CN107727614B - Space-time resolution spectral imaging system - Google Patents
Space-time resolution spectral imaging system Download PDFInfo
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- CN107727614B CN107727614B CN201710947393.0A CN201710947393A CN107727614B CN 107727614 B CN107727614 B CN 107727614B CN 201710947393 A CN201710947393 A CN 201710947393A CN 107727614 B CN107727614 B CN 107727614B
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- G—PHYSICS
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
- G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/12—Circuits of general importance; Signal processing
Abstract
The invention belongs to the technical field of optical microscopy instrument manufacturing technology and spectral imaging, and particularly relates to a space-time resolution spectral imaging system, which comprises a pulse laser light source system, a microscope system, an imaging module and an image processing module; the emission wavelength of the pulse laser light source system is lambda1And λ2A pulsed laser beam alternating therebetween; the microscope system and the imaging module are arranged on an output light path of the pulse laser light source system; the image processing module extracts a picture obtained by the imaging module, and obtains a picture representing the resonance wavelength of the plasma on the local surface of the target sample by performing operation processing on the scattered light intensity of the picture, so that space-time resolution spectral imaging is realized; the space-time resolution spectral imaging system can simultaneously detect the local surface plasma resonance scattering wavelength of a plurality of target samples in a visual field, the time resolution of the acquired spectrum is reduced from the second level to the millisecond level in the prior art, and the spatial resolution is obviously improved.
Description
Technical Field
The invention belongs to the technical field of optical microscopy instrument manufacturing technology and spectral imaging, and particularly relates to a space-time resolution spectral imaging system.
Background
Metal or semiconductor nanomaterials have received much attention because of their stable localized surface plasmon scattering properties. In order to utilize the refractive index ultrasensitive response capability of the scattered light to the surface of the nano material, various dark field microscopes have been developed to study the application of the plasma nanoprobe in chemical and biological analysis. The dark field microscopes available at present are mainly divided into two types: a spectral detection type and a scattered light intensity detection type. The spectrum detection type dark field microscope analyzes the plasma resonance scattering spectrum by using a broadband light source and a spectrometer, and the scattered light intensity type analyzes the scattering cross section of the nano particles under a specific wavelength by using laser with a single wavelength as a light source. The two dark field microscopes have respective advantages and disadvantages, for example, the spectrum detection type dark field microscope can stably collect the scattering spectrum of single-particle nano particles, but the spectrum collection of the nano particles needs to be carried out one by one, the spectrum collection of a plurality of particles cannot be carried out simultaneously, the sampling flux is low, and the detection sensitivity is influenced due to the strong scattering background in a biological system. The scattering light intensity type dark field microscope avoids the interference of biological background scattering, has extremely high time resolution and sensitivity, but is easy to be interfered by the fluctuation of light intensity of a light source, system mechanical error and direction change of an asymmetric target sample due to the fact that the scattering light intensity of the target sample under the wavelength is detected, and the spectral data of the target sample cannot be accurately measured.
Disclosure of Invention
The invention solves the technical problems in the prior art and provides a space-time resolution spectral imaging system.
In order to solve the problems, the technical scheme of the invention is as follows:
the space-time resolution spectral imaging system comprises a pulse laser light source system, a microscope system, an imaging module and an image processing module;
the emission wavelength of the pulse laser light source system is lambda1And λ2A pulsed laser beam alternating therebetween;
the microscope system and the imaging module are arranged on an output light path of the pulse laser light source system;
the image processing module extracts the picture obtained by the imaging module, and obtains the local surface plasma resonance wavelength of the target sample by performing operation processing on the scattered light intensity of the picture, so that space-time resolution spectral imaging is realized.
Preferably, the laser light source system comprises a wavelength λ1Of a first incident light source of wavelength lambda2The incident light of the first incident light source is reflected by the dichroic mirror and is converged into a beam with the wavelength at lambda through the incident light of the second incident light source penetrating the dichroic mirror1And λ2Alternating pulsed light beams in between.
Preferably, the space-time resolution spectral imaging system further comprises a waveform generator, wherein the waveform generator is connected with the first incident light source, the second incident light source and the imaging module; the waveform generator simultaneously outputs three different trigger signals to the first incident light source, the second incident light source and the imaging module; wherein the trigger signals output to the first incident light source and the second incident light source are a pair of trigger signals with opposite phases and same frequency f1The square wave signal of (1) and the trigger signal input to the imaging module is of frequency f2The pulse signal of f2=2f1。
Preferably, a polarizing plate is disposed between the dichroic mirror and the microscope system.
