CN109745010A - Positioning formula adsorbs microscope detection device and laser scanning microscope - Google Patents
Positioning formula adsorbs microscope detection device and laser scanning microscope Download PDFInfo
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Abstract
The embodiment of the present invention provides a kind of positioning formula absorption microscope detection device and laser scanning microscope.Wherein, above-mentioned positioning formula absorption microscope detection device includes absorption shell and mini microscope probe, absorption case inside, which is provided with, drives mini microscope to pop one's head in the telecontrol equipment that moves horizontally along X-Y axis, is provided in mini microscope probe for receiving and conducting output pulsed laser signal to the first optical path of the intracellular autofluorescence substance of life entity, acquisition and conduct autofluorescence substance and be excited the fluorescence signal of generation and the second optical path and zoom motor of second harmonic signal.Positioning formula absorption microscope detection device provided in an embodiment of the present invention and laser scanning microscope pass through the absorption shell of micromation, running gear and the mini microscope probe for being provided with the first optical path, the second optical path and zoom motor, realize that the precise positioning to the cyto-architectural three-dimensional different levels in life entity skin histology detects, structure is simple, easy to use.
Description
Technical field
The present embodiments relate to laser scanning microscope technical fields more particularly to a kind of positioning formula absorption microscope to visit
Survey device and laser scanning microscope.
Background technique
With the continuous development of medicine and biology, people are to cellular morphology, institutional framework or stomach in animal life
The research of middle fiber condition achieves marked improvement, particularly by the pulsed laser radiation excitation of near infrared region and by suitable
The detection of high sensitivity receiver, fluorescence signal and second harmonic signal are obtained, to obtain the biological cell form of living body
The relevant technologies achieve significant achievement.
And it is based on fluorescence signal, second harmonic signal and CARS (Coherent anti-Stokes Raman
Scattering, related anti-Stokes Raman scattering) signal, to obtain the correlation detection equipment of biological cell form, upper
It states and is play an important role in the application of technology.The existing imaging device for being used for human body cell or tissue detection, it is mainly three-dimensional
Non-linear laser flying-spot microscope, wherein the form of current above-mentioned laser scanning microscope is predominantly moved by mechanical arm
The detection device installation of the i.e. laser scanning microscope of the laser scanning microscope of microscope detection device on the robotic arm, passes through
The adjustment of mechanical arm carrys out movement detector, is then aligned with the different institutional framework of detection human body.
But the detection device based on mechanical arm in above-mentioned three dimensional non-linear laser scanning microscope, due to its volume compared with
Greatly, it pops one's head in the corresponding biggish skin area of human body, it, can not be into for the specific position of skin detection so that in concrete operations
Row is accurately positioned, and needs repeatedly to adjust mechanical arm, and multiple movement detector, locating effect is poor, is easy to produce offset deviation, from
And influence image quality.
Summary of the invention
For the technical problems in the prior art, the embodiment of the present invention provides a kind of positioning formula absorption microscope detection
Device and laser scanning microscope.
In a first aspect, the embodiment of the present invention provides a kind of positioning formula absorption microscope detection device, comprising:
It adsorbs shell and is set to the intracorporal mini microscope probe of the absorption shell, the absorption case inside setting
There is telecontrol equipment, the mini microscope probe is set on the telecontrol equipment, and the telecontrol equipment is described micro- for driving
Type microscope probe is moved horizontally along X-Y axis, in which:
The telecontrol equipment and mini microscope probe are all set in the absorption shell, the mini microscope
The first optical path, the second optical path are provided in probe and for driving nothing common to first optical path and second optical path
The zoom motor that poor remote object lens move up and down, in which:
First optical path is thin to life entity for receiving pulsed laser signal and conducting the output pulsed laser signal
Autofluorescence substance intracellular；
Second optical path is used to acquire and conduct the autofluorescence substance and is excited the fluorescence signal of generation and secondary
Harmonic signal.
Second aspect, the embodiment of the present invention provide a kind of absorption type three dimensional non-linear laser scanning microscope, comprising:
Phosphor collection device, air extractor, scanning collection controller, femtosecond pulse laser, fiber coupling module and
The positioning formula that first aspect of the embodiment of the present invention provides adsorbs microscope detection device, the phosphor collection device and the optical fiber
Coupling module is connect with positioning formula absorption microscope detection device fiber optic communication, the phosphor collection device and described fixed
Position formula absorption microscope detection device is electrically connected with the scanning collection controller, and the air extractor and the positioning formula are inhaled
Attached microscope detection device electrical connection, in which:
The femtosecond pulse laser, for exporting pulsed laser signal to the fiber coupling module；
The fiber coupling module, for coupling the pulsed laser signal of the femtosecond pulse laser output, and
It transmits described in mini microscope probe described in the pulsed laser signal to positioning formula absorption microscope detection device
Collimation lens；
The positioning formula absorption microscope detection device exports the pulse after receiving the pulsed laser signal
The laser signal autofluorescence substance intracellular to life entity, and the autofluorescence substance excitation is obtained by the object lens
The fluorescence signal and second harmonic signal generated afterwards, and the fluorescence signal and the second harmonic signal are exported to the fluorescence
Collection device；
The phosphor collection device converts institute after receiving the fluorescence signal and the second harmonic signal respectively
It states fluorescence signal and the second harmonic signal is corresponding electric signal；
The scanning collection controller swashs the pulse for controlling the scanning galvanometer in the mini microscope probe
Optical signal be scanned and synchronous acquisition described in electric signal；
The air extractor is evacuated for the outer adsorption space to positioning formula absorption microscope detection device,
With the negative pressure formed in the outer adsorption space.
Positioning formula absorption microscope detection device provided in an embodiment of the present invention and laser scanning microscope include absorption shell
Body is popped one's head in the intracorporal mini microscope of absorption shell is set to, and is detected absorption type microscope by the absorption shell of micromation and is filled
It sets and is adsorbed on life entity skin histology to be measured, so that mini microscope probe visits the eucaryotic cell structure in skin histology
It surveys, and adjustment, the horizontal location detection of Lai Jinhang eucaryotic cell structure single layer, zoom motor is moved horizontally by running gear X-Y axis
It drives infinity object lens common to first optical path and second optical path to move up and down, realizes to life entity skin
The detection of cyto-architectural different depth in tissue, and then realize to the cyto-architectural three-dimensional in life entity skin histology not
The precise positioning of same level detects, while mini microscope is popped one's head in through two built-in optical paths, and by optical output
Pulsed laser signal, the autofluorescence substance in activated cell, and obtain autofluorescence substance and be excited the fluorescence signal of generation
And second harmonic signal, so that the 3 D laser scanning microscope for being equipped with absorption type microscope detection device passes through acquisition
Fluorescence signal and second harmonic signal, to eucaryotic cell structure carry out three-dimensional imaging, structure is simple, easy to use.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is this hair
Bright some embodiments for those of ordinary skill in the art without creative efforts, can be with root
Other attached drawings are obtained according to these attached drawings.
