CN111122535A - Hyperspectral rapid imaging measurement system for molecular vibration mode - Google Patents
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Abstract
The invention provides a hyperspectral rapid imaging system for molecular vibration modes, which comprises: an optical comb light source for generating pump light, stokes light and probe light as a wide field coherent raman scattering process; the wide-field coherent Raman scattering microscopic light path is used for forming a large light spot light beam by the light generated by the optical comb light source to irradiate the sample, so that the sample generates an anti-Stokes light signal under the action of a coherent Raman scattering effect; and the high-speed area array photoelectric detector is used for collecting anti-Stokes light signals emitted by the sample and carrying out rapid hyperspectral imaging. The invention can simultaneously carry out spectral measurement and imaging, greatly improve the imaging speed and meet the imaging requirement of the living biological sample.
Description
Technical Field
The invention relates to the field of coherent Raman scattering microscopic imaging, in particular to a hyperspectral rapid imaging measurement system for a molecular vibration mode.
Background
Coherent Raman scattering is a nonlinear optical process based on molecular vibration energy level, has fingerprint identification capability on vibration and rotation of molecular functional groups, is very suitable for label-free biological imaging, and has higher spatial resolution compared with infrared spectrums with the same function. It mainly includes Coherent Anti-Stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS). Compared with spontaneous Raman scattering, the Raman signal is greatly enhanced through coherent excitation, and the detection sensitivity is higher.
Coherent anti-stokes raman scattering is a three-order nonlinear optical process, and the sensitivity of single molecule detection is greatly improved through resonance excitation. CARS microscope system usually uses a beam of wavelength tunable laser as pumping light omegapAnother beam of light with fixed wavelength is used as Stokes light omegaS. The vibrational spectrum of the molecule can be measured by scanning the pump wavelength, or the pump light can be tuned to the resonant frequency, at 2 ωp-ωSDetection for CARS imaging. The CARS signal intensity is high, and the wavelength of the CARS signal is blue-shifted relative to the exciting light, so the CARS micro-imaging technology can work under the high fluorescence background condition and has higher detection sensitivity. Compared with the micro-Raman imaging technology which uses a grating spectrometer to obtain Raman signals and adopts a point-by-point scanning mode to carry out imaging, the vibration spectrum is not measured in the CARS imaging process, and only a certain vibration frequency (omega) is measuredp-ωS) The imaging speed is greatly improved by imaging, and the method is widely applied to the imaging of living cells and tissues.
As two major classes of important optical analytical research techniques, spectroscopic techniques and optical microscopy have their own emphasis. Spectroscopic techniques are mainly used to analyze the function and variation of a sample, while optical microscopy is mainly used to observe its spatial distribution. Recently, a hyperspectral imaging technology combining the advantages of the two technologies has attracted much attention. Hyperspectral imaging is the detailed segmentation of an image in the spectral dimension. The vibration spectrum has the molecular fingerprint identification capability, so that the hyperspectral imaging of the molecular vibration mode can simultaneously detect the change of various functional molecules in a living body, and the innovation of the research methodology in the field of life science is greatly promoted. However, a microscopic imaging method which can perform hyperspectral imaging of a biomolecule vibration mode and can perform observation (in seconds) of a living biological sample at a speed has not existed so far. To achieve this, spectral and microscopic imaging methodological innovations are necessary.
The optical comb spectrum technology developed in recent years provides a new idea for the optical comb spectrum technology. The optical comb frequency domain is regularly distributed with a series of spectral lines with equal intervals and a large number, and the spectral lines are natural precise 'scribed lines' for spectral analysis, and the width of each 'scribed line' is extremely narrow and thin, so that extremely high spectral resolution is provided. The optical comb is used as a light source to provide pump light, Stokes light and detection light required by exciting a sample, and the hyperfine spectrum of the molecule can be obtained after Fourier transform data processing is carried out on a time domain interference pattern acquired by the photoelectric detector. In 2013, T.Ideguchi et al combined a dual optical comb with the CARS spectral measurement and imaging technology, and proved the feasibility of using the dual optical comb technology to image the hyperspectral of molecular vibration modes. In 2016, the K.Chen et al of Qinghua university combine with a microscopic imaging mode on the basis of the technology to realize hyperspectral coherent Raman microscopic imaging of the surface structure of the plant. However, because the point-by-point scanning imaging mode is adopted, the method still has the defects of few scanning pixels, long imaging time and the like, and cannot meet the requirement of quick imaging of the living biological sample.
