CN109187721A - Postposition is divided pupil confocal laser mass spectrum micro imaging method and device - Google Patents
Postposition is divided pupil confocal laser mass spectrum micro imaging method and device Download PDFInfo
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Abstract
本发明公开的后置分光瞳激光共焦质谱显微成像方法与装置，属于共焦显微成像及质谱成像测量技术领域。本发明将后置分光瞳激光共焦显微成像技术与质谱探测技术结合，利用经超分辨技术处理的后置分光瞳共焦显微镜的微小聚焦光斑对样品进行高空间分辨形态成像，利用质谱探测系统对样品微区带电分子、原子等进行质谱探测，利用激光质谱探测和后置分光瞳共焦探测结构融合实现样品微区完整组分信息与形态参数的高空间分辨和高灵敏成像与探测。本发明可为生物医学、材料科学等领域物质组分及形态成像探测提供一条全新的有效技术途径。
The invention discloses a post-pupil laser confocal mass spectrometry microscopy imaging method and device, belonging to the technical field of confocal microscopy imaging and mass spectrometry imaging measurement. The invention combines the post-pupil laser confocal microscopy imaging technology with the mass spectrometry detection technology, uses the tiny focusing spot of the post-pupil confocal microscope processed by the super-resolution technology to perform high spatial resolution morphological imaging of the sample, and uses the mass spectrometry detection system to detect the morphology of the sample. The charged molecules and atoms in the sample micro-area are detected by mass spectrometry. The fusion of laser mass spectrometry detection and post-pupil confocal detection structure is used to achieve high spatial resolution and high-sensitivity imaging and detection of the complete component information and morphological parameters of the sample micro-area. The invention can provide a brand-new and effective technical way for imaging detection of material components and morphology in the fields of biomedicine, material science and the like.
Description
Technical field
The invention belongs to confocal microscopic imaging technology imaging techniques and mass spectrum imaging technical field, and postposition is divided pupil laser
Confocal microscopic imaging technology is combined with mass spectrum imaging technology, is related to a kind of postposition light splitting pupil confocal laser mass spectrum micro-imaging side
Method and device have wide practical use in fields such as biomedicine, material science, physical chemistry, mineral products, minute manufacturings.
Background technique
Mass spectrograph (Mass Spectrometry) is to ionize the component in sample, makes the different charge-mass ratios generated
Charge atom, molecule or molecular fragment focused respectively under the action of electric and magnetic fields obtain by mass-to-charge ratio size order arrange
The map instrument of column.Mass spectrum imaging be tiny areas multiple in sample 2 dimensional region are analyzed by mass spectrometry respectively it is specific to detect
The distribution of mass-to-charge ratio (m/z) substance.
It is raw from the substance assistant laser desorpted ionized this highly sensitive and high quality detection range of last century the mid-80
The appearance of substance spectral imaging technology has opened up mass-spectrometry one brand-new field-biological mass spectrometry, has promoted mass-spectrometric technique application range
Expand to the various fields of life science, especially mass spectrum protein, nucleic acid, in terms of application,
New tool not only is provided for life science, but also also promotes the development of mass-spectrometric technique itself.
But there are problems following prominent for existing substance assistant laser desorpted ionized mass spectrograph:
1) due to being focused using simple laser come desorption ionization sample, thus its there are still laser focal beam spots big, matter
Compose the problems such as space exploration resolving power is not high；
2) long the time required to mass spectrum imaging, laser mass spectrometry instrument focal beam spot axial position often drifts about with respect to sample
Problem.
And the accurate acquisition of mineral products, " microcell " pattern of space substance and biological sample and complete component information is for section
It learns research and production detection is all extremely important.In fact, how to detect micro-area composition information with sensitivity is mesh
The important technological problems that the fields such as preceding mineral products analysis, biochemistry detection are urgently studied.
Postposition is divided pupil confocal laser technology and is detected using illumination and the non-line structure altogether of detection optical path, is not only significantly mentioned
The high azimuthal resolution and Focus accuracy of optical path, and the high-resolution imaging detection of sample topography may be implemented.
