CN105784674B - Double-colored stimulated Raman scattering imaging system based on binary channels quadrature detection - Google Patents
Double-colored stimulated Raman scattering imaging system based on binary channels quadrature detection Download PDFInfo
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- CN105784674B CN105784674B CN201610285663.1A CN201610285663A CN105784674B CN 105784674 B CN105784674 B CN 105784674B CN 201610285663 A CN201610285663 A CN 201610285663A CN 105784674 B CN105784674 B CN 105784674B
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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
- G01N21/62—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
- G01N21/65—Raman scattering
Abstract
The invention belongs to nonlinear optics technical field of imaging, specially a kind of double-colored stimulated Raman scattering imaging system based on binary channels quadrature detection.The present invention dexterously combines the orthogonal output of binary channels of spectrum focusing and lock-in amplifier, and light path design is simple, and improving cost is low, once it no longer needs to adjust any opto-electronic device after the completion of optimization, therefore system is highly stable.It may be implemented: carrying out double light spectrum image-formings in real time to lipid and protein, is expected to provide real-time image information for pathological examination；The mobile brought spectral error of sample is eliminated, provides technical support for living body and dynamic detection；When carrying out light spectrum image-forming in the sample to Multiple components, or large area sample being imaged, image taking speed is increased to original 2 times or more.The present invention will play great impetus to the clinical application of excited Raman imaging technique.
Description
Technical field
The invention belongs to nonlinear optics imaging field, specially it is a kind of can detect simultaneously two Raman vibration frequencies by
Swash Raman Imaging System.
Background technique
Excited Raman imaging technique is a kind of non-property line optics imaging means grown rapidly in recent years, it is Raman scattering
With the combination of stimulated radiation technology.Therefore, stimulated Raman scattering technology has the advantages of both the above technology: 1, it is excited to draw
The intensity of graceful scattering and measured object concentration are linear；2, signal strength is high, and signal-to-noise ratio is high, and picture quality is good；3, nothing but altogether
Vibration background interference, is not present spectrum distortion phenomenon.
But every single sweep operation of excited Raman imaging system can only be imaged for a Raman vibration frequency, i.e.,
Forming monochrome image.This is also the shortcoming that excited Raman imaging system endures puzzlement to the fullest extent all the time.In recent years, more in the world
A research group, which also has devised, several may be implemented polychrome excited Raman imaging system.But among a little schemes, what is had is a large amount of
Increase the quantity of experimental facilities；Some makes system light path become extremely complex, it is difficult to operate；Also become data processing
It is extremely complex.In comparison, EO-1 hyperion excited Raman imaging system is easily achieved, and low in cost.
Chirp is carried out to pump light and stokes light with the medium of high refractive index first, each of two bundle of pulsed laser
Wave packet is broadened in the time.Then an optical delay line is added in the optical path of wherein light beam, to the opposite of two-beam
Time delay is adjusted.It thus can (generally 200cm in a certain range-1Left and right) continuously change pump light and Si Tuo
Difference on the frequency between Ke Si light can so realize that polychrome excited Raman is imaged.But its major defect is optical delay
The adjustment of line position needs the regular hour, can not accomplish two or more Raman vibration frequency of real-time detection.This for
It needs dual colour imaging and for the measured object that fast moves, error certainly will or can be introduced.
The present invention is exactly a kind of double-colored excited Raman imaging system based on EO-1 hyperion excited Raman imaging system, can be real-time
Two Raman vibration frequencies are imaged, the mobile bring image error due to measured object is not only successfully avoided, but also
Substantially increase image taking speed.The reliability of the system is also verified.
Summary of the invention
The purpose of the present invention is to provide a kind of image taking speeds it is fast, high reliablity can be with two Ramans vibrations of real-time detection
The excited Raman imaging system of frequency.
It is proposed by the present invention can two Raman vibration frequencies of real-time detection excited Raman imaging system, be poly- based on spectrum
It is burnt and lock-in amplifier binary channels quadrature detection, it is improved EO-1 hyperion excited Raman imaging system.As shown in Figure 1,
System includes: the two beam femtosecond pulses (stokes light and pump light) of linear polarization, electrooptic modulator, signal generator, and 2
A optical delay line, polarizing beam splitter, half-wave plate, two-phase color mirror, microscope, short logical colour filter, photodiode, locking phase
Amplifier, computer；Wherein, light source of the two beam femtosecond lasers of linear polarization as system respectively will by the medium of high refractive index
Their chirps；Stokes light is modulated by electrooptic modulator, and modulating frequency is a quarter of laser pulse repetition frequency；The frequency
Rate synchronizing process is completed by signal generator, and signal generator repeats laser pulse collected in Stokes optical path
Frequency is as reference frequency；Modulated stokes light after the first optical delay line enter polarizing beam splitter, one
Part stokes light is directly mirrored, after another part stokes light then penetrates polarizing beam splitter, by containing
The light circuit of two optical delay lines, again passes by polarizing beam splitter, with the part stokes light weight being directly mirrored
Newly merge；Stokes light passes through half-wave plate, then by two-phase color mirror is it and pump light is overlapped and importing is shown together
Micro mirror；Signal is acquired with the mode of excited Raman light loss, a condenser is placed under microscope slide platform for collecting
Transmitted light, the light being collected into is translated into electric signal by photodiode after too short logical colour filter, then is input to locking phase and puts
In big device；Signal is after lock-in amplifier parses through being conveyed to computer by two orthogonal output channel.
