CN110441235A - A kind of Multiple modes coupling original position microspectrum imaging system - Google Patents

Abstract

The invention discloses a multi-mode coupling in-situ microspectral imaging system. The system is composed of an inverted microscope, an excitation light source module, an optical path switching module, a microscopic imaging and a spectral testing module, and involves multiple spectra in the same micron-level region of the sample. And imaging measurement functions, including micro-area Raman spectroscopy, micro-area fluorescence spectroscopy, fluorescence microscopic imaging, micro-area transmission spectroscopy, transmission microscopic imaging, micro-area reflectance spectroscopy, reflection microscopic imaging. The system has the characteristics of realizing multi-mode and multi-dimensional in-situ detection and analysis of the same micro-area of the sample without moving the sample to a variety of detection equipment. Multiple information such as Raman spectrum, fluorescence spectrum and imaging, transmission spectrum and imaging, reflection spectrum and imaging of the micro-area. And eliminate the measurement error caused by sample transfer, greatly improving the reliability of test results.

Description

A multi-mode coupled in-situ microspectral imaging system

technical field

The invention belongs to the field of optical analysis and detection, and in particular relates to a multi-mode coupling in-situ microspectral imaging system.

Background technique

Microspectroscopy and imaging technology mainly includes analysis and testing technologies such as Raman spectroscopy, fluorescence spectroscopy and imaging, transmission spectroscopy and imaging, and reflectance spectroscopy and imaging of sample micro-regions. The Raman spectrum of a substance can reflect the molecular structure information of the substance; the fluorescence spectrum and imaging can reflect the energy level information of the substance; the transmission spectrum and imaging and reflection spectrum and imaging can intuitively and accurately reflect the color and color change of the substance. Microspectroscopy and imaging technology not only has the ability of spatial resolution, but also has the ability of spectral resolution, and can know the composition and structure of the micro-region of the sample. This technology is widely used in industry, analysis and detection, scientific research and other fields.

However, in practical applications, various spectral analysis and testing technologies in microspectroscopy and imaging technology are independent of each other, and samples need to be moved to different instruments for analysis and testing, resulting in inevitable measurement errors. Existing instrument and equipment systems cannot meet the needs of multiple in-situ microspectroscopy and imaging measurements on the same micro-region of the sample. If the analysis and testing functions of Raman spectroscopy, fluorescence spectroscopy and imaging, transmission spectroscopy and imaging, and reflection spectroscopy and imaging can be realized simultaneously on a test system without transferring samples, it will be possible to characterize samples more comprehensively in situ The composition and structure of the micro-area will further expand the application of micro-spectroscopy and imaging technology, and promote the further development of the field of optical analysis and detection.

Contents of the invention

The object of the present invention is to provide a multi-mode coupling in-situ microspectral imaging system, which is composed of an inverted microscope, an excitation light source module, an optical path switching module, a microscopic imaging and a spectral testing module, and has the advantages of not needing to move samples to A variety of detection equipment can realize the characteristics of multi-mode and multi-dimensional in-situ detection and analysis of the same micro-area of the sample, and can obtain the information of the sample micro-area by in-situ characterization, and can obtain the Raman spectrum, Fluorescence spectrum and imaging, transmission spectrum and imaging, reflection spectrum and imaging and other multiple information. And eliminate the measurement error caused by sample transfer, greatly improving the reliability of test results.