Based on this, the present invention proposes that a kind of postposition is divided pupil confocal laser mass spectrum micro imaging method and device, innovation
It is: will mutually melts with the postposition of high-space resolution ability light splitting pupil confocal laser microtechnic with mass spectrometry detection technology for the first time
Close imaging and detection, it can be achieved that sample microcell high-space resolution and highly sensitive pattern, component.
A kind of postposition light splitting pupil confocal laser mass spectrum micro imaging method of the present invention and device can be biomedical, material supply section
Pattern, the component imaging detection in the fields such as, physical chemistry, mineral products, minute manufacturing provide a completely new effective technical way.
Summary of the invention
The purpose of the invention is to improve the spatial resolving power of mass spectrum imaging, inhibit focal beam spot phase in imaging process
Drift to sample proposes that a kind of postposition is divided pupil confocal laser mass spectrum micro imaging method and device, to obtain quilt simultaneously
Survey object micro-raman spectra information and component information.The purpose of the present invention is what is be achieved through the following technical solutions.
Postposition of the invention is divided pupil confocal laser mass spectrum micro imaging method, utilizes high-space resolution confocal microscope system
Focal beam spot axial fixed-focus and imaging are carried out to sample, the micro- system of pupil confocal laser is divided to postposition using mass spectrometry detection system
System focal beam spot desorption ionization sample and charged molecule, atom for generating etc. carry out microcell mass spectrum imaging, then pass through detection again
The imaging of sample microcell high-space resolution and highly sensitive pattern, component is then realized in the fusion of data information with analysis is compared
With detection, comprising the following steps:
Step 1: light-source system is collimated light beam by collimation lens collimation, collimated light beam passes through compression focal beam spot system
System is reflected through Amici prism transmission, dichroscope A and is focused on sample by measurement object lens；
Step 2: computer control precise three-dimensional working platform is made to drive sample along measuring surface normal direction in measurement object
Mirror foci nearby moves up and down, and reflects to form Returning beam by dichroscope A through sample reflection light, Returning beam passes through
After crossing light splitting prismatic reflection, through collection pupil, detection object lens, the relaying amplifying lens in postposition pupil, convergence is through after pin hole
It is received by light intensity detector, obtains postposition light splitting pupil confocal laser axial strength curve by light intensity signal processor；
Step 3: can be accurately positioned sample point axial direction using postposition light splitting pupil confocal laser axial strength curve
Elevation information；
Step 4: computer is accurate according to " extreme point " position control of postposition light splitting pupil confocal laser axial strength curve
Three-dimensional working platform drives sample to move along measuring surface normal direction, and the focal beam spot for measuring object lens is made to focus on sample
On；
Step 5: changing collimated light beam light illumination mode, the microcell desorption ionization of sample is excited to generate plasma plume；
Step 6: dividing in the plasma plume for being generated focal beam spot desorption ionization sample using ion suction pipe
Son, atom and ion suck to form detection ion, and detection ion, which enters, carries out mass spectrum imaging in mass spectrometry detection system, measure correspondence
The Information in Mass Spectra in focal beam spot region；
Step 7: the laser focal beam spot position that computer measures postposition light splitting pupil light splitting pupil confocal laser detection system
The Information in Mass Spectra that the laser that height of specimen information and mass spectrometry detection system measure focuses microcell carries out fusion treatment, is then gathered
The height Information in Mass Spectra of burnt hot spot microcell；
Step 9: computer control precise three-dimensional working platform makes to measure the next to be measured of object focal point alignment sample
Then region is operated by step 2~step 8, obtain the height Information in Mass Spectra of next focal zone to be measured；
Measured Step 10: repeating step 9 until all tested points on sample, then using computer into
Row processing obtains sample topographical information and complete component information.
Postposition of the invention is divided pupil confocal laser mass spectrum micro imaging method, makes collimated light beam shaping described in step 1
For annular beam, which focuses on sample through Amici prism transmission, dichroscope A reflection, measurement object lens again
Desorption ionization generates plasma plume.
Postposition of the invention is divided pupil confocal laser mass spectrum micro imaging method, and the pupil is D type postposition pupil or circle
Shape postposition pupil；Collecting pupil is that D type collects pupil or circular collection pupil；D type postposition pupil and D type collect pupil to be made jointly
With；Round postposition pupil and circular collection pupil are used in conjunction with.