There is therefore a need for a hyperspectral imaging measurement system of molecular vibrational modes that can perform spectral measurements and imaging quickly.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a hyperspectral rapid imaging measurement system in a molecular vibration mode, which utilizes an optical comb as a light source of a coherent Raman scattering microscopic imaging light path, adopts large light spots to irradiate a sample, and simultaneously performs rapid spectral measurement and two-dimensional imaging on an area array photoelectric detector in an optical interference mode. By combining an optical comb light source with a wide-field coherent Raman scattering imaging light path, two large light spot light beams with phase delay are used for simultaneously irradiating the surface of a sample, an anti-Stokes light signal is generated under the action of a coherent Raman effect, and detection and imaging are carried out through an area array photoelectric detector; each pixel point on the area array detector outputs an anti-stokes light signal, the change of the signal along with time is recorded, and the spectral information of the sample position corresponding to the pixel point can be obtained through Fourier transform; therefore, the hyperspectral rapid imaging measurement of the molecular vibration mode can be realized.
The specific technical scheme for realizing the invention is as follows:
a hyperspectral rapid imaging measurement system for molecular vibration modes comprises:
an optical comb light source for generating pump light, stokes light and probe light as a wide field coherent raman scattering process;
the wide-field coherent Raman scattering microscopic light path is used for forming a large light spot light beam by the light generated by the optical comb light source to irradiate the sample, so that the sample generates an anti-Stokes light signal under the action of a coherent Raman scattering effect; and
and the high-speed area array photoelectric detector is used for collecting anti-Stokes light signals from the sample and carrying out rapid hyperspectral imaging.
Preferably, the optical comb light source is composed of a single optical comb, a reflecting mirror, a beam splitter and a michelson interferometer including a phase-controlled delay line device.
Preferably, the optical comb light source is composed of a double optical comb, a reflecting mirror and a beam splitter.
Preferably, the wide-field coherent raman scattering microscope optical path is characterized in that according to different samples to be detected, an anti-stokes optical signal of the sample is obtained by adopting a forward or backward detection mode.
Preferably, in the forward detection mode, the wide-field coherent raman scattering microscope optical path is composed of a first microscope, a second microscope, a high-pass filter and a focusing lens in sequence, and the sample is located between the first microscope and the second microscope.
Preferably, in the backward detection mode, the wide-field coherent raman scattering microscope path is composed of a beam splitter, a microscope, a high-pass filter and a focusing lens in sequence, and the sample is located behind the microscope.
Preferably, fourier transform is performed on anti-stokes optical signals collected by each pixel of the high-speed area-array photodetector in the coherent raman scattering imaging at different times, so that a vibration spectrum corresponding to the pixel can be obtained, that is, hyperspectral imaging of a molecular vibration mode is realized.
The invention has the following beneficial effects:
1. compared with the traditional CARS microscopic imaging, the invention can realize rapid hyperspectral imaging by the optical comb light source and the wide-field coherent Raman scattering microscopic light path.
2. The invention carries out rapid imaging through the area array photoelectric detector, greatly improves the imaging speed and meets the imaging requirement of the living biological sample.
Drawings
Fig. 1 is a schematic structural diagram of a hyperspectral imaging measurement system for molecular vibration modes according to embodiment 1 of the present invention.
Fig. 2 is a schematic structural diagram of a hyperspectral imaging measurement system for molecular vibration modes according to embodiment 2 of the present invention.
Fig. 3 is a schematic structural diagram of a hyperspectral imaging measurement system for molecular vibration modes according to embodiment 3 of the invention.
Detailed Description
The following detailed description of the embodiments of the present invention is provided in connection with the drawings and examples, which are intended to better understand the contents of the present invention and not to limit the scope of the present invention.
Example 1
As shown in fig. 1, a hyperspectral imaging measurement system for molecular vibrational modes, which is an embodiment 1 of the present invention, includes: an optical comb light source for generating pump light, stokes light and probe light as a wide field coherent raman scattering process; the wide-field coherent Raman scattering microscopic light path is used for forming a large light spot light beam by the light generated by the optical comb light source to irradiate the sample, so that the sample generates an anti-Stokes light signal under the action of a coherent Raman scattering effect; and the high-speed area array photoelectric detector is used for collecting anti-Stokes light signals emitted by the sample and carrying out rapid hyperspectral imaging.