Fig. 1 is that positioning formula provided in an embodiment of the present invention adsorbs microscope detection device structural schematic diagram；
Fig. 2 is that the positioning formula that one embodiment of the invention provides adsorbs the cross-section structure signal after the combination of microscope detection device
Figure；
Fig. 3 be another embodiment of the present invention provides positioning formula adsorb microscope detection device combination after cross-section structure show
It is intended to；
Fig. 4 is that the cross-section structure that the positioning formula that yet another embodiment of the invention provides is adsorbed after the combination of microscope detection device shows
It is intended to；
Fig. 5 is that the cross-section structure that the positioning formula that further embodiment of this invention provides is adsorbed after the combination of microscope detection device shows
It is intended to；
Fig. 6 is absorption type three dimensional non-linear laser scanning microscope structural schematic diagram provided in an embodiment of the present invention；
Fig. 7 is phosphor collection apparatus structure schematic diagram provided in an embodiment of the present invention；
Fig. 8 is that absorption type three dimensional non-linear laser scanning microscope provided in an embodiment of the present invention detects human facial skin
Schematic illustration of tissue；
Fig. 9 is that absorption type three dimensional non-linear laser scanning microscope provided in an embodiment of the present invention detects human chest skin
Schematic illustration of tissue；
Figure 10 is that the multiple detection devices of absorption type three dimensional non-linear laser scanning microscope provided in an embodiment of the present invention are same
When detect human skin tissue schematic diagram；
Figure 11 is absorption type three dimensional non-linear laser scanning microscope detecting animal skin group provided in an embodiment of the present invention
Knit schematic diagram；
Figure 12 shows for the box composite structure that the embodiment of the present invention provides absorption type three dimensional non-linear laser scanning microscope
It is intended to；
Figure 13 is the box composite structure of absorption type three dimensional non-linear laser scanning microscope provided in an embodiment of the present invention
Joint sealing structural schematic diagram.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art
Every other embodiment obtained without creative efforts, shall fall within the protection scope of the present invention.
It is existing to be based on fluorescence signal and second harmonic signal, it is main to obtain the correlation detection equipment of biological cell form
If three dimensional non-linear laser scanning microscope, the form of current above-mentioned laser scanning microscope is predominantly moved by mechanical arm
The detection device installation of the i.e. laser scanning microscope of laser scanning microscope of dynamic microscope detection device on the robotic arm, is led to
The adjustment for crossing mechanical arm carrys out movement detector, is then aligned with the different institutional framework of detection human body.But above-mentioned three dimensional non-linear
The detection device based on mechanical arm in laser scanning microscope, since its volume is larger, the corresponding biggish skin of human body of popping one's head in
Area needs multiple adjustment machine so that can not be accurately positioned in concrete operations for the specific position of skin detection
Tool arm, multiple movement detector, locating effect is poor, is easy to produce offset deviation, to influence image quality.
For the ease of carrying out accurate positioning imaging to life body tissue, its cellular prion protein information is obtained, the present invention
Embodiment provides a kind of positioning formula absorption microscope detection device, and Fig. 1 is that positioning formula provided in an embodiment of the present invention absorption is aobvious
Micro mirror detection device structural schematic diagram, as shown in Figure 1, positioning formula absorption microscope detection device includes:
Absorption shell 11 and the mini microscope probe 12 being set in absorption shell 11, adsorb and set on the inside of shell 11
It is equipped with telecontrol equipment 13, mini microscope probe 12 is set on telecontrol equipment 13, and it is micro- that telecontrol equipment 13 is used for band actuating miniature
Mirror probe 12 is moved horizontally along X-Y axis, in which:
Telecontrol equipment 13 and mini microscope probe 12 are all set in absorption shell 11, are set in mini microscope probe 12
It is equipped with the first optical path, the second optical path and for driving infinity object lens common to the first optical path and the second optical path to carry out up and down
Mobile zoom motor, in which:
First optical path be used to receive pulsed laser signal and conduct output pulsed laser signal to life entity it is intracellular from
Fluoresce substance；
Second optical path is used to acquire and conduct autofluorescence substance and is excited the fluorescence signal and second harmonic signal of generation.
Specifically, positioning formula absorption microscope detection device provided in an embodiment of the present invention includes absorption shell 11 and inhales
Mini microscope probe 12 in attached shell 11, absorption shell 11 are miniaturized, and to be adsorbed on life entity skin surface, adsorb shell
Mini microscope probe 12 in body 11 is to detect the eucaryotic cell structure in life entity skin histology, wherein miniature aobvious
Micro mirror probe 12 is carried out X-Y axis and is moved horizontally by the running gear being set in absorption shell 11, to realize to life entity
The horizontal location of eucaryotic cell structure single layer in skin histology detects, and zoom motor drives common to the first optical path and the second optical path
Infinity object lens move up and down, and realize the detection to the cyto-architectural different depth in life entity skin histology, in turn
Realize that the precise positioning to the cyto-architectural three-dimensional different levels in life entity skin histology detects, wherein mini microscope is visited
It is provided in first 12 for receiving pulsed laser signal and conducting output pulsed laser signal to intracellular spontaneous glimmering of life entity
It first optical path of stimulative substance and acquires and conducts autofluorescence substance and be excited the fluorescence signal and second harmonic signal the of generation
Two optical paths, the setting of the first optical path and the second optical path complete absorption type microscope detection device to life entity skin
The excitation of intracellular autofluorescence substance, and the fluorescence signal for eucaryotic cell structure to be imaged that obtains that excitation generates and secondary
Harmonic signal.
Positioning formula absorption microscope detection device provided in an embodiment of the present invention includes absorption shell and is set to absorption shell
Intracorporal mini microscope probe, is adsorbed on life to be measured for absorption type microscope detection device by the absorption shell of micromation
On body skin histology, so that mini microscope probe detects the eucaryotic cell structure in skin histology, and pass through operation dress
It sets X-Y axis and moves horizontally adjustment, the horizontal location detection of Lai Jinhang eucaryotic cell structure single layer, zoom motor drives the first optical path and the
Infinity object lens common to two optical paths move up and down, and realize to cyto-architectural different deep in life entity skin histology
The detection of degree, and then realize that the precise positioning to the cyto-architectural three-dimensional different levels in life entity skin histology detects, together
When mini microscope probe by two built-in optical paths, and the pulsed laser signal Jing Guo optical output, in activated cell
Autofluorescence substance, and obtain autofluorescence substance and be excited the fluorescence signal and second harmonic signal of generation so that
The 3 D laser scanning microscope for being equipped with absorption type microscope detection device is believed by the fluorescence signal and second harmonic obtained
Number, three-dimensional imaging is carried out to eucaryotic cell structure, structure is simple, easy to use.
On the basis of the various embodiments described above, in positioning formula absorption microscope detection device provided in an embodiment of the present invention
Adsorbing shell includes outer housing, pedestal and coverslip, as shown in Figure 1, sucker 1121 is provided on pedestal 112, sucker 1121
In the sucker hole 1113 that insertion 111 bottom of outer housing opens up, outer housing 111 is detachably connected with pedestal 112 by magnetic field force,
In:
Coverslip 113 is fixed on the seal of sucker 1121, forms the built-in space and outer adsorption space of adsorption device,
Telecontrol equipment 13 and mini microscope probe 12 are all set in built-in space, and infinity object lens forward direction is directed at coverslip 113.I.e.
Positioning formula provided in an embodiment of the present invention adsorbs the outer housing 111 of the absorption shell in microscope detection device and pedestal 112 is
Can the material of mutually magnetic constitute, or the magnetic bodies of mutual magnetic is built-in with, so that each other can be detachable by magnetic field force
It links together, and is provided with sucker 1121 on pedestal 112,111 bottom of outer housing is provided with the suction for being embedded in sucker 1121
Disk hole 1113, sucker 1121 is equipped with the seal being connected to space in sucker 1121, when coverslip 113 covers on seal
When, built-in space is formed in the absorption type microscope detection device and adsorbent equipment is enabled to be adsorbed on life entity skin
Outer adsorption space；And telecontrol equipment 13 and mini microscope probe 12 are all set in built-in space, mini microscope probe
After 12 fix, infinity object lens forward direction is directed at coverslip 113, to export internal signal through coverslip 113 and receive outer
Portion's signal, thus realize microscope zoom, three-dimensional imaging, and detachably connected between outer housing 111 and pedestal 112 by magnetic field force
It connects, facilitates staff that coverslip 113 can be replaced by dismantling pedestal 112, it is easy to operate, easy to use.