Postposition of the invention is divided pupil confocal laser mass spectrum micro imaging method, compression focal beam spot system generation vector
The vector optical generator and iris filter of light beam substitute.
Postposition of the invention is divided pupil confocal laser mass spectrum microscopic imaging device: the light source system including generating excitation beam
System, Amici prism, the dichroscope A being sequentially placed along light source exit direction, measurement identical with dichroscope A reflection direction
The postposition of object lens, precision three-dimensional workbench, Amici prism reflection direction is divided pupil confocal laser measuring system, mass spectrograph and calculating
Machine processing system.
Postposition of the invention is divided pupil confocal laser mass spectrum microscopic imaging device, wherein the detection of postposition light splitting pupil confocal laser
Module, optional following two mode are realized:
Mode one, postposition light splitting pupil confocal laser detecting module are made of relaying amplifying lens, pin hole and light intensity detector,
Wherein pin hole is located in the image planes of relaying amplifying lens.
Mode two: postposition light splitting pupil confocal laser detecting module is made of relaying amplifying lens and ccd detector, wherein visiting
Survey the image plane center that region is located at ccd detector.
Postposition of the invention is divided pupil confocal laser mass spectrum microscopic imaging device, and light-source system is by pulse laser, optically focused
Lens, collector lens focal point Optic transmission fiber substitution simultaneously, in laser focusing system introduce outgoing beam attenuator, rear
It sets and introduces detection beam attenuator in light splitting pupil confocal laser detection system.By outgoing beam attenuator and detection beam attenuator
Light intensity regulating system is constituted, it is fixed to adapt to sample surfaces for the spot intensity of decay focal beam spot and light intensity detector detection
Light intensity demand when position.
The utility model has the advantages that
1) " extreme point " of pupil confocal laser axial strength curve and the focus of high-acruracy survey object lens are divided by postposition
This characteristic is accurately corresponded to, accurate fixed-focus is realized to sample, is able to suppress existing mass spectrograph because in long-time mass spectrum imaging
Drifting problem of the focal beam spot with respect to sample；
2) the preparatory fixed-focus of sample is carried out using " extreme point " of postposition light splitting pupil confocal laser axial strength curve, makes minimum
Focal beam spot focuses on sample surfaces, can be realized sample microcell high-space resolution mass spectrometry detection and microcell micro-imaging, effectively
Ground plays the potential differentiated between postposition light splitting pupil confocal laser system altitude；
3) using compression focal beam spot technology, it can be improved the spatial resolving power of laser mass spectrometry instrument；
4) signal is obtained due to the method using division focal spot, focal plane can be detected in image detection system by changing
The parameter of set tiny area is gone up to match the reflectivity of different samples, so as to extend its application field；It can also
It is enough that the matching that the object lens of the measurement to different NA values can be realized only is handled by computer system software, without again to system
Any hardware adjustment is carried out, the versatility of instrument is advantageously implemented.
Detailed description of the invention
Fig. 1 is that postposition of the present invention is divided pupil confocal laser mass spectrum micro imaging method schematic diagram；
Fig. 2 is that the postposition of the embodiment of the present invention 2 is divided pupil confocal laser mass spectrum micro imaging method and schematic device；
Fig. 3 is that the postposition of the embodiment of the present invention 3 is divided pupil confocal laser mass spectrum micro imaging method and schematic device；
Fig. 4 is that postposition is divided pupil confocal laser axial strength curve；
Wherein: 1- light-source system, 2- collimation lens, 3- collimated light beam, 4- compression focal beam spot system, 5- Amici prism,
6- dichroscope A, 7- measure object lens, 8- sample, 9- plasma plume, 10- precision three-dimensional workbench, 11-D type postposition pupil,
12-D type collects pupil, 13- detection object lens, 14- postposition light splitting pupil confocal laser detection system, 15- and relays amplifying lens, 16-
Pin hole, 17- light intensity detector, 18- amplification Airy, 19- search coverage, 20- postposition light splitting pupil confocal laser axial strength are bent
Line, 21- modulation light beam, 22- detection ion, 23- Returning beam, 24- postposition light splitting pupil measuring beam, 25- decaying light beam, 26-
Optical fiber incidence end, 27- fiber exit end, 28- ion suction pipe, 29- mass spectrograph, 30- computer, 31- vector optical generator, 32-
Iris filter, 33- circle postposition pupil, 34- circular collection pupil, 35-CCD detector, 36- pulse laser, 37- optically focused
Lens, 38- Optic transmission fiber, 39- outgoing beam attenuator, 40- detect beam attenuator.