The optical comb light source is composed of a single optical comb 11, a beam splitter 13 and a Michelson interferometer 14 containing a delay line device for controlling the phase. The light generated by the single optical comb 11 is split by the beam splitter 13 and enters the michelson interferometer 14. Wherein the michelson interferometer 14 includes a reference arm and a scan arm. The light on the reference arm of the michelson interferometer 14 is reflected by the first mirror 12a and the light on the scanning arm passes through the phase-controlled delay line device 141. The phase control delay line device 141 includes a dispersion element 142, a focusing element 143, a scanning mirror 144, and a second reflecting mirror 12 b. The dispersive element 142 is for example a grating and the focusing element is for example a focusing lens. The dispersive element 142, the focusing element 143 and the scanning mirror 144 constitute a 4f system. In a 4f system, the pivot positions of the dispersive element 142 and the scanning mirror 144 are each located one focal length f of the focusing element 143, and the distance of the dispersive element 142 from the pivot position of the scanning mirror 144 is exactly 2 f. The plane in which the scanning mirror 144 lies is also referred to as the fourier transform plane, so that each spectral component of the dispersed beam can be spatially separated at the location of the scanning mirror 144. The spatially separated beams pass through the focusing element 143 again and are focused on the dispersive element 142, forming a complete 4f system. The light passing through the 4f system is reflected by the second mirror 12b and returns along the same path. By varying the pivot position and the scan angular frequency of the scan mirror 144, the added phase and group delays of the delay line arrangement 141 can be adjusted to achieve arbitrarily varying scanning speeds, spectral bandwidth and spectral resolution under the trade-off of the nyquist theorem.
The two beams of light with certain delay time on the reference arm and the scanning arm of the michelson interferometer 12 are respectively used as pump light, stokes light and probe light of a wide-field coherent raman scattering microscopic imaging light path, enter the wide-field coherent raman scattering microscopic light path 2, irradiate a sample in a forward detection coherent raman scattering light path mode, and generate optical signals which are overlapped in space at the sample.
In this embodiment, the wide-field coherent raman scattering microscopic optical path is a forward detection wide-field coherent raman scattering microscopic optical path. The optical path is composed of a first microscope 15, a second microscope 16, a high-pass filter 17 and a focusing lens 18 in sequence. The sample 19 is located between the first microscope 15 and the second microscope 16. The first microscope 15 focuses two parallel beams of light at the sample 19. According to the lens imaging rule, the light spot can be amplified and imaged at the sample position by adjusting the amplification factor of the microscope and the distance between the sample 19 and the first microscope 15, so that the irradiation of the sample by the large light spot light beam is realized. The anti-stokes light signal generated by the sample 19 is converted into parallel light by the second microscope 16 under the effect of coherent raman scattering. The anti-stokes optical signal has a higher frequency than the original optical signal, and thus can be separated from the original optical signal by the high-pass filter 17. The anti-stokes optical signal further passes through a focusing lens 18, and is focused and incident on a high-speed area array photoelectric detector 20 for fast imaging. The anti-Stokes optical signal carries information of a vibration mode of sample molecules, and can realize vibration spectrum measurement and coherent Raman scattering imaging of the sample.
The high-speed area array photodetector 20 collects the anti-stokes optical signal and performs fast imaging. Fourier transformation is carried out on anti-Stokes light signals collected by each pixel of the high-speed area array photoelectric detector at different time, corresponding vibration spectrums can be obtained, and hyperspectral imaging of a molecular mode is achieved.
Compared with a confocal mode for imaging a sample, the wide-field coherent Raman scattering microscopic light path can realize irradiation of a large-spot light beam on the sample. In addition, compared with a dot matrix photoelectric detector or a linear array photoelectric detector, the rapid area array photoelectric detector can obtain higher luminous flux and pixel resolution. Each pixel point can collect anti-stokes light signals changing along with time, so that the invention can collect and image signals of the whole area of the sample irradiated by the large-spot light beam in a multi-channel manner in a short time, greatly improves the imaging speed and meets the imaging requirement of the living biological sample.