On the basis of the various embodiments described above, in positioning formula absorption microscope detection device provided in an embodiment of the present invention
First optical path includes the first light, the second light and third light successively to connect, in which:
Pulsed laser signal transmits collimation lens, analyzer, polarization spectroscope, the first quarter-wave in the first optical path
Piece forms the first light to scanning galvanometer；
After forming the first light, pulsed laser signal transmits the first quarter-wave plate after scanning galvanometer reflects again
To polarization spectroscope, the second light is formed；
After forming the second light, pulsed laser signal is after polarization spectroscope reflects, the first scanning mirror of transmission, the second scanning
Mirror, dichroscope to infinity object lens form third light；
Second optical path successively includes the infinity object lens and dichroic for acquiring and conducting fluorescence signal and second harmonic signal
Mirror.That is Fig. 2 is that the positioning formula that one embodiment of the invention provides adsorbs the schematic diagram of the section structure after the combination of microscope detection device,
The optical path being directed in mini microscope probe, as shown in Fig. 2, positioning formula provided in an embodiment of the present invention absorption microscope is visited
The first optical path in device is surveyed when handling pulsed laser signal, pulsed laser signal is in the optical device Jing Guo the first optical path
When, since optical path is turned back, it will form three light, respectively the first light, the second light and third light form the first light
There are collimation lens 121, analyzer 122, polarization spectroscope 123, the first quarter-wave plate 124 to scanning to shake in the optical path of line
Mirror 125, being formed in the optical path of the second light has scanning galvanometer 125, the first quarter-wave plate 124 and polarization spectroscope
123, formed third light optical path in have polarization spectroscope 123, transmission the first scanning mirror 126, the second scanning mirror 127, two to
Look mirror 128 and infinity object lens 129；
Wherein, pulsed laser signal enters analyzer 122 after the collimation of collimation lens 121, makes standard after fiber exit
Direct light becomes linearly polarized light, and the polarization direction of linearly polarized light is consistent with the transmission polarization direction of polarization spectroscope 123, therefore line is inclined
Vibration light is directly transmitted through polarization spectroscope 123 after entering polarization spectroscope 123, into positioned at 123 left side of polarization spectroscope
First quarter-wave plate 124, wherein the polarization direction of the fast axis direction of the first quarter-wave plate and line polarisation is at ± 45 degree
Angle, therefore after first quarter-wave plate 124, which becomes circularly polarized light and is incident on scanning galvanometer 125,
Form above-mentioned first light；After forming the first light, the circularly polarized light for being scanned the reflection of galvanometer 125 is again introduced into the one or four point
One of wave plate 124 become linearly polarized light, the wherein transmission polarization direction of the polarization direction of the linearly polarized light and polarization spectroscope 123
Vertically, therefore reflection occurs on the light splitting surface of polarization spectroscope 123 and goes out from the lower section of polarization spectroscope 123 for the linearly polarized light
It penetrates, forms the second light；After forming the second light, the collimated light of outgoing, which enters, scans mirror convergence, into infinity object lens 129,
Its focus is located at relaying image planes, forms third light；Scanning galvanometer 125 scans on XY axis can make pulsed laser signal focus
It is scanned in entirely relaying image planes.Pulsed laser signal is transmitted through the parallel flat as dichroscope 128, dichroscope
128 fluorescence signals and second harmonic signal for dividing pulsed laser signal and autofluorescence substance to be excited generation according to wavelength zone,
It is transmitted through pulsed laser signal and reflects fluorescence signal and second harmonic signal.Pulsed laser signal is scanned Jing Hui
The poly- relaying image planes generated are overlapped with the rear image planes of infinity object lens 129.Therefore the relaying picture that pulsed laser signal scanning generates
It can be zoomed on sample according to the amplification factor of infinity object lens 129.Focus Club of the pulsed laser signal on sample generates glimmering
Optical signal and second harmonic signal, the fluorescence signal and second harmonic signal of generation collected by infinity object lens 129 and two to
Being reflected into collection fiber optic bundle for Look mirror 128 is collected, and wherein zoom motor 15 drives and moves down on infinity object lens 129
It is dynamic；Wherein scanning galvanometer can be the scanning galvanometer based on mechanical, motor or micro electronmechanical principle, below each embodiment be as
This.
On the basis of the various embodiments described above, in positioning formula absorption microscope detection device provided in an embodiment of the present invention
First optical path includes successively connect the first light, the second light, third light, the 4th light and the 5th light, in which:
Pulsed laser signal is incident on polarization spectroscope by collimation lens, the analyzer in the first optical path, forms first
Light；
After forming the first light, pulsed laser signal is after polarization spectroscope reflects, and the second quarter-wave plate of transmission is extremely
Plane mirror forms the second light；
After forming the second light, pulsed laser signal transmits the second quarter-wave after plane mirror reflects again
Piece, polarization spectroscope, third quarter-wave plate to scanning galvanometer form third light；
After forming third light, pulsed laser signal transmits third quarter-wave plate after scanning galvanometer reflects again
To polarization spectroscope, the 4th light is formed；
After forming the 4th light, pulsed laser signal is after polarization spectroscope reflects, the first scanning mirror of transmission, the second scanning
Mirror, dichroscope to infinity object lens form the 5th light；
Second optical path successively include acquire and conduct fluorescence signal and second harmonic signal infinity object lens and two to
Look mirror.That is Fig. 3 be another embodiment of the present invention provides positioning formula adsorb microscope detection device combination after cross-section structure show
It is intended to, the optical path being directed in mini microscope probe, as shown in figure 3, positioning formula provided in an embodiment of the present invention absorption is aobvious
For the first optical path in micro mirror detection device when handling pulsed laser signal, pulsed laser signal is in the optics Jing Guo the first optical path
It when device, since optical path is turned back, will form five light, respectively include successively connect the first light, the second light, third light
Line, the 4th light and the 5th light, being formed in the optical path of the first light has collimation lens 140, analyzer 141 and polarization point
Light microscopic 142, being formed in the optical path of the second light has polarization spectroscope 142, the second quarter-wave plate 143 and plane mirror
144, being formed in the optical path of third light has plane mirror 144, the second quarter-wave plate 143, polarization spectroscope 142, the
Three quarter-wave plates 145 and scanning galvanometer 146, being formed in the optical path of the 4th light has 146, the 3rd 4 points of scanning galvanometer
One of wave plate 145 and polarization spectroscope 142, formed the 5th light optical path in have polarization spectroscope 142, the first scanning mirror
147, the one or two scanning mirror 148, dichroscope 149 and infinity object lens 160；
Wherein pulsed laser signal enters analyzer 141 after the collimation of collimation lens 140, makes standard after fiber exit
Direct light becomes linearly polarized light, and the polarization direction of the linearly polarized light is vertical with the transmission polarization direction of polarization spectroscope 142, therefore line
Polarised light occurs reflection after entering polarization spectroscope 142 and is emitted from the right side of polarization spectroscope 142, forms the first light；Shape
After the first light, the linearly polarized light after outgoing enters the second quarter-wave plate 143, wherein the second quarter-wave plate is fast
Axis direction and line polarisation polarization direction are at ± 45 degree of angles, therefore linearly polarized light becomes circular polarization after the second quarter-wave plate
Light, and it is incident on plane mirror 144, form the second light；After forming the second light, circularly polarized light is through plane mirror 144
Back reflection, again passing by the second quarter-wave plate 143 along original optical path return becomes linearly polarized light, the polarization side of the linearly polarized light
To consistent with the transmission polarization direction of polarization spectroscope 142, therefore linearly polarized light enters after polarization spectroscope 142 directly for the second time
It is transmitted through polarization spectroscope 142, into the third quarter-wave plate 145 for being located at the left side of polarization spectroscope 142, wherein third
The fast axis direction of quarter-wave plate and the polarization direction of line polarisation are at ± 45 degree of angles, therefore line polarisation passes through third a quarter
Become circularly polarized light after wave plate to be incident on scanning galvanometer 146, forms third light；After forming third light, it is scanned vibration
The circularly polarized light that mirror 146 reflects, which is again introduced into third quarter-wave plate 145, becomes linearly polarized light, and wherein the linearly polarized light is inclined
Vibration direction is vertical with the transmission polarization direction of polarization spectroscope 142, therefore the polarised light is on the light splitting surface of polarization spectroscope 142
It reflects, and is emitted from the lower section of polarization spectroscope 142, form the 4th light；After forming the 4th light, the collimated light of outgoing
Into mirror convergence is scanned, into infinity object lens 160, focus is located at relaying image planes, forms the 5th light；Scanning galvanometer
146 on XY axis scanning can make pulsed laser signal focus entirely relaying image planes on scan.Pulsed laser signal is transmitted through
As the parallel flat of dichroscope 149, dichroscope 149 divides pulsed laser signal and autofluorescence substance quilt according to wavelength zone
The fluorescence signal and second harmonic signal generated is excited, pulsed laser signal is transmitted through and makes fluorescence signal and second harmonic
Signal reflex.Pulsed laser signal is scanned the relaying image planes that mirror convergence generates and is overlapped with the rear image planes of infinity object lens 160.