Preferably, the incident angles of the first incident light source and the second incident light source are independently adjustable.
Preferably, the microscope system is any one of a total internal reflection microscope, a transmission dark field microscope, a reflection dark field microscope, a light sheet microscope, a confocal microscope and an endoscope.
Preferably, the image processing module splits the picture sequence obtained by the imaging module into a sequence 1 and a sequence 2, the sequence 1 being defined by the target sample for a wavelength λ1The sequence 2 consists of the target sample for a wavelength λ2A scatter photograph of the light source of (1).
The photos of the sequence 1 and the sequence 2 generate photos of the sequence 3 through the following relational operation:
wherein
I1The intensity value of each pixel point of the sequence 1 of photographs,
I2the intensity value of each pixel point of the sequence 2 of photographs,
I3expressing the intensity value of each pixel point of the sequence 3 photo, wherein the unit is nm, namely the local surface plasma resonance scattering wavelength of the target nano-particles;
B=(λ1+λ2)/2,
A=-C0k, said C0Is the scattering cross section when the local surface plasmon resonance scattering wavelength of the target sample is B, and k is the local surface plasmon resonance scattering wavelength of the target sample at lambda1And λ2In time between, it is at λ1Or λ2The scattering cross section at (a) is the absolute value of the slope of the curve as a function of the scattering wavelength of the local surface plasmon resonance.
Preferably, the targetThe local surface plasmon resonance scattering wavelength of the sample falls within lambda1、λ2In the meantime.
Preferably, said λ1、λ2Selected from any one of values from 300nm to 2000 nm.
Preferably, said f1Selected from any one of values from 0.01Hz to 100 MHz.
A space-time resolved spectroscopic imaging system is used to detect a target sample comprising nanoparticles having localized surface plasmon resonance scattering properties.
Preferably, the space-time resolved spectroscopic imaging system is used for detecting a scattered light wavelength signal of a nanoparticle within a probe cell or a nanoparticle scattered light wavelength signal in tumor tissue.
When the microscope system of the space-time resolution spectral imaging system is a total internal reflection microscope, the pulse light beam irradiates the sample stage at a high angle of more than 70 degrees through an oil lens with a high numerical aperture; producing high slant angle lamellar light sheet (HILO); and irradiating the nano particles by using HILO to generate local surface plasmon resonance scattering, wherein the scattered light signals are collected by the oil lens and enter the imaging module to perform dark field scattering imaging.
Compared with the prior art, the invention has the advantages that,
according to the time-space resolution spectral imaging system, the scattering intensity of two wavelengths of a pulse laser beam is directly converted into spectral data through the image processing module, the local surface plasma resonance scattering wavelengths of a plurality of target samples in a visual field can be detected simultaneously, and the defect of low sampling flux of a spectral detection type dark field microscope is overcome; meanwhile, the laser light source is adopted to replace a broadband white light source, so that the defect that the spectrum detection type dark field microscope has strong scattering background in a biological system is overcome;
the invention directly converts the scattering intensity of two wavelengths of the pulse laser beam into the local surface plasma resonance scattering wavelength through the image processing module, rather than detecting the scattering intensity of the target sample under the wavelength like the traditional single-wavelength scattering light intensity type dark field microscope, thereby eliminating the interference caused by the fluctuation of the light source intensity, the system mechanical error and the direction change of the asymmetric target sample;
the invention is a novel dark field micro-spectrum imaging means, compared with the current dark field micro-imaging technology, the invention has obvious improvement in function and performance: the time resolution of the acquired spectrum of the space-time resolution spectrum imaging system is reduced from the second level to the millisecond level in the prior art; the spatial resolution is significantly improved, the prior art can only detect a few particles in the spectrometer slit, while the present invention can detect all particles in the field of view.
Drawings
FIG. 1 is a schematic view of a space-time-resolved spectral imaging system of example 1;
FIG. 2 is a schematic view of a space-time-resolved spectral imaging system according to example 2;
fig. 3 is a trigger signal diagram of the pulsed laser light source system and the imaging module of embodiment 2;
FIG. 4 is a schematic diagram of a space-time-resolved spectral imaging system based on a total internal reflection microscope of example 3;
fig. 5 is a trigger signal diagram of the pulsed laser light source system and the imaging module of embodiment 3;
FIG. 6 is a partial surface plasmon resonance wavelength spectrum of gold nanorods of example 3;
FIG. 7 is a partial surface plasmon resonance wavelength variation curve of the gold nanorods modified by the thermosensitive polymer polyisoprenamide of example 4;
in the figure, 1 is a total internal reflection microscope, 2 is a first incident light source, 3 is a second incident light source, 4 is an imaging module, 5 is an image processing module, 6 is a dichroic mirror, and 7 is a gold nanorod.