In present system, the restrictive condition of dual colour imaging is determined are as follows: on time dimension, measure stokes light
Interference pattern and pump light and stokes light between interaction property, to ensure the pump light and Si Tuo in imaging
Time delay between Ke Si light is no more than the time range of the interaction between them, also, the letter of two output channels
It number does not interfere with each other.
In present system, the process of double-colored excited Raman imaging is realized are as follows: control using same computer micro-
Scarnning mirror unit and the signal for receiving lock-in amplifier, the time of integration of appropriate setting signal and microscopical sweep amplitude and speed
Degree, so that it may realize the imaging to sample.
As EO-1 hyperion excited Raman imaging system, double-colored excited Raman imaging system of the invention can pass through adjusting
In system the position of optical delay line come position want detection Raman vibration frequency.In the restrictive condition of determined dual colour imaging
Under, the position that cooperation adjusts two optical delay lines shown in Fig. 1 can successfully be realized to two different Raman vibration frequencies
The detection of rate, that is, all kinds of to different molecules can be imaged, i.e. dual colour imaging.
The present invention dexterously combines the orthogonal output of binary channels of spectrum focusing and lock-in amplifier, light path design
Simply, improving cost is low, once it no longer needs to adjust any opto-electronic device after the completion of optimization, therefore system is highly stable.It can
To realize:
(1) double light spectrum image-formings in real time are carried out to lipid and protein, is expected to provide real-time image information for pathological examination；
(2) the mobile brought spectral error of sample is eliminated, provides technical support for living body and dynamic detection；
(3) when carrying out light spectrum image-forming in the sample to Multiple components, or large area sample being imaged, by imaging speed
Degree is increased to original 2 times or more.The present invention will play great impetus to the clinical application of excited Raman imaging technique.
Detailed description of the invention
Fig. 1 is double-colored excited Raman imaging system schematic diagram.
Fig. 2 is stokes light interference pattern and stokes light and pump light cross correlation curve.
Fig. 3 is the spontaneous Raman spectrum and binary channels stimlated Raman spectrum of oleic acid.
The stimlated Raman spectrum and the HeLa cell figure under different scanning mode that Fig. 4 is oleic acid and bovine serum albumin(BSA)
Picture.
Fig. 5 is that Mice brain tissues are sliced entirety and partial enlarged view.
Specific embodiment
The step of building with test double-colored excited Raman imaging system is as follows:
(1) stokes light is transformed.
As shown in Figure 1, the double-colored excited Raman imaging system that the present invention is built is established and is imaged in EO-1 hyperion excited Raman
On the basis of system.It will be that the pulse of pump light and stokes light of linearly polarized light is opened up with chirp medium in time respectively
It is wide.The repetition rate that laser pulse is acquired in Stokes optical path is used as the reference frequency of signal generator to generate photoelectricity
The modulating frequency of modulator and parsing reference frequency (laser repetition rate 80MHz, Electro-optical Modulation and the locking phase of lock-in amplifier
The frequency, demodulation frequency of amplifier is 20MHz).The polarization characteristic of stokes light can be periodically changed in electrooptic modulator.This
Sample, stokes light, will by the light intensity of reflective portion when encountering polarizing beam splitter after the first optical delay line
Change with the modulation period of electrooptic modulator；The light intensity of transmissive portion.Due to reflecting and penetrating polarizing beam splitter
Stokes light be shifting relationship, so they can have 1/2 modulation period of a delay in time, that is,
180 degree has been differed in Electro-optical Modulation phase.Next, allow the stokes light of transmissive portion by the second optical delay line, then
It is secondary to pass through polarizing beam splitter, allow it to be overlapped with the stokes light of reflective portion.As long as suitably setting the second optical delay
The position of line, 90 degree or 270 degree of phase modulation difference when two parts stokes light can be made to meet again.That is,
Being switched on and off modulated stokes light is including the light of the second optical delay line by a polarizing beam splitter and one
The stokes light containing two components is transformed into behind circuit, and the two components are orthogonal on phase modulation.However,
Their polarization direction be also it is mutually perpendicular, just need to be added polarization of the half-wave plate to adjust them at this time in the optical path
Direction keeps the linear polarization component on its each comfortable pump light polarization direction equal.Stokes light improved in this way passes through one
A dichroscope imports microscope after merging with the pump light after chirp, by object lens get to sample will generate two it is orthogonal
Excited Raman signal.One condenser of light after generation excited Raman process is collected, using one short logical colour filter handle
Stokes light is got rid of, and the intensity of photodiode detection pump light is utilized by the way of excited Raman light loss.Photoelectricity
The electric signal of diode is input into lock-in amplifier, and the orthogonal output channel of its two (X and Y) can parse two just
The excited Raman signal of friendship.The two signals, which are finally input to the microscopical computer of control, may be implemented real-time binary channels
Excited Raman is imaged.