Therefore the relaying that pulsed laser signal scanning generates according to the amplification factor of infinity object lens 160 as that can zoom on sample.Arteries and veins
Focus Club of the impulse optical signal on sample generates fluorescence signal and second harmonic signal, the fluorescence signal and second harmonic of generation
Signal is collected by infinity object lens 160 and being reflected into collection fiber optic bundle for dichroscope 149 is collected.
On the basis of the various embodiments described above, absorption type microscope detection device provided in an embodiment of the present invention further includes
Electric adjustable curvature lens, Fig. 4 are that the positioning formula that yet another embodiment of the invention provides adsorbs cuing open after microscope detection device combines
Face structural schematic diagram, as shown in figure 4, electric adjustable curvature lens 120 are between collimation lens 121 and analyzer 122, pulse swashs
Optical signal transmits collimation lens 121, electric adjustable curvature lens 120, analyzer 122, polarization spectroscope 123, the first a quarter
Wave plate 124 forms the first new light to scanning galvanometer 125.Optical element in the first i.e. new light successively includes collimation
Lens 121, electric adjustable curvature lens 120, analyzer 122, polarization spectroscope 123, the first quarter-wave plate 124 and scanning
Galvanometer 125.Wherein electric adjustable curvature lens 120 setting so that, can by electric adjustable curvature lens 120 apply voltage or
Electric current makes electric 120 surface of adjustable curvature lens generate the bending accordingly arrived, and then the directional light that collimation lens 121 are emitted generates
Different focal powers.Specific optical path are as follows: it is adjustable to be incident on electricity from fiber exit in parallel after collimation lens 121 for laser signal
Curvature lens 120 generate corresponding focal power, outgoing from electric adjustable curvature lens 120 according to the voltage or current signal of load
Be converging or diverging with light by the optical element in the first optical path, new the first light, the second light and the third light of formation
Line converges on sample after being transmitted to infinity object lens 129.Wherein, the power variation meeting that electric adjustable curvature lens 120 introduce
Move up and down the focus of the laser signal of 129 mouthfuls of infinity object lens outgoing in longitudinal direction, and electric adjustable curvature lens 120
Response speed it is very fast, the scanning imagery of quick longitudinal direction may be implemented in KHz magnitude in scan frequency.Its
In, electric adjustable curvature lens 120 are equivalent to parallel plate glass when not applying voltage or current signal, unglazed to laser signal
Focal power and the focus after infinity object lens 129 will not be made to generate any offset, to realize three-dimensional imaging.Specifically make
Used time, the electricity adjustable curvature lens 120 are complementary with zoom motor 13, adjust 129 position of infinity object lens by zoom motor 13,
After coarse adjustment to corresponding depth position, system is switched to electric 120 zoom scan mode of adjustable curvature lens, carries out to sample quick
Three-dimensional imaging, it is adjustable only by electricity wherein when absorption type microscope detection device is not when installing zoom motor 13
Curvature lens 120 can also carry out zoom adjustment.
On the basis of the various embodiments described above, absorption type microscope detection device provided in an embodiment of the present invention further includes
Electric adjustable curvature lens, Fig. 5 are that the positioning formula that further embodiment of this invention provides adsorbs cuing open after microscope detection device combines
Face structural schematic diagram, as shown in figure 5, electric adjustable curvature lens 150 are between collimation lens 140 and analyzer 141, pulse swashs
Optical signal transmission collimation lens 140, electric adjustable curvature lens 150, analyzer 141 are incident on polarization spectroscope 142, are formed new
First light.Optical element in the first i.e. new light successively includes collimation lens 140, electric adjustable curvature lens 150, analyzing
Device 141 and polarization spectroscope 142.Wherein electric adjustable curvature lens 150 setting so that, can be by electric adjustable curvature lens
150 application voltage or currents make electric 150 surface of adjustable curvature lens generate the bending accordingly arrived, and then collimation lens 140
The directional light of outgoing generates different focal powers.Specific optical path are as follows: laser signal is from fiber exit, after collimation lens 140
It is incident on electric adjustable curvature lens 150 in parallel, generates phase from electric adjustable curvature lens 150 according to the voltage or current signal of load
The focal power answered, outgoing are converging or diverging with light by the optical element in the first optical path, the first new light of formation, second
After light, third light, the 4th light and the 5th light, converged on sample after being transmitted to infinity object lens 160.Wherein,
The power variation that electric adjustable curvature lens 150 introduce can make the focus of the laser signal of 160 mouthfuls of infinity object lens outgoing vertical
Moved up and down on deep direction, and the response speed of electric adjustable curvature lens 150 is very fast, scan frequency in KHz magnitude, because
The scanning imagery of quick longitudinal direction may be implemented in this.Wherein, electric adjustable curvature lens 150 are not applying voltage or electric current letter
Number when be equivalent to parallel plate glass, without focal power and the focus after infinity object lens 160 will not be made to generate laser signal
Any offset, to realize three-dimensional imaging.When specifically used, the electricity adjustable curvature lens 150 are complementary with zoom motor,
160 position of infinity object lens is adjusted by zoom motor, after coarse adjustment to corresponding depth position, system is switched to electric adjustable curvature
150 zoom scan mode of lens carries out quick three-dimensional imaging to sample, wherein when absorption type microscope detection device is in uneasiness
When filling zoom motor, zoom adjustment can also be carried out only by electric adjustable curvature lens 150.