Detailed Description
Example 1:
referring to fig. 1, the space-time resolution spectral imaging system comprises a pulsed laser light source system, a microscope system, an imaging module and an image processing module;
the emission wavelength of the pulse laser light source system is lambda1And λ2Pulsed laser light alternating betweenBundling;
the microscope system and the imaging module are arranged on an output light path of the pulse laser light source system;
the image processing module extracts the picture obtained by the imaging module, and obtains the picture representing the resonance wavelength of the plasma on the local surface of the target sample by performing operation processing on the scattered light intensity of the picture, so that space-time resolution spectral imaging is realized.
Example 2:
referring to fig. 2, the space-time resolution spectral imaging system comprises a waveform generator, a pulse laser light source system, a microscope system, an imaging module and an image processing module;
the laser light source system comprises a wavelength lambda1Of a first incident light source of wavelength lambda2The incident light of the first incident light source is reflected by the dichroic mirror and is converged into a beam with the wavelength at lambda through the incident light of the second incident light source penetrating the dichroic mirror1And λ2A pulsed light beam alternating therebetween;
the waveform generator is connected with the first incident light source, the second incident light source and the imaging module; the waveform generator simultaneously outputs three different trigger signals to the first incident light source, the second incident light source and the imaging module; wherein the trigger signals output to the first incident light source and the second incident light source are a pair of trigger signals with opposite phases and same frequency f1The square wave signal of (1) and the trigger signal input to the imaging module is of frequency f2The pulse signal of f2=2f1;
The incident angles of the first incident light source and the second incident light source can be independently adjusted;
the microscope system and the imaging module are arranged on an output light path of the pulse laser light source system;
the image processing module splits a picture sequence obtained by the imaging module into a sequence 1 and a sequence 2, wherein the sequence 1 is formed by a target sample with a wavelength of lambda1The sequence 2 consists of the target sample for a wavelength λ2A scatter photograph of the light source of (1).
The photos of the sequence 1 and the sequence 2 generate photos of the sequence 3 through the following relational operation:
wherein
I1The intensity value of each pixel point of the sequence 1 of photographs,
I2the intensity value of each pixel point of the sequence 2 of photographs,
I3expressing the intensity value of each pixel point of the sequence 3 photo, wherein the unit is nm, namely the local surface plasma resonance scattering wavelength of the target nano-particles;
B=(λ1+λ2)/2,
A=-C0k, said C0Is the scattering cross section when the local surface plasmon resonance scattering wavelength of the target sample is B, and k is the local surface plasmon resonance scattering wavelength of the target sample at lambda1And λ2In time between, it is at λ1Or λ2The scattering cross section at (a) is the absolute value of the slope of the curve as a function of the scattering wavelength of the local surface plasmon resonance.
Wherein the local surface plasmon resonance scattering wavelength of the target sample falls within λ1、λ2More preferably, said λ1、λ2And may range from 300nm to 2000 nm.
F is1May range from 0.01Hz to 100 MHz.
Example 3:
the difference from example 1 or 2 is that the microscope system of the space-time resolution spectral imaging system employs a total internal reflection microscope, a transmission dark field microscope, a reflection dark field microscope, a light sheet microscope, a confocal microscope or an endoscope.
The space-time resolution spectral imaging system based on the total internal reflection microscope is shown in figure 3, and the pulse light beam passes through an oil lens with high numerical aperture and irradiates a sample stage at a high angle of more than 70 degrees; producing high tilt angle lamellar light (HILO); and irradiating the nano particles by using HILO to generate local surface plasmon resonance scattering, wherein the scattered light signals are collected by the oil lens and enter the imaging module to perform dark field scattering imaging.
In order to obtain two beams of light with parallel polarization directions, a polarizing plate may be arranged between the dichroic mirror and the microscope system.
The space-time resolution spectral imaging system based on the total internal reflection microscope is used for imaging the resonance scattering wavelength of the local surface plasma of the gold nanorod, and the adopted lambda1=735nm,λ2=785nm,f1The frequency is 50Hz, the result is shown in figure 6, and the local surface plasma resonance scattering wavelength (in the figure, lambda) of all the gold nanorods in the visual field can be detected simultaneouslypeakRepresenting the local surface plasmon resonance scattering wavelength).
Example 4:
a space-time resolved spectroscopic imaging system is used to detect a target sample comprising nanoparticles having localized surface plasmon resonance scattering properties.