(2) interference pattern for measuring stokes light, realizes dual colour imaging.
In upper step, if two components of stokes light are strict orthogonal on phase modulation, they
Single laser pulse is strictly overlapped in time, necessarily leads to interference phenomenon.As shown in Figure 2 (A) shows, when two beam Stokes
When delay time between light reaches 800 femtoseconds or more, just almost without interference between them.That is, can be by micro-
The position of the second optical delay line is adjusted to avoid the interference between two components of stokes light, lock output channel X is avoided with this
With the interference of Y output channel.From Fig. 2 (B) as can be seen that the delay between pump light and stokes light is in 4 picoseconds
It can still interact, this delay duration is much larger than 800 femtoseconds, and this guarantees the feasibilities of system.
In addition, in EO-1 hyperion excited Raman imaging system, the position of optical delay line can be detected when decision systems
Raman vibration frequency.In the present system and equally: the position of the first optical delay line is determined by polarizing beam splitter
The Raman vibration frequency that excited Raman signal caused by the part stokes light directly reflected is detected；Second optics prolongs
The position of slow line can determine the Raman vibration frequency of the Stokes optical detection through polarizing beam splitter.In conclusion
Mainly there are three functions for second optical delay line: 1, the phase modulation of 90 degree of generation is poor；2, avoid two parts stokes light it
Between interference；3, select the Raman vibration frequency to be detected different from the stokes light of reflective portion.
(3) crosstalk between two output channels is detected.
In the previous step, in order to avoid two different Raman vibration frequencies of interference and detection between two signals need
The position of the second optical delay line is adjusted, so, the signal in two channels cannot keep the pass of stringent quadrature in phase
System.But, a quarter modulation period, (corresponding 90 degree phase delays) were about 12.5 nanoseconds, be far longer than for avoid interference with
Select the optical delay (at most several picoseconds) introduced needed for different Raman vibration frequencies.That is, selection Raman vibration
For dynamic frequency with while avoiding interference with generation, the quadrature in phase characteristic of two channel signals only receives minimum influence.Such as Fig. 3 institute
Show, Fig. 3 (A) is the spontaneous Raman spectrum of oleic acid, and Fig. 3 (B) is two channels of double-colored excited Raman system while the oleic acid that measures
Stimlated Raman spectrum, it can be seen from the figure that two stimlated Raman spectrums are almost, and all with spontaneous Raman light
It composes essentially identical.This illustrates that the signal in the channel X and Y there's almost no crosstalk.
(4) with different sample verifying test system performances.
Single component sample, living cells sample and mouse brain slice sample test system performance are used respectively.
Fig. 4 (A) gives the oleic acid (representing lipid) of X passage and the excited Raman of bovine serum albumin(BSA) (representing protide)
The observation of stimulated Raman scattering of scattering spectrum and the oleic acid in the channel Y.Two grey column stripeds in figure have been marked to cell
The Raman peak position to be detected is imaged.The position of first optical delay line (Fig. 1) is in 8.78(d1) and 9.11(d2) time-division at millimeter
2850 and 2930cm of spontaneous Raman spectrum is not corresponded to-1Peak position, in this two tools position respectively it can be concluded that the signal and rouge of lipid
The signal that class is superimposed with protide.That is, can be in 2850cm-1Place obtains the signal of lipid, uses 2930cm-1The letter at place
The signal for number subtracting lipid can be obtained by the signal of albumen, and then again synthesizing rouge with the signal of albumen can in a figure
To obtain the distribution situation of lipid and albumen in sample.Fig. 4 (B) gives with EO-1 hyperion excited Raman imaging system of the present invention
The cell image photographed under the mode scanned frame by frame, this mode need to make it be fixed on d by mobile optical delay line1D2
Two positions can just obtain the distribution map of two kinds of ingredients.It is this due to there is the movement of uncertain various composition inside biopsy samples
The image obtained under the mode scanned frame by frame certainly will introduce artificial error in post-processing.Arrow in Fig. 4 (B) just marks
Due to fat drips drift and the error that introduces.However in corresponding Fig. 4 (C), it is imaged using the double-colored excited Raman of the present invention
System, it is only necessary to which the position of the first optical delay line is fixed on d2As soon as the cell image that can be photographed on position, this
Avoid the movement bring image error due to various composition in living cells.