On the basis of the various embodiments described above, in positioning formula absorption microscope detection device provided in an embodiment of the present invention
Telecontrol equipment includes fixed bracket, the first drive block and the second drive block, as shown in Figure 1, the first drive block 132 and the second driving
Block 133 is fixedly connected with fixed bracket 131 respectively, and the second drive block 133 is fixed on the side wall of absorption shell 11, in which:
Second drive block 133 is for driving the first drive block 132 and fixed bracket 131 to move along the y axis together, and first
Drive block 132 is for driving mini microscope probe to move along the x axis.I.e. on the basis of the various embodiments described above, the present invention
The telecontrol equipment in positioning formula absorption microscope detection device that embodiment provides includes fixed bracket 131, the first drive block 132
With the second drive block 133, the first drive block 132 and the second drive block 133 include sliding block and fixed block, the first drive block 132
The first fixed block be fixed on fixed bracket 131, the first sliding block of the first drive block 132 under the driving of first motor, with
First fixed block is opposite to be slided；Second sliding block of the second drive block 133 is fixed on fixed bracket 131, the second drive block 133
Second fixed block is fixed on the side wall of outer housing 11, the second sliding block of the second drive block 133 under the driving of the second motor, with
Second fixed block is opposite to be slided；Mini microscope probe 12 can be fixed on the first sliding block of the first drive block 132, to make
The first drive block 132 is able to drive mini microscope probe and 12 moves along the x axis, the second drive block 133 is able to drive the
One drive block 132 and fixed bracket 131 and the mini microscope probe 12 being fixed on the first drive block 132 are together along Y-axis
Direction is mobile, to can be driven by telecontrol equipment after realizing adsorbent equipment on coarse positioning to life entity skin histology to be measured
Mini microscope probe 12 is dynamic along X-axis and Y direction micro-shifting, and Lai Shixian is in the horizontal plane to the accurate of mini microscope probe 12
Positioning, while being realized by motor driven and being moved along X axis and Y direction, so that in the same of the microscope adsorbent equipment not moved
When, it realizes the imaging of different zones, can preferably observe the imaging region for wanting observation.Simultaneously can by process control,
Make the identical distance of displacement platform stepping, and the image of imaging is spliced, the imaging results in the N times of visual field can be obtained.
On the basis of the various embodiments described above, in positioning formula absorption microscope detection device provided in an embodiment of the present invention
Fixed bracket includes X-axis bracket and Y-axis bracket, and X-axis bracket is mutually perpendicular to be fixedly connected with Y-axis bracket, in which:
X-axis bracket with the first drive block is detachable is fixedly connected, Y-axis bracket and the second drive block it is detachable it is fixed even
It connects.Fixation bracket in positioning formula absorption microscope detection device i.e. provided in an embodiment of the present invention is L-type comprising X-axis branch
Frame and Y-axis bracket, X-axis bracket are mutually perpendicular to be fixedly connected with Y-axis bracket, the first fixed frame of X-axis bracket and the first drive block
It is detachably fixed connection, Y-axis bracket is detachably fixed with the second sliding block of the second drive block and connect, to realize mini microscope
Probe can move under the driving of telecontrol equipment along XY axis direction.
It is provided in an embodiment of the present invention for the positioning of mini microscope probe to be arranged on the basis of the various embodiments described above
Sucker in formula adsorbent equipment further includes adsorption orifice and bleeding point, and adsorption orifice is connected with seal, to empty by outer absorption
Between be adsorbed on life entity to be measured；
Bleeding point is connected with outer adsorption space, to extract the gas in outer adsorption space.I.e. the embodiment of the present invention mentions
The sucker being set on pedestal in the positioning formula absorption microscope detection device of confession is in addition to seal further includes adsorption orifice and pumping
Mouthful, and seal is connected with adsorption orifice, forms the inner space of sucker, when coverslip sealing is fixed on seal, inhales
The inner space of disk just forms outer adsorption space, and adsorption orifice is adsorbed on life entity to be measured by the outer adsorption space；It is above-mentioned
Bleeding point is connected to outer adsorption space, by the bleeding point, can extract the gas in outer adsorption space, to form outer absorption
Negative pressure in space.Under the action of external pressure, positioning formula absorption microscope detection device is adsorbed on the skin of life entity to be measured
The tissues such as skin.
It is provided in an embodiment of the present invention for the positioning of mini microscope probe to be arranged on the basis of the various embodiments described above
Outer housing in formula adsorbent equipment includes first shell and second shell, as shown in Figure 1, in which:
Accommodation space is provided in first shell 1111, telecontrol equipment is set in accommodation space, 1111 He of first shell
Second shell 1112 is detachable to be fixedly connected.The positioning i.e. provided in an embodiment of the present invention popped one's head in for mini microscope to be arranged
Outer housing in formula adsorbent equipment includes two parts, respectively first shell 1111 and second shell 1112, first shell 1111
There is the accommodation space for telecontrol equipment to be arranged, i.e., the sucker hole in above-described embodiment is also disposed at the bottom of first shell 1111
Portion, sucker are embedded in the sucker hole of first shell 1111, in conjunction with coverslip and second shell 1112, can form above-mentioned reality
Apply outer adsorption space and the built-in space in example, wherein first shell 1111 and second shell 1112 can be detachable by screw
It is fixedly connected, so facilitates the assembling, disassembly and the replacement of component of whole device.
The embodiment of the present invention also provides a kind of absorption type three dimensional non-linear laser scanning microscope, and Fig. 6 is that the present invention is implemented
The absorption type three dimensional non-linear laser scanning microscope structural schematic diagram that example provides, as shown in fig. 6, the laser scanning microscope packet
It includes:
Phosphor collection device 56, air extractor 52, scanning collection controller 531, femtosecond pulse laser 541, optical fiber coupling
It molds block 542 and positioning formula provided by the above embodiment adsorbs microscope detection device 51, phosphor collection device 56 and optical fiber
Coupling module 542 is connect with positioning formula absorption 51 fiber optic communication of microscope detection device, phosphor collection device 56 and positioning formula
Absorption microscope detection device 51 is electrically connected with scanning collection controller 531, and air extractor 52 and positioning formula adsorb microscope
Detection device 51 is electrically connected, in which:
Femtosecond pulse laser 541, for exporting pulsed laser signal to fiber coupling module 542；
Fiber coupling module 542 for coupling the pulsed laser signal of the output of femtosecond pulse laser 541, and transmits arteries and veins
Impulse optical signal mini microscope probe into positioning formula absorption microscope detection device；
Positioning formula adsorbs microscope detection device, after receiving pulsed laser signal, output pulsed laser signal to life
The autofluorescence substance in body cell is ordered, and obtains the fluorescence signal and two generated after the excitation of autofluorescence substance by object lens
Rd harmonic signal, and fluorescence signal and second harmonic signal are exported to phosphor collection device 56；
Phosphor collection device 56, after receiving fluorescence signal and second harmonic signal, difference conversion fluorescence signal and two
Rd harmonic signal is corresponding electric signal；
Scanning collection controller 531, for control mini microscope probe in scanning galvanometer to pulsed laser signal into
Row scanning and synchronous acquisition electric signal；
Air extractor 52 is evacuated, to be formed for the outer adsorption space to positioning formula absorption microscope detection device
Negative pressure in outer adsorption space.