Example 2
As shown in fig. 2, a hyperspectral imaging measurement system for molecular vibrational modes, which is an embodiment 2 of the present invention, includes: an optical comb light source for generating pump light, stokes light and probe light as a wide field coherent raman scattering process; the wide-field coherent Raman scattering microscopic light path is used for forming a large light spot light beam by the light generated by the optical comb light source to irradiate the sample, so that the sample generates an anti-Stokes light signal under the action of a coherent Raman scattering effect; and the high-speed area array photoelectric detector is used for collecting anti-Stokes light signals emitted by the sample and carrying out rapid hyperspectral imaging.
Unlike embodiment 1, the optical comb light source in this embodiment is composed of a double optical comb 21 (i.e., optical comb 1 and optical comb 2), a mirror 22, and a beam splitter 23. Repetition frequency f of double-optical comb light source1And f2There is a slight difference Δ f, satisfies f2＝f1And +/-deltaf, wherein each optical comb can be used as pump light, Stokes light and probe light of a wide-field coherent Raman scattering microscopic imaging light path. Because the repetition frequencies of the two optical comb pulse sequences are different, the two optical comb pulse sequences are in time delay scanning of one pulse sequence to the other sequence, the effect of the optical comb pulse sequence is equivalent to that of a Michelson interference spectrometer, and the difference is that mechanical scanning and pulse delay control are not needed at the moment, so that quick beat frequency can be realized. The light generated by the double optical comb passes through the reflecting mirror 22 and the beam splitter 23, respectively, to generate spatially overlapped optical signals. The two beams of light with certain time delay and spatial overlapping enter a wide-field coherent Raman scattering microscopic light path 2, illuminate a sample in a forward detection coherent Raman scattering light path mode, and generate spatial overlapping light signals at the sample.
In this embodiment, the wide-field coherent raman scattering microscopic optical path is a forward detection wide-field coherent raman scattering microscopic optical path. The optical path is composed of a first microscope 15, a second microscope 16, a high-pass filter 17 and a focusing lens 18 in sequence. The sample 19 is located between the first microscope 15 and the second microscope 16. The first microscope 15 focuses two parallel beams of light at the sample 19. By adjusting the distance between the sample 19 and the first microscope 15, it is possible to achieve a large spot beam illuminating the sample. The anti-stokes light signal generated by the sample 19 is converted into parallel light by the second microscope 16 under the effect of coherent raman scattering. The anti-stokes optical signal has a higher frequency than the original optical signal, and thus can be separated from the original optical signal by the high-pass filter 17. The anti-stokes optical signal further passes through a focusing lens 18, and is focused and incident on a high-speed area array photoelectric detector 20 for fast imaging. The anti-Stokes optical signal carries information of a vibration mode of sample molecules, and can realize vibration spectrum measurement and coherent Raman scattering imaging of the sample.
The high-speed area array photodetector 20 collects the anti-stokes optical signal and performs fast imaging. Fourier transformation is carried out on anti-Stokes light signals collected by each pixel of the high-speed area array photoelectric detector at different time, corresponding vibration spectrums can be obtained, and hyperspectral imaging of a molecular mode is achieved.
In the CARS spectral fast imaging measurement system with the dual optical comb of this embodiment, the appearance period of the coherent signal (i.e. the inverse of the spectral data update rate) is: 1/. DELTA.f. After the change of the coherent signal of the double optical combs along with time is recorded, the CARS spectrum can be obtained through Fourier change, and the resolution ratio of the corresponding spectrum is A (f)1,/Δ f)/t/c, in cm-1Where c is the speed of light, t is the time length of the coherent signal, and a is the coefficient (constant) corresponding to the apodization window function selected for fourier transform. In the dual-optical comb CARS, if the spectral width of the optical comb is delta omega (unit is cm)-1) Then the measurable raman signal spectral range is: 0- Δ ω.
Example 3
As shown in fig. 3, a hyperspectral imaging measurement system for molecular vibrational modes, which is embodiment 3 of the present invention, includes: an optical comb light source for generating pump light, stokes light and probe light as a wide field coherent raman scattering process; the wide-field coherent Raman scattering microscopic light path is used for forming a large light spot light beam by the light generated by the optical comb light source to irradiate the sample, so that the sample generates an anti-Stokes light signal under the action of a coherent Raman scattering effect; and the high-speed area array photoelectric detector is used for collecting anti-Stokes light signals emitted by the sample and carrying out rapid hyperspectral imaging.