A multi-mode coupling in-situ microspectral imaging system described in the present invention is composed of an inverted microscope, an excitation light source module, an optical path switching module, a microscopic imaging and a spectrum testing module, and the excitation light source module is It consists of three parts: a Raman light source unit, a fluorescent excitation light source unit, and a reflected illumination light source unit; among them, the Raman light source unit is composed of a 532nm high-brightness semiconductor laser (1), a first lens (2), a second lens (3), The first variable diaphragm (4), the band-pass filter (5), the dichroic beam splitter (6), the long-pass filter (7), the first fiber coupler (8), the first mirror (9) and The second reflector (10) is composed; the fluorescent excitation light source unit is composed of a second iris diaphragm (15), a third reflector (16), a second adjustable reflector (17) and a xenon lamp light source (19); The reflective lighting light source unit is composed of a fourth reflector (18) and a white light source (20); the optical path switching module is composed of an optical path switcher, a first optical path switcher (11) and a second optical path switcher (24), Wherein the first optical path switcher (11) includes the third lens (12), the first adjustable reflector (13) and the fourth lens (14); the second optical path switcher (24) includes the fifth lens (25), The 3rd adjustable reflector (26) and the 6th lens (27); Described microscopic imaging and spectrum testing module are made up of first microscopic imaging with detector (23), the second optical fiber coupler (28), spectrometer (29), the second microscopic imaging is made up of detector (30) and computer (31), wherein the first microscopic imaging detector (23) is a CMOS area array camera, and the second microscopic imaging detector (30) ) is a CCD area array camera; the Raman light source unit in the excitation light source module is connected with the first optical path switcher (11) and the second optical path switcher (24) in the optical path switching module respectively, and the fluorescence excitation light source unit and reflected lighting The light source unit is respectively connected with the first optical path switcher (11); the first optical path switcher (11) and the second optical path switcher (24) in the optical path switching module are respectively connected with the inverted microscope (22), and the second optical path switcher (24) is connected with the second optical fiber coupler (28) in the microscopic imaging and spectral test module; The spectrometer (29) in the microscopic imaging and spectral test module is provided with the detector (30) for the second microscopic imaging , the computer (31) is respectively connected in series with the first detector for microscopic imaging (23) and the second detector for microscopic imaging (30).

A C-mount interface of the inverted microscope (22) is connected with the first microscopic imaging detector (23), another C-mount interface is connected with the second optical path switcher (24), and the rear side interface is connected with the third The iris (21) is connected.

The dichromatic beam splitter (6) in the excitation light source module reflects the Raman excitation light of 532nm, and simultaneously transmits the Raman scattering signal of 500-2500nm; the long-pass filter (7) transmits the Raman scattering signal of 510-2500nm; the first Reflector (9), the second reflector (10), the third reflector (16) and the fourth reflector (18) are all reflective mirrors; the second adjustable reflector (17) is a 90 ° adjustable surface Reflector.

Both the first adjustable reflector (13) and the third adjustable reflector (26) in the optical path switching module are 45° adjustable sliding reflectors.

A multi-mode coupling in-situ microspectroscopy and imaging system described in the present invention, the specific operation of the system is carried out according to the following steps:

a. Adjust the first optical path switcher (11), so that the Raman light source enters the stage sample of the inverted microscope (22), focuses on the sample micro-area, excites the sample micro-area and generates Raman scattering signals, Raman scattering The signal returns to the first optical path switcher (11) from the inverted microscope (22), passes through the second reflector (10), the first reflector (9), the dichroic beam splitter (6), the long-pass filter (7), the second A fiber coupler (8), a second optical path switcher (24), and a second fiber coupler (28) are incident to the spectrometer (29) and the second microscopic imaging detector (30), and the computer (31) displays the sample Raman spectrum of the micro-area;

b. Adjust the first optical path switcher (11), so that the fluorescence excitation light source, reflected illumination light source, and transmitted light excitation light source respectively enter the sample on the stage of the inverted microscope (22), focus on the sample micro-area, and excite the sample micro-area , according to the difference of the excitation light source, the sample micro-area generates corresponding spectral signals, and when it is received by the first microscopic imaging detector (23), the computer (31) independently displays the fluorescence, reflection and transmission images of the sample micro-area respectively ; When the spectral signal generated by the sample is switched to pass through the second optical path switcher (24), the fluorescence, reflection, and transmission spectral signals are incident on the spectrometer (29) and the second microscopic imaging through the second optical fiber coupler (28) Using the detector (30), according to the corresponding spectral signal, the computer (31) independently displays the fluorescence spectrum, reflection spectrum and transmission spectrum of the micro-region of the sample.

A multi-mode coupling in-situ microspectral imaging system described in the present invention, compared with the prior art, has the following beneficial effects:

(1) In-situ Raman spectroscopy, fluorescence spectroscopy and imaging, transmission spectroscopy and imaging, reflection spectroscopy and imaging and other multi-mode analysis, detection and characterization of the micron-scale area of the sample to be tested can effectively avoid measurement deviation caused by moving samples, It has irreplaceable advantages in in-situ characterization of samples;

(2) In addition, the present invention can realize the independent acquisition of multiple information such as Raman spectrum, fluorescence spectrum and imaging, transmission spectrum and imaging, reflection spectrum and imaging, and can break through the limitation of only single signal collection in previous instrument and equipment design, realizing In situ acquisition of multi-level information of samples.

