, the light beam output precision 17 is dichroscope, and dichroscope effect is the transmission beam emissions portion
11 exciting lights issued, the fluorescence that reflected sample surface is excited.
As another embodiment of the present invention, the light beam output precision 17 can be set to beam splitter, and light beam passes through
The latter half reflection half transmission of beam splitter, may be implemented the effect of reflective co-focusing imaging.
Refering to Fig. 4 and Fig. 5, pinhole array turntable 16 includes pin hole battle array of the several groups along 16 circumference uniform distribution of pinhole array turntable
Column 160, every group of pinhole array 160 include several evenly distributed pin holes 161, refering to Fig. 3, two groups of adjacent pinhole array of sequence
160 correspondence pin hole dip distance is half pinhole size Da, it can be understood as pin hole 161 is from first pinhole array
Start, each array of pin hole deviates 0.5Da in pinhole array backward, and certainly, the present invention does not do this offset distance
Specific to limit, offset distance is determined according to Scanning size and precision.The correspondence of two groups of sequentially adjacent pinhole array 160
Pin hole 161 is set as offset a certain distance, and in conjunction with the rotation of pinhole array turntable 16, light beam successively irradiates pinhole array turntable
Each pinhole array 160 on 16, light beam pass through second pinhole array 160 when across pin hole light beam relative to pass through first
Across the beam deviation of pin hole 0.5Da when pinhole array 160, so analogize, light beam is worn when passing through third pinhole array 160
The light beam of needle passing hole is relative to by, across the beam deviation of pin hole 0.5Da, pinhole array turns when second pinhole array 160
Disk 16 constantly rotates, and light beam successively passes through each pinhole array 160, can scan object whole surface.Every group of pinhole array 160
Including several evenly distributed pin holes 161, scanning light beam passes through multiple pin holes 161 simultaneously, realizes that multifocal acquires simultaneously, accelerates
Scanning speed is to realize fast imaging.
Preferably, it is applied to detect the crosstalk between light beam when confocal scanning is micro- to reduce to the greatest extent, improves scanning matter
It measures, in same group of pinhole array 160, the distance between adjacent pin hole 161 is at least 5 pinhole size Da, certainly, the present invention
Specific restriction is not made to the distance between adjacent pin hole 161, this distance can have according to the cross talk conditions between detection light
Body setting.
Further, the image pickup part 5 is CCD camera.
The present invention also provides it is a kind of based on volume holographic record multifocal confocal scanning microscopic system microscopic method,
In, comprising the following steps:
Step A00: writing scan light beam starts beam emissions portion 11, starts motor 19, and motor 19 drives volume holographic record
Turntable 14 and pinhole array turntable 16 rotate synchronously, and the excitation beam that beam emissions portion 11 emits is by beam splitting component 12 by part
Transmissive portion reflection, the body that reflective portion light beam is incident to rotation as reference light after the reflecting mirror 13 again secondary reflection are complete
On breath record turntable 14, the pinhole array on pinhole array turntable 16 of the transmissive portion light beam Jing Guo the rotation forms point light source
Array, pointolite array light beam collimate after penetrating light beam output precision 17 by the first lens 18, are incident to the volume holographic of rotation
Record turntable 14 is simultaneously interfered and is recorded on volume holographic record turntable 14 with reflective portion light beam；
Step B00: scanning photo-reduction, the light beam that beam emissions portion 11 emits are irradiated in the volume holographic record turntable of rotation
14, volume holographic record turntable 14 restores the pointolite array after being irradiated by light beam, and pointolite array light beam passes through the first lens
18, it is focused after light beam output precision 17 and passes through the pin hole on the pinhole array turntable 16, pointolite array light beam is described
Second lens 3 form focal beam spot after focusing on sample；
Step C00: sample light is collected and imaging, and pointolite array excitation beam excites sample surfaces to issue fluorescence, swashed
The fluorescence of hair is collected by the second lens 3, is reflected after focusing on pinhole array turntable 16 by light beam output precision 17, what is reflected is glimmering
Light light beam focuses on the image pickup part 5 through the third lens 4.
The present invention by provide it is a kind of based on volume holographic record multifocal confocal scanning microscopic system and microscopic method,
Multiple groups pointolite array can be recorded in the different positions that volume holographic records turntable by carousel mode, it is reversed using reference light
When being incident on volume holographic record turntable, corresponding pointolite array can be restored, forms multifocal illumination on sample, and pass through
The mode of rotation realizes that copolymerization burnt multi-point scanning increases substantially sharp while the present invention can be realized the imaging of rapid copolymerization coke
Luminous utilization rate, the Laser Scanning Confocal Microscope image taking speed for solving the prior art is slow, and significant component of exciting light is by turntable
Other parts other than middle pin hole, which are blocked, to be caused to excite light utilization efficiency low, and luminous energy wastes serious technical problem.
It should be understood that the application of the present invention is not limited to the above for those of ordinary skills can
With improvement or transformation based on the above description, all these modifications and variations all should belong to the guarantor of appended claims of the present invention
Protect range.