Specifically, absorption type three dimensional non-linear laser scanning microscope provided in an embodiment of the present invention includes phosphor collection dress
Set 56, air extractor 52, scanning collection controller 531, femtosecond pulse laser 541, fiber coupling module 542 and positioning formula
Microscope detection device 51 is adsorbed, so that human skin can be adsorbed on or to go deep into the three-dimensional that human body stomach detected non-by being formed
Linear laser flying-spot microscope, wherein femtosecond pulse laser 541 can be used for exciting human skin with emission pulse laser signal
Autofluorescence substance in cell generates multiphoton fluorescence signal and second harmonic signal, the femtosecond pulse including using 920nm
FAD and collagen in 541 activated cell of laser, excite the fluorescence signal of 500-600nm and the second harmonic of 460nm
Signal, and pass through 780nm 541 activated cell of femtosecond pulse laser in FAD or the autofluorescences substance such as NADH, to produce
Raw corresponding fluorescence signal and second harmonic signal；
Wherein, phosphor collection device 56 is integrated with two paths of signals and collects optical path, respectively fluorescence signal collection optical path and two
Rd harmonic signal collects optical path, the collection respectively of Lai Shixian fluorescence signal and second harmonic signal；Scanning collection controller 531 is controlled
Scanning galvanometer in mini microscope probe processed is scanned pulsed laser signal and autofluorescence substance is excited to generate fluorescence
Signal and second harmonic signal, and acquire 56 conversion fluorescence signal of phosphor collection device and second harmonic signal obtain first
Electric signal and the second electric signal；Air extractor 52 includes mainly aspiration pump, is connected with exhaust pipe, exhaust pipe and above-mentioned implementation
Bleeding point in example is connected, and extraction valve is arranged in exhaust pipe, and extraction valve is electrically connected with air extractor 52, and air extractor 52 is logical
The switch of adjustment extraction valve and the size of opening and closing are crossed, the extraction flow of exhaust pipe is controlled, to realize external adsorption space
Pumping control, and then the negative pressure in outer adsorption space is adjusted, so that adsorbent equipment is adsorbed on life by the effect of atmospheric pressure
The tissues such as body skin, stomach, and the absorption type three dimensional non-linear laser scanning microscope may include that two-photon is swept according to classification
Retouch microscope and multi-photon flying-spot microscope etc., wherein when femtosecond pulse laser is can be by common continuous light laser
In the case where substitution, increase aperture, it is burnt aobvious for copolymerization also to can adjust the absorption type three dimensional non-linear laser scanning microscope
Micro mirror.Wherein, the resolution ratio of the absorption type three dimensional non-linear laser scanning microscope may be configured as 800nm, and visual field can be
200 microns * 200 microns, image taking speed can be 26 frames (256*256 pixel) or 13 frames (512*512 pixel).
Absorption type three dimensional non-linear laser scanning microscope provided in an embodiment of the present invention is using phosphor collection device, pumping
Device, scanning collection controller, femtosecond pulse laser, fiber coupling module and positioning formula adsorb microscope detection device,
It can be adsorbed on human skin to be formed or go deep into the three dimensional non-linear laser scanning microscope that human body stomach is detected, lead to
It crosses adjustment mini microscope probe and is adjusted focal length at a distance from coverslip, realize the 3-D scanning of laser scanning microscope,
Multiphoton fluorescence signal and second harmonic signal are obtained by autofluorescence substance in femtosecond pulse laser activated cell, is realized
Laser scanning microscope is non-linear, collects fluorescence signal and second harmonic signal by phosphor collection device, and is converted to opposite
The electric signal answered, and then the corresponding fluorescent image etc. for reflecting cell tissue structure is obtained by the electric signal, wherein positioning formula
The use for adsorbing microscope detection device can pop one's head on mini microscope to generate to vibrate and influence to avoid life entity activity, to keep away
Exempt from vibration and influence image quality, and above-mentioned laser scanning microscope can realize that a variety of imaging patterns include XY imaging, XZ imaging
And 3D imaging, wherein XY is imaged as carrying out transversal scanning imaging in a certain level of eucaryotic cell structure certain depth, and XZ is imaged as
From the XZ cross-sectional imaging of surface layer certain depth down, 3D is imaged as carrying out XY from surface layer certain depth down, each depth
Imaging, is reconstructed into 3D rendering, equipment operation is simple, easy to use.
On the basis of the various embodiments described above, Fig. 7 is phosphor collection apparatus structure schematic diagram provided in an embodiment of the present invention,
As shown in fig. 7, phosphor collection device provided in an embodiment of the present invention includes fiber optic universal interface 881, the first photomultiplier tube
882, the second photomultiplier tube 883 and the first receipts between fiber optic universal interface 881 and the first photomultiplier tube 882
Collect optical path, the second collection optical path between fiber optic universal interface 881 and the second photomultiplier tube 883, in which:
First collection optical path successively includes coupling collecting lens 81, infrared fileter 82, the first dichroscope 83, first
Optical filter 84 and the first collecting lens 85, wherein the first collection optical path is for collecting the fluorescence that phosphor collection device receives
Signal, the first photomultiplier tube 882 are the first electric signal for conversion fluorescence signal；
Second collection optical path successively includes coupling collecting lens 81, infrared fileter 82, the first dichroscope 83, second
Dichroscope 86, the second optical filter 87 and the second collecting lens 88, wherein the second collection optical path is for collecting phosphor collection dress
The second harmonic signal received is set, the second photomultiplier tube 883 is for converting second harmonic signal as the second electric signal.I.e.
Phosphor collection device provided in an embodiment of the present invention has simple two-way signal collecting function, is integrated with two-way optical path, wherein first receives
Collecting the first dichroscope 83 in optical path is transmission fluorescence signal, the dichroscope of reflected second harmonics, the second dichroscope 86
It is same dichroscope with the first dichroscope 83, is used for reflected second harmonics, the first optical filter 84 is for transmiting fluorescence letter
Number, remaining interference signal is filtered out, the second optical filter 87 filters out remaining interference signal, example for transmiting corresponding second harmonic signal
Such as, it when using 880nm femto second optical fiber laser exciting human surface skin intracellular autofluorescence substance, can be obtained
The second harmonic signal of 390nm and the two-photon auto flourescence signals of 450-600nm, are passed through, 420 by 420nm above wavelength
The dichroscope of following wavelength reflection i.e. the first dichroscope 83 can separate two-way fluorescence, respectively using the of 390 ± 20nm
The second optical filter 87 available clean second harmonic signal and fluorescence signal of one optical filter 84 and 450-600nm.
On the basis of the various embodiments described above, absorption type three dimensional non-linear scan laser microphotograph provided in an embodiment of the present invention
Mirror further includes industrial personal computer, as shown in fig. 6, industrial personal computer 532 is electrically connected with scanning collection controller 531, in which:
Industrial personal computer 532 is for obtaining collected first electric signal of scanning collection controller 531 and the second electric signal, and base
The first fluorescent image is generated in the first electric signal and the second fluorescent image is generated based on the second electric signal.That is the embodiment of the present invention
The absorption type three dimensional non-linear laser scanning microscope of offer further includes the industrial personal computer being electrically connected with scanning collection controller 531
532, which is based on the first electric signal and generates the first fluorescent image and generate the second fluorogram based on the second electric signal
Picture can be respectively used to display eucaryotic cell structure and fibre structure information, control software is wherein equipped on industrial personal computer, soft by controlling
Part sends control instruction to scanner, to control scanning collection controller, to obtain above-mentioned first electric signal and the second telecommunications
Number.
On the basis of the various embodiments described above, absorption type three dimensional non-linear scan laser microphotograph provided in an embodiment of the present invention
Mirror further includes display, as shown in fig. 6, display 55 is electrically connected with industrial personal computer 532, for showing the first fluorescent image and the
Two fluorescent images.Absorption type three dimensional non-linear laser scanning microscope i.e. provided in an embodiment of the present invention further includes for showing the
The display 55 of one fluorescent image and the second fluorescent image, by display 55, staff can directly acquire the first fluorescence
The relevant information of image and the second fluorescent image.
