CN106918580B - Super-resolution fluorescence microscope system with nano infrared imaging function - Google Patents
Super-resolution fluorescence microscope system with nano infrared imaging function Download PDFInfo
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- CN106918580B CN106918580B CN201710095962.3A CN201710095962A CN106918580B CN 106918580 B CN106918580 B CN 106918580B CN 201710095962 A CN201710095962 A CN 201710095962A CN 106918580 B CN106918580 B CN 106918580B
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- 238000003331 infrared imaging Methods 0.000 title claims abstract description 32
- 238000003384 imaging method Methods 0.000 claims abstract description 22
- 238000009423 ventilation Methods 0.000 claims abstract 2
- 239000000523 sample Substances 0.000 claims description 94
- 239000000919 ceramic Substances 0.000 claims description 8
- 230000008859 change Effects 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 4
- 230000004075 alteration Effects 0.000 claims description 3
- 230000005284 excitation Effects 0.000 claims description 3
- 238000010857 super resolution fluorescence microscopy Methods 0.000 description 9
- 238000010586 diagram Methods 0.000 description 8
- 238000012876 topography Methods 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 238000000386 microscopy Methods 0.000 description 3
- 238000012805 post-processing Methods 0.000 description 3
- 238000004451 qualitative analysis Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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Abstract
本申请公开了一种具有纳米红外成像功能的超分辨荧光显微系统及其级联平台。所述具有纳米红外成像功能的超分辨荧光显微系统除了具有普通超分辨荧光显微系统的成像功能外，还可对样品进行纳米红外成像。所述级联平台至少包括：级联平台本体；样品室，由所述级联平台本体的轴向延伸的闭合侧壁和所述级联平台本体的上表面围合而成；通气孔，设置于所述侧壁上，用于填充气体，以保证样品室恒温；样品夹持器，设置于所述样品室内，用于固定待测样品；调节旋钮，设置于所述级联平台本体上，用于在水平方向上调节所述待测样品。
The present application discloses a super-resolution fluorescence microscope system with nano-infrared imaging function and a cascade platform thereof. The super-resolution fluorescence microscope system with nano-infrared imaging function can perform nano-infrared imaging on the sample in addition to the imaging function of the ordinary super-resolution fluorescence microscope system. The cascade platform at least includes: a cascade platform body; a sample chamber, which is enclosed by an axially extending closed side wall of the cascade platform body and an upper surface of the cascade platform body; a ventilation hole, provided with On the side wall, it is used to fill the gas to ensure the constant temperature of the sample chamber; the sample holder is arranged in the sample chamber to fix the sample to be tested; the adjustment knob is arranged on the main body of the cascade platform, Used to adjust the sample to be tested in the horizontal direction.
Description
Technical Field
The application relates to a super-resolution fluorescence microscope system with a nanometer infrared imaging function and further relates to a cascade platform.
Background
With the continuous development of the microscopic technology, the super-resolution fluorescence microscope is favored by users because the imaging resolution is improved by one order of magnitude by breaking through the limit of diffraction limit. With the continuous improvement of user demands, only obtaining a super-resolution image cannot meet experimental demands, if a sample is imaged, the super-resolution image in the sample can be obtained, a nano infrared topography map of the surface of the sample can also be obtained, and meanwhile, the capability of the sample for absorbing laser with different wavelengths can be researched to become an ideal choice for users.
Disclosure of Invention
The invention provides a super-resolution fluorescence microscope system with a nanometer infrared imaging function. The function is complete. The system can obtain a nano infrared topography map and a photoinduction force map on the surface of a sample, can perform qualitative analysis on the internal information of the sample to obtain a super-resolution map, and can highly coincide various acquired images while integrating the system so as to deeply and finely study the sample. The system overcomes the defect that the super-resolution fluorescence microscope can only realize qualitative analysis of samples for a long time. The functional modules of the invention expand the application field and the application range of the microscope system.
The cascade platform for the super-resolution fluorescence microscope system with the nanometer infrared imaging function comprises:
a cascading platform body;
the sample chamber is formed by enclosing an axially extending closed side wall of the cascade platform body and the upper surface of the cascade platform body;
the vent hole is arranged on the side wall and is used for filling gas so as to ensure the constant temperature of the sample chamber;
the adjusting knob is arranged on the cascading platform body and used for adjusting the sample to be detected in the horizontal direction;
and the sample holder is used for fixing the sample to be measured.
Wherein the sample chamber, in use, forms a sealed closed space region. The gas can be filled through the vent holes according to requirements, and a constant temperature environment is provided for the sample to be detected.
In addition, two air vents can be generally arranged, wherein one air vent is used for air inlet, and the other air vent is used for air outlet. Thus, in one embodiment, there are at least two vent holes in the side wall.
In a specific embodiment, the side wall of the sample chamber is further provided with a light through hole. The laser emitted by the laser can irradiate the target area of the sample to be measured through the light through hole.