Claims (9)
1. a kind of multifocal confocal scanning microscopic system based on volume holographic record, which is characterized in that including
Scanning light beam recording device emits excitation beam and records multiple groups pointolite array；
Scanning light beam reduction apparatus restores the multiple groups pointolite array being recorded, and multifocal illumination is formed on sample；
Second lens focus the multiple groups pointolite array being reduced, form focal beam spot on sample focal plane；
Image pickup part carries out real time imagery to sample image；
The third lens focus on the fluorescence signal that sample surfaces issue on the photosensitive element of image pickup part
The scanning light beam recording device includes beam emissions portion, beam splitting component, reflecting mirror, pinhole array turntable, light beam output
Component, the first lens and volume holographic record turntable, and the excitation beam of the beam emissions portion transmitting is by beam splitting component by part
Transmissive portion reflection, reflective portion light beam are incident to the volume holographic note as reference light after the reflecting mirror again secondary reflection
It records on turntable, transmissive portion light beam forms pointolite array, point light source battle array by the pinhole array on the pinhole array turntable
Column light beam penetrates after light beam output precision by the first collimated, be incident to the volume holographic record turntable and with the reflection
Segment beam is interfered and is recorded on volume holographic record turntable, further includes that control unit and the driving pinhole array turn
Disk and the volume holographic record the motor that turntable rotates synchronously, pinhole array turntable and institute described in the output axis connection of the motor
Volume holographic record turntable is stated, described control unit controls the frequency of the motor rotation.
2. the multifocal confocal scanning microscopic system according to claim 1 based on volume holographic record, which is characterized in that
The scanning light beam recording device further includes one for limiting the diaphragm of record area area, and it is complete that the diaphragm is set to the body
The light beam incidence end of breath record turntable.
3. the multifocal confocal scanning microscopic system according to claim 1 based on volume holographic record, which is characterized in that
The scanning light beam reduction apparatus includes beam emissions portion, volume holographic record turntable, the first lens, light beam output precision and pin hole
Array turntable, the light beam of the beam emissions portion transmitting are irradiated in the volume holographic record turntable, and the volume holographic records turntable
The pointolite array is restored after being irradiated by light beam, pointolite array light beam is after first lens, light beam output precision
Focus and pass through the pin hole on the pinhole array turntable, pointolite array light beam by after second lens focus on sample
Focal beam spot is formed, the scanning light beam reduction apparatus further includes control unit and the driving pinhole array turntable and the body
The motor that holographic recording turntable rotates synchronously, pinhole array turntable described in the output axis connection of the motor and volume holographic note
Turntable is recorded, described control unit controls the frequency of the motor rotation.
4. the multifocal confocal scanning microscopic system according to any one of claims 1 to 3 based on volume holographic record,
It is characterized in that, the light beam output precision is dichroscope, transmits the exciting light that the beam emissions portion issues, reflected sample table
The fluorescence that face is excited.
5. the multifocal confocal scanning microscopic system according to any one of claims 1 to 3 based on volume holographic record,
It is characterized in that, the light beam output precision is beam splitter.
6. the multifocal confocal scanning microscopic system according to claim 1 or 3 based on volume holographic record, feature exist
In the pinhole array turntable includes the several groups pinhole array uniformly distributed along pinhole array disk peripheral, the pinhole array packet
Several evenly distributed pin holes are included, the correspondence pin hole distance of two groups of adjacent pinhole array of sequence is half pin hole ruler
It is very little.
7. the multifocal confocal scanning microscopic system according to claim 1 based on volume holographic record, which is characterized in that
The image pickup part is CCD camera.
8. a kind of multifocal confocal scanning microscopic system as described in any one of claim 1 to 7 based on volume holographic record
Microscopic method, which comprises the following steps:
Step A00: writing scan light beam starts beam emissions portion, and the excitation beam of beam emissions portion transmitting passes through beam splitting component
It is partially transmitted part reflection, reflective portion light beam is incident to rotation as reference light after the reflecting mirror again secondary reflection
Volume holographic records on turntable, and the pinhole array on pinhole array turntable of the transmissive portion light beam Jing Guo the rotation forms point light source
Array, pointolite array light beam is through, by the first collimated, the volume holographic for being incident to rotation records after light beam output precision
Turntable is simultaneously interfered and is recorded on volume holographic record turntable with reflective portion light beam；
Step B00: scanning photo-reduction, the light beam of beam emissions portion transmitting are irradiated in the volume holographic record turntable of rotation, volume holographic
Record turntable restores the pointolite array after being irradiated by light beam, pointolite array light beam passes through the first lens, light beam output group
Focused after part and by the pin hole on the pinhole array turntable, pointolite array light beam by after second lens focus in sample
Focal beam spot is formed on product；
Step C00: sample light is collected and imaging, and pointolite array excitation beam excites sample surfaces to issue fluorescence, is excited
Fluorescence is collected by the second lens, is reflected after focusing on pinhole array turntable by light beam output precision, the fluorescent light beam warp reflected
The third lens focus on the image pickup part.
9. the microscopic method of the multifocal confocal scanning microscopic system according to claim 8 based on volume holographic record,
It is characterized in that, starting motor after starting beam emissions portion, motor drives volume holographic record turntable and pin hole in step A00
Array turntable rotates synchronously.
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| CN107121422A (en) * | 2017-06-21 | 2017-09-01 | 中国科学院苏州生物医学工程技术研究所 | A kind of parallel confocal microscopic imaging device and method based on digital micromirror array |
| CN108802989B (en) * | 2018-08-17 | 2020-06-02 | 华中科技大学 | Parallel multizone image device |
| CN112525871A (en) * | 2020-10-28 | 2021-03-19 | 汕头大学 | Non-scanning confocal microscopic system based on micro-LED |
| CN113063967B (en) * | 2021-02-18 | 2023-02-10 | 上海大学 | Rotary sample table device capable of realizing three-dimensional atom probe microtip array sample rotation |
| CN117369106B (en) * | 2023-12-05 | 2024-03-08 | 北京大学 | Multi-point confocal image scanning microscope and imaging method |
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| CN101795339A (en) * | 2010-01-25 | 2010-08-04 | 赖博 | Confocal optical scanner |
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