Fig. 5 is the excited Raman image of mouse brain slice, and wherein Fig. 5 (A) is the general image of brain section, Fig. 5 (B) to 5
It (F) is cortex, terminal mucro, corpora hypothalamicum, the partial enlargement image of hippocampus and corpus callosum respectively.Present invention dual-color mode
Successfully the in vitro brain tissue of mouse is imaged, and image taking speed has been increased to EO-1 hyperion excited Raman imaging system
Twice or more of scan pattern frame by frame.
Claims (3)
1. a kind of double-colored stimulated Raman scattering imaging system based on binary channels quadrature detection is to focus to put with locking phase based on spectrum
Big device binary channels quadrature detection, it is characterised in that system includes: the two beam femtosecond lasers i.e. stokes light and pump of linear polarization
Pu light, electrooptic modulator, signal generator, 2 optical delay lines, polarizing beam splitter, half-wave plate, two-phase color mirror are micro-
Mirror, short logical colour filter, photodiode, lock-in amplifier, computer；Wherein, two beam femtosecond lasers of linear polarization are as system
Light source, by the medium of high refractive index respectively by their chirps；Stokes light is modulated by electrooptic modulator, and modulating frequency is laser
The a quarter of pulse recurrence frequency；The frequency modulation procedure is completed by signal generator, and signal generator is in stoke
Collected laser pulse repetition frequency is as reference frequency in this optical path；Modulated stokes light prolongs by the first optics
Enter polarizing beam splitter after slow line, a part of stokes light is directly mirrored, and another part stokes light then penetrates
After polarizing beam splitter, by the circuit containing the second optical delay line, polarizing beam splitter is again passed by, and it is direct
The part stokes light of reflection reconsolidates together；Stokes light passes through half-wave plate, then by two-phase color mirror it
It is overlapped with pump light and imports microscope together；Signal is acquired with the mode of excited Raman light loss, in microscope slide platform
One condenser of lower placement, for collecting transmitted light, the light being collected into is after too short logical colour filter by photodiode by its turn
Electric signal is turned to, then is input in lock-in amplifier；Signal is after lock-in amplifier parses through by two orthogonal output channel
It is conveyed to computer.
2. the double-colored stimulated Raman scattering imaging system according to claim 1 based on binary channels quadrature detection, feature
It is, determines the condition of dual colour imaging are as follows: on time dimension, measures the interference pattern and pump light of stokes light
Interaction property between stokes light, to ensure the time delay in imaging between pump light and stokes light
Signal no more than the time range of the interaction between them, also, two output channels does not interfere with each other.
3. the double-colored stimulated Raman scattering imaging system according to claim 1 based on binary channels quadrature detection, feature
It is, realizes the process of double-colored excited Raman imaging are as follows: control microscope scanning element and reception using same computer
The signal of lock-in amplifier, the time of integration of appropriate setting signal and microscopical sweep amplitude and speed, so that it may realization pair
The imaging of sample.
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| CN106645081B (en) * | 2016-10-26 | 2019-11-12 | 复旦大学 | Stimlated Raman spectrum imaging system based on quick scanning light path |
| CN109038204B (en) * | 2018-07-05 | 2020-08-04 | 上海理工大学 | Stimulated Raman scattering imaging light source based on photonic crystal fiber |
| CN110836876B (en) * | 2018-08-15 | 2021-05-14 | 浙江大学 | Super-resolution microscopy method and system based on saturated pumping-stimulated radiation detection |
| CN109060767A (en) * | 2018-10-19 | 2018-12-21 | 清华大学 | A kind of double frequency comb spectrum focusing coherent antistockes Raman spectroscopy detection systems |
| CN109632762B (en) * | 2018-12-21 | 2020-08-11 | 清华大学 | Stimulated Raman difference method and device |
| CN113792568B (en) * | 2020-11-05 | 2023-12-19 | 北京航空航天大学 | Method for determining key features for determining cell status |
| CN113390850B (en) * | 2021-06-02 | 2022-06-21 | 复旦大学 | Gastric Raman femtosecond picosecond image mapping method based on U-shaped convolution neural network |
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