In one embodiment, the cascade platform can be precisely controlled by an upper computer to perform nanoscale adjustment on the sample to be measured in the horizontal direction.
The second application provides a super-resolution fluorescence microscope system with a nano infrared imaging function, wherein the microscope system comprises the cascade platform, a nano infrared imaging function module and a super-resolution fluorescence microscope module; the nano infrared imaging functional module and the super-resolution fluorescence microscopic module are respectively arranged on two sides of the cascade platform.
In a specific embodiment, the microscopy system further comprises a light-induced force module; the light induction force module and the nano infrared imaging functional module are positioned on the same side of the cascade platform. After the laser with different wavelengths irradiates the sample to be detected, the sample to be detected can display a curve in real time due to different absorption capacities of the laser with different wavelengths, and finally a light absorption spectrum line and a light induction force diagram are obtained.
In one embodiment, the nano infrared imaging functional module comprises an infrared feedback laser, a probe, a piezoelectric ceramic scanner and a high-sensitivity detector; wherein infrared feedback laser instrument is used for launching laser, the probe is used for scanning the sample surface that awaits measuring, piezoceramics scanner is used for coarse tuning and/or fine setting the probe, high sensitive detector is used for receiving via the laser beam that the probe reflection was returned.
In a specific embodiment, the super-resolution fluorescence microscopy module comprises a laser, a beam expander, a diaphragm, a focusing lens, a dichroic mirror, an objective lens, a convergence unit and an imaging camera;
the laser is used for providing needed exciting light for the super-resolution imaging experiment;
the beam expander is used for expanding the light beam into an ideal experimental light spot size;
the diaphragm is used for filtering out stray light so as to reduce aberration;
the focusing mirror is used for focusing the light beam on an imaging focal plane of a target of the sample to be detected;
a dichroic mirror for reflecting the excitation light emitted from the laser via the lens (the dichroic mirror) and transmitting the fluorescence emitted from the sample to be measured via the lens (the dichroic mirror);
and the convergence unit is used for converging the fluorescence emitted by the sample to be detected onto the imaging camera.
In one embodiment, the light-induced force module comprises:
the quantum cascade laser is used for emitting laser beams;
a deflector for adjusting the parabolic mirror to change the direction of the laser beam;
and the paraboloidal mirror is used for converging the laser beam with the direction changed by the deflector to a focal plane of a target area of the imaging sample to be measured.
In a particular embodiment, the deflector and the parabolic mirror are fixed by a holder arranged on a sidewall of the sample chamber.
In one embodiment, the side wall of the sample chamber is provided with a fixer for fixing the deflector and the paraboloidal mirror on the inner side wall of the sample chamber.
In one embodiment, the area imaged by the nano infrared imaging functional module is identical to the area imaged by the super-resolution fluorescence microscopy module.
In one embodiment, the sample to be tested is a fluorescently labeled sample. The fluorescently labeled sample can be a sample labeled with a fluorescent dye.
In a preferred embodiment, the area imaged by the nano infrared imaging function module, the area imaged by the super-resolution fluorescence microscopy module and the area imaged by the light-induced force module are the same.
Drawings
FIG. 1 shows a schematic diagram of a super-resolution fluorescence microscopy system with nano-infrared imaging.
Fig. 2 shows a schematic image path of the microscope system of fig. 1.
FIG. 3 shows a three-dimensional block diagram of a cascade platform for the microscopy system of FIG. 1.
FIG. 4 shows a schematic diagram of another super-resolution fluorescence microscopy system with nano-infrared imaging.
Fig. 5 shows a schematic image path of the microscope system of fig. 4.
FIG. 6 shows a three-dimensional block diagram of a cascade platform for the microscopy system of FIG. 4.
List of parts and reference numerals:
10, an infrared feedback laser; 12, a high-sensitivity detector; 14, a piezo ceramic scanner; 16, a probe; 20, a quantum cascade laser; 22, a deflector; 24, a parabolic mirror; 30, a sample bin; 32, a cascade platform; 40, an objective lens; 42, a dichroic mirror; 44, a focusing mirror; 46, a beam expander; 48, a diaphragm; 410, a laser; 412, a convergence unit; 414, an imaging camera; 2-1, cascading a platform body; 2-2, a sample chamber; 2-3, vent holes; 2-4, adjusting a knob; 2-5, a sample holder; 2-6, a light through hole; 2-7, a fixer.
Detailed Description
The present application will be described in detail with reference to examples, but the present application is not limited to these examples.
Example 1
As shown in fig. 1, a schematic diagram of a super-resolution fluorescence microscopy system with nano-infrared imaging function according to the present application is shown, which includes a nano-infrared imaging function module, a super-resolution fluorescence microscopy module, and a sample cascade platform.