Description of drawings

Fig. 1 is the schematic diagram of multimode coupled in situ microspectral imaging system of the present invention;

Fig. 2 is a schematic structural diagram of the multi-mode coupling in-situ microspectral imaging system of the present invention;

Fig. 3 is the structure schematic diagram of Raman laser unit of the present invention;

Fig. 4 is a structural schematic diagram of the fluorescent excitation light source unit of the present invention;

Fig. 5 is a structural schematic diagram of a reflective lighting light source unit according to the present invention;

6 is a schematic structural diagram of the optical path switching module of the present invention, wherein a is the first optical path switcher, and b is the second optical path switcher;

Fig. 7 is a schematic structural diagram of the micro-imaging and spectral testing module of the present invention.

Detailed ways

The technical solution of the present invention will be specifically described below in conjunction with the accompanying drawings.

Example

A multi-mode coupling in-situ microspectral imaging system described in the present invention is composed of an inverted microscope, an excitation light source module, an optical path switching module, a microscopic imaging and a spectrum testing module, and the excitation light source module is It consists of three parts: a Raman light source unit, a fluorescent excitation light source unit, and a reflected illumination light source unit; among them, the Raman light source unit is composed of a 532nm high-brightness semiconductor laser 1, a first lens 2, a second lens 3, and a first iris diaphragm 4. Band-pass filter 5, dichroic beam splitter 6, long-pass filter 7, first fiber coupler 8, first reflector 9 and second reflector 10; Diaphragm 15, the third reflector 16, the second adjustable reflector 17 and xenon light source 19; the reflective illumination light source unit is composed of the fourth reflector 18 and white light source 20; the optical path switching module is composed of the first optical path switcher An optical path switcher 11 and a second optical path switcher 24, wherein the first optical path switcher 11 includes a third lens 12, a first adjustable reflector 13 and a fourth lens 14; the second optical path switcher 24 includes a fifth lens 25. The third adjustable reflector 26 and the sixth lens 27; the microscopic imaging and spectrum testing module is composed of the first microscopic imaging detector 23, the second optical fiber coupler 28, the spectrometer 29, the second microscopic Imaging is composed of a detector 30 and a computer 31, wherein the first detector 23 for microscopic imaging is a CMOS area array camera, and the second detector 30 for microscopic imaging is a CCD area array camera; the Raman light source in the excitation light source module The units are respectively connected to the first optical path switcher 11 and the second optical path switcher 24 in the optical path switching module, and the fluorescent excitation light source unit and the reflective illumination light source unit are respectively connected to the first optical path switcher 11; the first optical path in the optical path switching module The switcher 11 and the second optical path switcher 24 are connected with the inverted microscope 22 respectively, and the second optical path switcher 24 is connected with the second optical fiber coupler 28 in the microscopic imaging and spectrum testing module; The spectrometer 29 is provided with a second microscopic imaging detector 30, and the computer 31 is respectively connected in series with the first microscopic imaging detector 23 and the second microscopic imaging detector 30;

One C-mount interface of the inverted microscope 22 is connected to the first microscopic imaging detector 23, the other C-mount interface is connected to the second optical path switcher 24, and the rear side interface is connected to the third iris diaphragm 21 ;

In the excitation light source module, the two-color spectroscope 6 reflects the Raman excitation light of 532nm, and simultaneously transmits the Raman scattering signal of 500-2500nm; the long-pass filter 7 transmits the Raman scattering signal of 510-2500nm; the first reflector 9, The second reflecting mirror 10, the third reflecting mirror 16, and the fourth reflecting mirror 18 are all mirrors; the second adjustable reflecting mirror 17 is a 90 ° adjustable surface reflecting mirror;

Both the first adjustable reflector 13 and the third adjustable reflector 26 in the optical path switching module are 45° adjustable sliding reflectors;

When in use, by adjusting the excitation light source module, optical path switching module, and microscopic imaging and spectral testing module, the independent acquisition of multiple information of the micron-scale area of the sample under different excitation light sources is realized;

Select fluorescent carbon quantum dot nanofiber membrane sample as an example:

Measurement of Raman spectrum in any micron-scale region of fluorescent carbon quantum dot nanofiber film sample:

Put the sample on the stage of an inverted microscope 22, turn on the 532nm high-brightness semiconductor laser, and the 532nm green laser passes through the beam expander composed of the first lens 2 and the second lens 3, the first variable diaphragm 4, and the bandpass filter. Slice 5 reflects the incident light of 532nm on the dichroic beam splitter 6, passes through the first reflector 9 and the second reflector 10, and adjusts the first adjustable reflector 13 and the third variable light in the first optical path switcher 11 Diaphragm 21 finally enters the inverted microscope 22 to focus on the micro area of the sample, adjust the first iris diaphragm 4 and the third iris diaphragm 21 to adjust the laser intensity and spot size, and adjust the optical path switching module of the inverted microscope 22 to make the sample The Raman scattering signal returns along the original optical path and directly passes through the dichroic beam splitter. After being filtered and purified by the long-pass filter 7, the first optical fiber coupler 8 is used to guide the second optical path switcher 24, and the third adjustable reflector 26 is adjusted. The Raman scattering signal enters the spectrometer 29 through the sixth lens 27 through the second fiber coupler 28 to realize the collection of the Raman spectrum signal of the micro-area of the sample, and the Raman spectrum of the micro-area of the sample can be presented on the computer;

Analysis and measurement of the fluorescence spectrum and imaging of the same micro-area of the sample:

Turn on the xenon lamp light source 19, adjust the first adjustable reflector 13 and the second adjustable reflector 17 in the first optical path converter 11, so that the 532nm Raman excitation light cannot enter the inverted microscope 22 and the xenon lamp excitation light enters the inverted microscope 22 , focus on the same micro-area of the sample, adjust the optical path switching module of the inverted microscope 22, so that the fluorescence spectrum signal of the sample enters the first microscopic imaging detector 23, and realize the collection of the fluorescent image of the micro-area of the sample; adjust the optical path switching module of the inverted microscope 22 , so that the fluorescence spectrum signal of the sample passes through the fifth lens 25, and adjusts the third adjustable reflector 26 in the second optical path switcher 24, then passes through the sixth lens 27, and enters the spectrometer 29 and the second optical fiber coupler 28 through the second optical fiber coupler 28. 2. The detector 30 for microscopic imaging realizes the collection of the fluorescence spectrum signal of the same micro-area of the sample, and at this time displays the fluorescence spectrum and imaging of the same micro-area of the sample on the computer;

Analysis and measurement of reflection spectrum and imaging of the same micro-area of the sample:

Turn on the white light source 20, adjust the first adjustable reflector 13 and the second adjustable reflector 17 in the first optical path converter 11, so that neither the 532nm Raman excitation light nor the reflected illumination light can enter the inverted microscope 22, and only white light enters. The inverted microscope 22 is focused on the same micro-area of the sample, and the optical path switching module of the inverted microscope 22 is adjusted so that the reflection spectrum signal of the sample enters the first micro-imaging detector 23 to realize the acquisition of the reflection image of the micro-area of the sample; adjust the inverted microscope 22 Optical path switching module, so that the reflection spectrum signal of the sample passes through the fifth lens 25, and adjusts the third adjustable reflector 26 in the second optical path switcher 24, then passes through the sixth lens 27, and enters the spectrometer through the second fiber coupler 28 29 and the second micro-imaging detector 30 to realize the acquisition of the reflection spectrum signal of the same micro-area of the sample, and at this time, the reflection spectrum and imaging of the same micro-area of the sample are displayed on the computer;

Analysis and measurement of the transmission spectrum and imaging of the same micro-area of the sample:

Adjust the first adjustable reflector 13 and the second adjustable reflector 17 in the first optical path converter 11, so that external light sources such as 532nm Raman excitation light, fluorescence excitation light, and reflected illumination light cannot enter the inverted microscope 22. The inverted microscope 22 has its own light source and focuses on the same micro-area of the sample. Adjust the optical path switching module of the inverted microscope 22 so that the transmission spectrum signal of the sample enters the first micro-imaging detector 23 to realize the acquisition of the transmission image of the micro-area of the sample; Adjust the optical path switching module of the inverted microscope 22 so that the transmitted spectrum signal of the sample passes through the fifth lens 25, adjust the third adjustable mirror 26 in the second optical path switcher 24, pass through the sixth lens 27, and couple through the second optical fiber The detector 28 enters the spectrometer 29 and the second micro-imaging detector 30 to realize the acquisition of the transmission spectrum signal of the same micro-area of the sample. At this time, the transmission spectrum and imaging of the same micro-area of the sample are displayed on the computer.