On the basis of the various embodiments described above, absorption type three dimensional non-linear scan laser microphotograph provided in an embodiment of the present invention
Positioning formula absorption microscope detection device in mirror is multiple.Phosphor collection device and optical fiber coupling i.e. provided in an embodiment of the present invention
Molding block can be connect with the absorption microscope detection device fiber optic communication of multiple positioning formulas simultaneously, i.e., non-thread in an absorption type three-dimensional
Property laser scanning microscope system in integrate multiple detection devices, with realize to life entity different tissues position while detect,
To compare and analyze.
On the basis of the various embodiments described above, absorption type three dimensional non-linear scan laser microphotograph provided in an embodiment of the present invention
Mirror further includes adjusting optical fiber, is filled respectively with positioning formula absorption microscope detection for phosphor collection device and fiber coupling module
Optical fiber between setting transmits connection, in which:
Adjust the adjustable in length of optical fiber.Absorption type three dimensional non-linear scan laser microphotograph i.e. provided in an embodiment of the present invention
Phosphor collection device and fiber coupling module in mirror adsorb microscope by length-adjustable adjusting optical fiber and positioning formula respectively
Detection device carries out optical fiber transmission connection and carries out flexible movement detector to realize according to different experiments scene needs, avoid
The limitation of limited fiber lengths, wherein the adjustable in length of optical fiber is adjusted, to realize each by the optical fiber for replacing different length
The application of kind occasion can carry out the optical fiber replacement of different length at any time as needed.
In order to more clearly illustrate absorption type three dimensional non-linear laser scanning microscope provided in an embodiment of the present invention
Application scenarios are now described further with legend, and Fig. 8 is absorption type three dimensional non-linear laser scanning provided in an embodiment of the present invention
Microscope detection human facial skin's schematic illustration of tissue, by the air extracting function of air extractor 52, will position formula as shown in 8
Absorption microscope detection device 51 is adsorbed on human face, wherein scanning collection controller and work are integrated in first device 53
Control machine, industrial personal computer are electrically connected with display 55, and second device 54 is integrated with femtosecond pulse laser, fiber coupling module and fluorescence
Collection device, fiber coupling module and phosphor collection device connect with positioning formula absorption 51 optical fiber of microscope detection device transmission
It connects, wherein the absorption type three dimensional non-linear laser scanning microscope working principle is identical as the various embodiments described above, no longer superfluous herein
It states.
Wherein, Fig. 9 is that absorption type three dimensional non-linear laser scanning microscope provided in an embodiment of the present invention detects human body chest
Portion's skin histology schematic diagram, as shown in 9, by the air extracting function of air extractor 52, by positioning formula absorption microscope detection dress
It sets 51 and is adsorbed on human chest, wherein be integrated with scanning collection controller and industrial personal computer, industrial personal computer and display in first device 53
Device 55 is electrically connected, and second device 54 is integrated with femtosecond pulse laser, fiber coupling module and phosphor collection device, fiber coupling
Module and phosphor collection device are connected with positioning formula absorption 51 optical fiber of microscope detection device transmission, wherein the absorption type three
Dimension non-linear laser flying-spot microscope working principle is identical as the various embodiments described above, and details are not described herein again.
Wherein, Figure 10 is the multiple detection dresses of absorption type three dimensional non-linear laser scanning microscope provided in an embodiment of the present invention
It sets while detecting human skin tissue schematic diagram, as shown in 10, by the air extracting function of air extractor 52, by multiple positioning
Formula absorption microscope detection device 51 is adsorbed on human face, chest and leg simultaneously respectively, wherein integrates in first device 53
Scanning collection controller and industrial personal computer, industrial personal computer are electrically connected with display 55, and second device 54 is integrated with femtosecond pulse
Device, fiber coupling module and phosphor collection device, fiber coupling module and phosphor collection device adsorb microscope with positioning formula
The transmission connection of 51 optical fiber of detection device, thus, it realizes under the action of multiple positioning formulas adsorb microscope detection device, visits simultaneously
Survey human body different parts skin histology structure, it is easy to operate, easy to use, and due to positioning formula absorption microscope detection device with
Using optical fiber transmission connection between second device, fiber lengths can be adjusted, and human body to be measured is move freely.Its
In, the absorption type three dimensional non-linear laser scanning microscope working principle is identical as the various embodiments described above, and details are not described herein again.Figure
11 be absorption type three dimensional non-linear laser scanning microscope detecting animal skin histology schematic diagram provided in an embodiment of the present invention, such as
Shown in Figure 11, the air extracting function of air extractor 52 again may be by, positioning formula absorption microscope detection device 51 is adsorbed on
On the skin histology of life entity, wherein be integrated with scanning collection controller and industrial personal computer, industrial personal computer and display in first device 53
Device 55 is electrically connected, and second device 54 is integrated with femtosecond pulse laser, fiber coupling module and phosphor collection device, fiber coupling
Module and phosphor collection device are connected with positioning formula absorption 51 optical fiber of microscope detection device transmission, working principle with it is above-mentioned
Each embodiment is identical.
For the absorption type three dimensional non-linear laser scanning microscope that the various embodiments described above provide, the embodiment of the present invention is also mentioned
Another specific embodiment is supplied, Figure 12 provides absorption type three dimensional non-linear laser scanning microscope for the embodiment of the present invention
Box combining structure schematic diagram, as shown in figure 12, the scanning collection control of the absorption type three dimensional non-linear laser scanning microscope
Device 531, industrial personal computer 532, air extractor 52, phosphor collection device 56 and femtosecond pulse laser processed and fiber coupling are integrated in
Integration module 540 together, is integrated in Portable suitcase together, there is the industrial personal computer with display screen in case, and suitcase
Display 55 is integrated on case lid；Positioning formula absorption microscope detection device 51 is adsorbed on the skin histology of human body to be measured, with
The intracorporal fiber coupling of case is connected with 56 fiber optic communication of phosphor collection device, is connect with aspiration pump by exhaust pipe, and power supply is inserted
Head is electrically connected with scanning collection controller 531 and industrial personal computer 532.Wherein Figure 13 is that absorption type provided in an embodiment of the present invention is three-dimensional
The joint sealing structural schematic diagram of the box composite structure of non-linear laser flying-spot microscope is integrated on case lid and shows as shown in figure 13
Show that device 55 is integrated with the cabinet for being equipped with modules, facilitate whole equipment mobile, and replacement workplace, and should
Display 55 when in use, can be placed on cabinet in addition, to facilitate staff to obtain the information on display.When having used
After the absorption type three dimensional non-linear laser scanning microscope, staff can portable equipment case, convenient changing workplace, especially
It, can be more convenient using the equipment in hospital, laboratory or outdoor location.
It should be further noted that the absorption type three dimensional non-linear laser scanning microscope that the various embodiments described above provide,
In the wavelength for changing femtosecond pulse laser and after adjusting the filter range of each optical filter, fluorescence and non-SHG (Second in part
Harmonic Generation, second harmonic generate) the active tissue of signal, CARS signal can be collected, so as to adjust for
The miniature CARS microscope of absorption type, specific adjusting parameter are configured according to specific needs.
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not to invention protection scope
Limitation, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not required to
The above is only preferred embodiment of the present application, are not intended to restrict the invention, for those skilled in the art, this
Application can have various modifications and variations.Within the spirit and principles of this application, made any modification, equivalent replacement,
Improve etc., it should be included within the scope of protection of this application.