Specifically, the nano infrared imaging functional module comprises: an infrared feedback laser 10, a high-sensitivity detector 12, a piezoelectric ceramic scanner 14 and a probe 16. The imaging path is shown in fig. 2. Laser emitted by the infrared feedback laser 10 is reflected by a probe 16 on a piezoelectric ceramic scanner 14 and then enters the high-sensitivity detector 12, the reflected light changes along with the scanning of the probe 16 on a sample and then irradiates different positions of the high-sensitivity detector 12, and the high-sensitivity detector 12 processes the position change information of the reflected light to obtain a nano infrared topography of the sample. The probe 16 for scanning the sample can be fine tuned for its position change by the piezo ceramic scanner 14 to scan the desired target imaging area of the sample.
The super-resolution fluorescence microscopy module comprises: laser 410, beam expander 46, stop 48, focusing lens 44, dichroic mirror 42, objective lens 40, converging unit 412, and imaging camera 414. The imaging path is shown in fig. 2. Laser emitted by the laser 410 is expanded by the beam expander 46 and then reaches the surface of the sample through the focusing lens 44, the dichroic mirror 42 and the objective lens 40, the sample marked by the fluorescent dye is excited by the laser to emit fluorescence, the fluorescence is imaged at the imaging camera 414 through the objective lens 40, the dichroic mirror 42 and the converging unit 412, and the super-resolution image is obtained after post-processing of the image.
And obtaining a super-resolution image and a nano infrared topography image of the same target area of the sample after post-processing of the image.
The sample cascade platform comprises: a sample bin 30 and a cascade platform 32. The sample chamber 30 is used for placing experimental samples, and the samples to be tested are prevented from being polluted by the outside.
As shown in FIG. 3, a specific configuration of the cascade platform 32 includes a cascade platform body 2-1; the sample chamber 2-2 is formed by enclosing an axially extending closed side wall of the cascade platform body 2-1 and the upper surface of the cascade platform body 2-1, and a sealed closed space region can be formed in use; furthermore, the sample chamber 2-2 is also provided with at least two vent holes 2-3 for the gas to enter and exit, and the constant temperature experiment can be carried out by injecting gas according to the experiment requirement; an adjusting knob 2-4 for roughly adjusting the horizontal movement of the sample; the sample holders 2-5 can accurately hold the sample to prevent the sample from drifting, and meanwhile, the holder with the optimal elastic coefficient is selected to hold the sample to avoid crushing the sample.
Example 2
As shown in fig. 4, a schematic diagram of another super-resolution fluorescence microscopy system with nano-infrared imaging function of the present application is shown. In embodiment 2, the same components as those in embodiment 1 are assigned the same reference numerals, and description thereof is omitted, and only the differences between embodiment 2 and embodiment 1 will be described.
The super-resolution fluorescence microscope system with the nano infrared imaging function shown in fig. 4 comprises a nano infrared imaging function module, a super-resolution fluorescence microscope module, a sample cascade platform and a light induction force module. The functions of the nano infrared imaging functional module and the super-resolution fluorescence microscopic module are the same as those of the embodiment 1, and are not described again.
The light-induced force module includes: quantum cascade laser 20, deflector 22, parabolic mirror 24. The induced light emitted by the quantum cascade laser 20 is reflected and converged by the parabolic mirror 24 and then strikes the sample, the laser is used as the induced light to act on the sample, the absorption capacity of the sample to the laser with different wavelengths is different, and finally, a light absorption spectrum line and a light induction force diagram corresponding to the wavelengths are formed. When the laser deviates from the ideal target area, the deflector 22 will precisely adjust the turning of the parabolic mirror 24 and thus change the direction of the induced light propagation. The imaging path of the microscope system is shown in fig. 5.
The working flow of the microscope system is as follows in combination with fig. 4 and 5: placing a sample chamber 30 containing a sample on a cascade platform 32, adjusting the piezo ceramic scanner 14 to move the probe 16 to a desired imaging area of the sample; the deflector 22 is adjusted to twist to drive the paraboloidal mirror 24 to rotate, and laser emitted by the quantum cascade laser 20 is converged to a target imaging area after being reflected by the paraboloidal mirror 24; when the probe 16 and the inducing light act on the same area of the sample, the target area starts to be scanned, and the sample generates different absorption curves according to the change of the laser wavelength due to the different absorption capacities of different samples to the laser with different wavelengths.
The laser 410 is turned on, the laser emitted by the laser irradiates the target area on the sample, which is the same as the nano infrared topography imaging, the sample marked by the fluorescent dye generates fluorescence after being irradiated by the laser, and the fluorescence is finally imaged at the imaging camera 414. During the propagation of the laser beam, each optical element needs to be adjusted so that the laser light strikes the center of the optical element, so that the laser light does not deviate from the main optical axis of each optical element.
After the images are subjected to post-processing, a super-resolution map, a photoinduction force map, a nano infrared morphology map and a light absorption spectrum line of the same target area of the sample can be obtained.
The cascade platform in the sample cascade platform has all functions of the embodiment 1, and also comprises light through holes 2-6 specially reserved for the quantum cascade laser and a fixer 2-7 for fixing the deflector, and only the deflector 22 with the paraboloidal mirror 24 is fixed on the fixer 2-7.
Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.
Claims (6)
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