Detection of local surface plasmon resonance scattering wavelength of thermosensitive polymer polyisopropenyl amide modified gold nanorods by using space-time resolution spectral imaging system based on total internal reflection microscope (TIR) described in example 3, and adopted lambda1=735nm,λ2=785nm,f1The frequency is 500Hz, the time resolution of the collected spectrum is 2ms, and the result is shown in FIG. 7, two gold nanorods of which the surfaces are modified by thermosensitive polymer polyisopropenyl amide are heated by laser, namely the gold nanorods 1 and the gold nanorods 2 in the figure. The local surface plasmon resonance scattering wavelength of the gold nanorods was red-shifted within 4ms after the laser heating (heating was started at a time of 20 ms), and blue-shifted within 4ms after the heating was stopped (heating was stopped at a time of 370 ms).
Based on the above experiment, the space-time resolution spectral imaging system of the present invention can be used to detect the scattered light wavelength signal of the nanoparticle in the probe cell or the scattered light wavelength signal of the nanoparticle in the tumor tissue.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and all equivalent substitutions or substitutions made on the above-mentioned embodiments are included in the scope of the present invention.
Claims (8)
1. The space-time resolution spectral imaging system is characterized by comprising a pulse laser light source system, a microscope system, an imaging module and an image processing module;
the emission wavelength of the pulse laser light source system is lambda1And λ2A pulsed laser beam alternating therebetween;
the microscope system and the imaging module are arranged on an output light path of the pulse laser light source system;
the image processing module extracts the picture obtained by the imaging module, and obtains the local surface plasma resonance wavelength of the target sample by performing operation processing on the scattered light intensity of the picture, so as to realize space-time resolution spectral imaging;
the laser light source system comprises a wavelength lambda1Of a first incident light source of wavelength lambda2The incident light of the first incident light source is reflected by the dichroic mirror and is converged into a beam with the wavelength at lambda through the incident light of the second incident light source penetrating the dichroic mirror1And λ2A pulsed light beam alternating therebetween;
the space-time resolution spectrum imaging system also comprises a waveform generator, wherein the waveform generator is connected with the first incident light source, the second incident light source and the imaging module; the waveform generator simultaneously outputs three different trigger signals to the first incident light source, the second incident light source and the imaging module; wherein the trigger signals output to the first incident light source and the second incident light source are a pair of trigger signals with opposite phases and same frequency f1The square wave signal of (1) and the trigger signal input to the imaging module is of frequency f2The pulse signal of f2=2f1。
2. The spatiotemporal-resolving spectral imaging system of claim 1, wherein a polarizer is disposed between the dichroic mirror and the microscope system.
3. The space-time resolved spectral imaging system of claim 1 wherein the incident angles of the first incident light source and the second incident light source are independently adjustable.
4. The spatio-temporal resolution spectral imaging system according to claim 1, wherein said microscope system is any one of a total internal reflection microscope, a transmission dark field microscope, a reflection dark field microscope, a light sheet microscope, a confocal microscope, an endoscope.
5. The spatio-temporal resolution spectral imaging system according to claim 1, wherein the image processing module splits the sequence of pictures obtained by the imaging module into a sequence 1 and a sequence 2, the sequence 1 being defined by a target sample pair having a wavelength λ1The sequence 2 consists of the target sample for a wavelength λ2The light source of (1);
the photos of the sequence 1 and the sequence 2 generate photos of the sequence 3 through the following relational operation:
wherein
I1The intensity value of each pixel point of the sequence 1 of photographs,
I2the intensity value of each pixel point of the sequence 2 of photographs,
I3representing the intensity value of each pixel point of the sequence 3 photo, namely the local surface plasma resonance scattering wavelength of the target nano-particles;
B=(λ1+λ2)/2,
A=-C0k, said C0Is the scattering cross section when the local surface plasmon resonance scattering wavelength of the target sample is B, and k is the local surface plasmon resonance scattering wavelength of the target sample at lambda1And λ2In time between, it is at λ1Or λ2The scattering cross section at (a) is the absolute value of the slope of the curve as a function of the scattering wavelength of the local surface plasmon resonance.
6. The spatio-temporally resolved spectral imaging system of claim 1, wherein λ1、λ2Selected from any one of values from 300nm to 2000 nm.
7. The spatio-temporally resolved spectral imaging system of claim 1 wherein f1Selected from any one of values from 0.01Hz to 100 MHz.
8. The spatio-temporal resolution spectroscopic imaging system of any one of claims 1-7 for detecting a target sample comprising nanoparticles having localized surface plasmon resonance scattering properties.
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