The apparatus embodiments described above are merely exemplary, wherein unit can be as illustrated by the separation member
Or may not be and be physically separated, component shown as a unit may or may not be physical unit, i.e.,
It can be located in one place, or may be distributed over multiple network units.It can select according to the actual needs therein
Some or all of the modules achieves the purpose of the solution of this embodiment.Those of ordinary skill in the art are not paying creative labor
In the case where dynamic, it can understand and implement.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
Claims (10)
1. a kind of positioning formula adsorbs microscope detection device characterized by comprising
It adsorbs shell and is set to the intracorporal mini microscope probe of the absorption shell, the absorption case inside is provided with fortune
Dynamic device, the mini microscope probe are set on the telecontrol equipment, and the telecontrol equipment is described miniature aobvious for driving
Micro mirror probe is moved horizontally along X-Y axis, in which:
The telecontrol equipment and mini microscope probe are all set in the absorption shell, the mini microscope probe
Inside it is provided with the first optical path, the second optical path and for driving infinity common to first optical path and second optical path
The zoom motor that object lens move up and down, in which:
First optical path is intracellular to life entity for receiving pulsed laser signal and conducting the output pulsed laser signal
Autofluorescence substance；
Second optical path is used to acquire and conduct the autofluorescence substance and is excited the fluorescence signal and second harmonic of generation
Signal.
2. positioning formula according to claim 1 adsorbs microscope detection device, which is characterized in that the absorption shell includes
Outer housing, pedestal and coverslip are provided with sucker on the pedestal, and the sucker is embedded in the suction that the outer housing bottom opens up
In disk hole, the outer housing is detachably connected with the pedestal by magnetic field force, in which:
The coverslip is fixed on the seal of the sucker, and built-in space and the outer absorption for forming the adsorption device are empty
Between, the telecontrol equipment and mini microscope probe are all set in the built-in space, and the infinity object lens are positive
It is directed at the coverslip.
3. positioning formula according to claim 1 adsorbs microscope detection device, which is characterized in that first optical path includes
The first light, the second light and the third light successively to connect, in which:
The pulsed laser signal transmit collimation lens in first optical path, analyzer, polarization spectroscope, the one or four/
One wave plate forms first light to scanning galvanometer；
After forming first light, the pulsed laser signal transmits described first after scanning galvanometer reflection again
Quarter-wave plate forms second light to the polarization spectroscope；
After forming second light, the pulsed laser signal through the polarization spectroscope reflection after, transmission the first scanning mirror,
Second scanning mirror, dichroscope to the infinity object lens, form the third light；
Second optical path successively includes the infinity for acquiring and conducting the fluorescence signal and the second harmonic signal
Object lens and the dichroscope.
4. positioning formula according to claim 1 adsorbs microscope detection device, which is characterized in that first optical path includes
Successively connect the first light, the second light, third light, the 4th light and the 5th light, in which:
The pulsed laser signal is incident on polarization spectroscope by collimation lens, the analyzer in first optical path, is formed
First light；
After forming first light, the pulsed laser signal through the polarization spectroscope reflection after, transmission the two or four/
One wave plate forms second light to plane mirror；
After forming second light, the pulsed laser signal transmits described the after plane mirror reflection again
Two quarter-wave plates, the polarization spectroscope, third quarter-wave plate to scanning galvanometer form the third light；
After forming the third light, the pulsed laser signal transmits the third after scanning galvanometer reflection again
Quarter-wave plate forms the 4th light to the polarization spectroscope；
After forming the 4th light, the pulsed laser signal through the polarization spectroscope reflection after, transmission the first scanning mirror,
Second scanning mirror, dichroscope to the infinity object lens form the 5th light；
Second optical path successively includes the infinity for acquiring and conducting the fluorescence signal and the second harmonic signal
Object lens and the dichroscope.
5. positioning formula according to claim 3 adsorbs microscope detection device, which is characterized in that further include electric adjustable curvature
Lens, the electricity adjustable curvature lens are between the collimation lens and the analyzer, the pulsed laser signal transmission
The collimation lens, the electric adjustable curvature lens, the analyzer, the polarization spectroscope, first quarter-wave
Piece forms the first new light to the scanning galvanometer.
6. positioning formula according to claim 4 adsorbs microscope detection device, which is characterized in that further include electric adjustable curvature
Lens, the electricity adjustable curvature lens are between the collimation lens and the analyzer, the pulsed laser signal transmission
The collimation lens, the electric adjustable curvature lens, the analyzer are incident on the polarization spectroscope, form the first new light
Line.
7. positioning formula according to claim 1 adsorbs microscope detection device, which is characterized in that the telecontrol equipment includes
Fixed bracket, the first drive block and the second drive block, first drive block and second drive block respectively with the fixation
Bracket is fixedly connected, and second drive block is fixed on the side wall of the absorption shell, in which:
Second drive block is described for driving first drive block and the fixed bracket to move along the y axis together
First drive block is for driving the mini microscope probe to move along the x axis.
8. positioning formula according to claim 7 adsorbs microscope detection device, which is characterized in that the fixed bracket includes
X-axis bracket and Y-axis bracket, the X-axis bracket are mutually perpendicular to be fixedly connected with the Y-axis bracket, in which:
The X-axis bracket with first drive block is detachable is fixedly connected, the Y-axis bracket and second drive block can
It is detachably fixed connection.
9. a kind of absorption type three dimensional non-linear laser scanning microscope characterized by comprising
Phosphor collection device, air extractor, scanning collection controller, femtosecond pulse laser, fiber coupling module and right
It is required that the described in any item positioning formulas of 1-8 adsorb microscope detection device, the phosphor collection device and the fiber coupling mould
Block is connect with positioning formula absorption microscope detection device fiber optic communication, and the phosphor collection device and the positioning formula are inhaled
Attached microscope detection device is electrically connected with the scanning collection controller, and the air extractor adsorbs micro- with the positioning formula
The electrical connection of mirror detection device, in which:
The femtosecond pulse laser, for exporting pulsed laser signal to the fiber coupling module；
The fiber coupling module for coupling the pulsed laser signal of the femtosecond pulse laser output, and transmits
Mini microscope probe of the pulsed laser signal into positioning formula absorption microscope detection device；
The positioning formula absorption microscope detection device exports the pulse laser after receiving the pulsed laser signal
The signal autofluorescence substance intracellular to life entity, and produced after obtaining the autofluorescence substance excitation by the object lens
Raw fluorescence signal and second harmonic signal, and the fluorescence signal and the second harmonic signal are exported to the phosphor collection
Device；
The phosphor collection device is converted described glimmering respectively after receiving the fluorescence signal and the second harmonic signal
Optical signal and the second harmonic signal are corresponding electric signal；
The scanning collection controller believes the pulse laser for controlling the scanning galvanometer in the mini microscope probe
Number be scanned and synchronous acquisition described in electric signal；
The air extractor is evacuated, with shape for the outer adsorption space to positioning formula absorption microscope detection device
At the negative pressure in the outer adsorption space.
10. absorption type three dimensional non-linear laser scanning microscope according to claim 9, which is characterized in that the fluorescence
Collection device includes fiber optic universal interface, the first photomultiplier tube, the second photomultiplier tube and connects positioned at the fiber optic universal
First between mouth and first photomultiplier tube collects optical path, is located at the fiber optic universal interface and second photoelectricity times
Second increased between pipe collects optical path, in which:
It is described first collection optical path successively include coupling collecting lens, infrared fileter, the first dichroscope, the first optical filter with
And first collecting lens, wherein the first collection optical path for collecting the fluorescence signal that phosphor collection device receives,
First photomultiplier tube is for converting the fluorescence signal as the first electric signal；
It is described second collection optical path successively include the coupling collecting lens, the infrared fileter, first dichroscope,
Second dichroscope, the second optical filter and the second collecting lens, wherein the second collection optical path is for collecting phosphor collection
The second harmonic signal that device receives, second photomultiplier tube is for converting the second harmonic signal as second
Electric signal.
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