The optical comb light source in this embodiment is a dual optical comb light source, which is the same as embodiment 2 and is not described herein again.
In this embodiment, different from embodiments 1 and 2, according to different samples to be measured, the sample is irradiated in a backward detection manner in the wide-field coherent raman scattering microscopic imaging optical path. The optical path of forward detection in embodiments 1 and 2 can excite the sample with smaller optical power, and is suitable for imaging molecules capable of generating larger vibration signals. And the light path for back detection has strong detection sensitivity to the sample to be detected with the size close to or smaller than the wavelength of light. The wide-field coherent Raman scattering microscopic light path for back detection is composed of a second beam splitter 31, a microscope 32, a high-pass filter 33 and a focusing lens 34 in sequence. The sample is located behind the microscope 32. Unlike forward probing, the anti-stokes light signal generated by the sample reaches the microscope behind the sample in transmission. In the light path of the back-detection, the anti-stokes light signal generated by the sample will pass in reflection again through the microscope 32 in front of the sample.
Specifically, two beams of light generated by the dual-optical comb light source are reflected to the microscope 32 via the second beam splitter 31, and the light transmitted by the microscope 32 reaches the sample 35. Under the action of coherent raman effect, the anti-stokes light signal generated by the sample 35 and the original light signal transmit through the second beam splitter 31, and then the anti-stokes light signal and the original light signal are separated through the high pass filter 33. And the anti-stokes optical signal is focused on the high-speed area array photoelectric detector 20 through the focusing lens 34 for fast imaging. The fourier transform is performed on the anti-stokes optical signals collected by each pixel of the high-speed area array photodetector 20 at different times, so that a corresponding vibration spectrum can be obtained, and therefore hyperspectral imaging of the molecular mode can be achieved.
It is apparent that those skilled in the art will recognize that the above-described embodiments are illustrative only and not intended to be limiting, and that changes and modifications of the above-described embodiments are not excluded from the scope of the claims of the present invention as long as they are within the spirit and scope of the invention.
Claims (7)
1. A hyperspectral rapid imaging measurement system for molecular vibration modes is characterized by comprising:
an optical comb light source for generating pump light, stokes light and probe light as a wide field coherent raman scattering process;
the wide-field coherent Raman scattering microscopic light path is used for forming a large light spot light beam by the light generated by the optical comb light source to irradiate the sample, so that the sample generates an anti-Stokes light signal under the action of a coherent Raman scattering effect; and
and the high-speed area array photoelectric detector is used for collecting anti-Stokes light signals emitted by the sample and carrying out rapid hyperspectral imaging.
2. The hyperspectral rapid imaging measurement system for molecular vibration modes according to claim 1, wherein the optical comb light source is composed of a single optical comb, a beam splitter and a michelson interferometer with phase controlled delay line devices.
3. The hyperspectral rapid imaging measurement system for molecular vibration modes according to claim 1, wherein the optical comb light source consists of a double optical comb, a reflector and a beam splitter.
4. The hyperspectral rapid imaging measurement system for the molecular vibration mode according to any one of claims 1 to 3 is characterized in that the wide-field coherent Raman scattering microscopic light path obtains the anti-Stokes light signals of the sample in a forward or backward detection mode according to the difference of the samples to be measured.
5. The hyperspectral rapid imaging measurement system for molecular vibration modes according to claim 4, wherein in the forward detection mode, the wide-field coherent Raman scattering microscope path consists of a first microscope, a second microscope, a high-pass filter and a focusing lens in sequence, and the sample is positioned between the first microscope and the second microscope.
6. The hyperspectral rapid imaging measurement system for molecular vibration modes according to claim 4,
in the backward detection mode, the wide-field coherent Raman scattering microscopic light path is sequentially composed of a beam splitter, a microscope, a high-pass filter and a focusing lens, and the sample is positioned behind the microscope.
7. The hyperspectral rapid imaging measurement system for molecular vibration modes according to claim 1,
fourier transformation is carried out on anti-Stokes light signals collected by each pixel of the high-speed area array photoelectric detector in the coherent Raman scattering imaging at different time, so that a vibration spectrum corresponding to the pixel can be obtained, and hyperspectral imaging of a molecular vibration mode is realized.
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