Specific embodiment
Invention is further described in detail with reference to the accompanying drawings and examples.
Embodiment 1
As shown in Figure 1, placing D type postposition on detection 13 pupil plane of object lens collects pupil 11, light-source system 1 selects point light
Source, point light source outgoing excitation beam by collimation lens 2, compression focal beam spot system 4, Amici prism 5, dichroscope A6 with
It after measuring object lens 7, is focused on sample 8, computer 30 controls precision three-dimensional workbench 10 and sample 8 is driven to measure
7 near focal point of object lens moves up and down, and the light through sample reflection passes through D type by dichroscope A6 reflection, the reflection of Amici prism 5
D type in postposition pupil 11 is collected pupil, detection object lens 13 and relaying amplifying lens 14, convergence and is visited after penetrating pin hole 16 by light intensity
It surveys device 17 to receive, obtains postposition light splitting pupil confocal laser axial strength curve 20 by light intensity signal processor；
It can be accurately positioned the axial height of sample 8 using postposition light splitting pupil confocal laser axial strength curve 20
Information；
Change point light source operating mode, improve illumination intensity, the microcell desorption ionization of excitation sample 8 generates plasma
Body feathers 9；
The molecule in plasma plume 9 that is generated focal beam spot desorption ionization sample 8 using ion suction pipe 28, original
Son and ion suck to form particle detection 22, and particle detection 22, which enters in mass spectrometry detection system 29, carries out mass spectrum imaging, measure pair
Answer the Information in Mass Spectra in focal beam spot region；
The laser that postposition light splitting pupil confocal laser detection system measures is focused micro-raman spectra information by computer 30, mass spectrum is visited
The Information in Mass Spectra that the laser that examining system 29 measures focuses microcell carries out fusion treatment, obtains the height and mass spectrum of focal beam spot microcell
Information；
Computer 30, which controls precision three-dimensional workbench 10, to be made to measure next region to be measured that object lens 7 are directed at sample 8,
Then the height and Information in Mass Spectra of next focal zone to be measured are obtained；
Until all tested points on sample 8 are measured, then computer 30 is utilized to carry out data fusion and figure
As reconstruction processing, sample topographical information and complete component information can be obtained.
Embodiment 2
As shown in Fig. 2, compressing focal beam spot system 4 by swearing in postposition light splitting pupil confocal laser mass spectrum microscopic imaging device
Beam production system 31, the substitution of iris filter 32 are measured, the D type in D type postposition pupil 11 collects pupil 12 can be by round postposition
Circular collection pupil 34 in pupil 33 substitutes, and postposition is divided pin hole 16 and light intensity detector in pupil confocal laser detecting module
17 can be replaced by ccd detector 35, and wherein search coverage is located at the image plane center of ccd detector.
Remaining imaging method and process are same as Example 1.
Embodiment 3
As shown in figure 3, postposition light splitting pupil confocal laser mass spectrum microscopic imaging device in, point light source by pulse laser 36,
The substitution of Optic transmission fiber 38 of collector lens 37,37 focal point of collector lens, the light beam that laser 36 is emitted pass through collector lens 37
It assembles, the optical fiber incidence end 26 by being located at 37 focal point of collector lens receives, by fiber exit after the transmission of Optic transmission fiber 38
End 27 issues and forms point light source；Meanwhile outgoing beam attenuator 39 is introduced in laser focusing system, pupil laser is divided in postposition
Detection beam attenuator 40 is introduced in confocal detection system.Light is constituted by outgoing beam attenuator 39 and detection beam attenuator 40
Strong regulating system, illuminating bundle is modulated to decaying light beam 25 and is used to illuminate by irradiating light beam attenuator 43, for focal beam spot of decaying
The spot intensity detected with light intensity detector 17, to adapt to light intensity demand when sample surfaces positioning.
Remaining imaging method and process are same as Example 1.