The invention provides a multi-mode coupling in-situ microspectroscopy and imaging system, which can realize in-situ detection of microscopic Raman spectroscopy, microfluorescence spectroscopy and imaging, microtransmission spectroscopy and imaging, Microreflectance spectroscopy and imaging functions, so as to realize multi-dimensional and multi-mode rapid detection of sample micro-regions.

The above are the preferred embodiments of the present invention, and all changes made according to the technical solution of the present invention, when the functional effect produced does not exceed the scope of the technical solution of the present invention, all belong to the protection scope of the present invention.

Claims (4)

1. A multimode coupling in-situ microspectral imaging system is characterized in that the system is composed of an inverted microscope, an excitation light source module, an optical path switching module, a microscopic imaging and a spectrum testing module, and the excitation light source module It is composed of three parts: Raman light source unit, fluorescent excitation light source unit, and reflected illumination light source unit; among them, the Raman light source unit is composed of a 532nm high-brightness semiconductor laser (1), a first lens (2), and a second lens (3) , the first iris diaphragm (4), the band-pass filter (5), the dichroic beam splitter (6), the long-pass filter (7), the first fiber coupler (8), the first reflector (9) and the second reflector (10); the fluorescence excitation light source unit is composed of the second iris diaphragm (15), the third reflector (16), the second adjustable reflector (17) and the xenon light source (19) The reflective lighting light source unit is composed of a fourth reflector (18) and a white light source (20); the optical path switching module is composed of an optical path switcher, a first optical path switcher (11) and a second optical path switcher (24) , wherein the first optical path switcher (11) includes a third lens (12), a first adjustable mirror (13) and a fourth lens (14); the second optical path switcher (24) includes a fifth lens (25) , the third adjustable reflector (26) and the sixth lens (27); the microscopic imaging and spectral testing module is composed of the first microscopic imaging detector (23), the second optical fiber coupler (28), A spectrometer (29), a second microscopic imaging detector (30) and a computer (31), wherein the first microscopic imaging detector (23) is a CMOS area array camera, and the second microscopic imaging detector ( 30) is a CCD area array camera; the Raman light source unit in the excitation light source module is respectively connected to the first optical path switcher (11) and the second optical path switcher (24) in the optical path switching module, and the fluorescence excitation light source unit and the reflection The illumination light source unit is respectively connected to the first optical path switcher (11); the first optical path switcher (11) and the second optical path switcher (24) in the optical path switching module are respectively connected to the inverted microscope (22), and the second optical path switcher The detector (24) is connected with the second optical fiber coupler (28) in the microscopic imaging and spectral testing module; the spectrometer (29) in the microscopic imaging and spectral testing module is provided with a second microscopic imaging detector (30) ), the computer (31) is respectively connected in series with the first detector for microscopic imaging (23) and the second detector for microscopic imaging (30). 2. A multi-mode coupled in-situ microspectral imaging system according to claim 1, characterized in that a C-mount interface of the inverted microscope (22) is connected to the first detector for microscopic imaging (23 ), the other C-mount interface is connected with the second optical path switcher (24), and the rear interface is connected with the third iris diaphragm (21). 3. A multi-mode coupled in-situ microspectral imaging system according to claim 1, characterized in that the dichroic beamsplitter (6) in the excitation light source module reflects 532 nm Raman excitation light and simultaneously transmits 500- Raman scattering signal at 2500nm; long-pass filter (7) transmits Raman scattering signal at 510-2500nm; first reflector (9), second reflector (10), third reflector (16) and fourth reflector The reflectors (18) are all reflective mirrors; the second adjustable reflector (17) is a 90° adjustable surface reflector. 4. A multi-mode coupling in-situ microspectral imaging system according to claim 1, characterized in that the first adjustable mirror (13) and the third adjustable mirror (26) in the optical path switching module are both It is a 45° adjustable sliding mirror.