A specific embodiment of the invention is described in conjunction with attached drawing above, but these explanations cannot be understood to limit
The scope of the present invention.Protection scope of the present invention is limited by appended claims, any in the claims in the present invention base
Change on plinth is all protection scope of the present invention.
Claims (8)
1. postposition is divided pupil confocal laser mass spectrum micro imaging method, it is characterised in that: utilize high-space resolution confocal microscopy system
The focal beam spot of system carries out axial fixed-focus and imaging to sample (8), is divided pupil laser to postposition using mass spectrometry detection system
Confocal microscope system focal beam spot desorption ionization sample (8) and charged molecule, the atom etc. generated carry out microcell mass spectrum at
Picture, then again by the fusion of detection data information and compare analysis then realize sample (8) microcell high-space resolution and
The imaging and detection of highly sensitive pattern, component, comprising the following steps:
Step 1: light-source system (1) is collimated light beam (3) by collimation lens (2) collimation, collimated light beam (3) is poly- by compression
Burnt spot system (4) reflects through Amici prism transmission (5), dichroscope A (6) and focuses on sample by measurement object lens (7)
(8) on；
Step 2: computer (30) control precision three-dimensional workbench (10) is made to drive sample (8) along measuring surface normal direction
It is moved up and down in measurement object lens (7) near focal point, reflects to form and return by dichroscope A (6) through sample (8) reflection light
Light echo beam (23), Returning beam (23) are after Amici prism (5) are reflected, through collection pupil, the detection object lens in postposition pupil
(13), amplifying lens (15) are relayed, convergence is received through after pin hole (16) by light intensity detector (17), is handled by light intensity signal
Device obtains postposition light splitting pupil confocal laser axial strength curve (20)；
Step 3: can be accurately positioned sample (8) point using postposition light splitting pupil confocal laser axial strength curve (20)
Axial height information；
Step 4: " extreme point " position control of computer (30) according to postposition light splitting pupil confocal laser axial strength curve (20)
Precision three-dimensional workbench (10) drives sample (8) to move along measuring surface normal direction, makes the focal beam spot for measuring object lens (7)
It focuses on sample (8)；
Step 5: changing collimated light beam (3) light illumination mode, the microcell desorption ionization of excitation sample (8) generates plasma
Plumage (9)；
Step 6: in the plasma plume (9) generated focal beam spot desorption ionization sample (8) using ion suction pipe (28)
Molecule, atom and ion suck to be formed detection ion (22), detection ion (22) enter mass spectrometry detection system in carries out mass spectrum
Imaging, measures the Information in Mass Spectra in corresponding focal beam spot region；
Step 7: the laser focal beam spot that computer (30) measures postposition light splitting pupil light splitting pupil confocal laser detection system (14)
The Information in Mass Spectra that the laser that position height of specimen information and mass spectrometry detection system (29) measure focuses microcell carries out fusion treatment, after
And obtain the height Information in Mass Spectra of focal beam spot microcell；
Step 9: computer (30) control precision three-dimensional workbench (10) makes to measure object lens (7) focus alignment sample (8)
Next region to be measured, is then operated by step 2~step 8, and the height mass spectrum of next focal zone to be measured is obtained
Information；
Step 10: repetition step 9 is measured until all tested points on sample (8), then computer (30) are utilized
It is handled and obtains sample topographical information and complete component information.
2. postposition according to claim 1 is divided pupil confocal laser mass spectrum micro imaging method, it is characterised in that: make described
Collimated light beam (3) be shaped as annular beam, the annular beam again through Amici prism (5) transmission, dichroscope A (6) reflection, survey
Amount object lens (7) focuses on desorption ionization on sample (8) and generates plasma plume (9).
3. postposition according to claim 1 is divided pupil confocal laser mass spectrum micro imaging method, it is characterised in that: after described
Setting pupil is D type postposition pupil (11) or round postposition pupil (33)；Collecting pupil is that D type collects pupil (12) or circular collection
Pupil (34)；D type postposition pupil (11) and D type are collected pupil (12) and are used in conjunction with；Round postposition pupil (33) and circular collection
Pupil (34) is used in conjunction with.
4. postposition according to claim 1 is divided pupil confocal laser mass spectrum micro imaging method, it is characterised in that: compression is poly-
Burnt spot system (4) vector optical generator (31) and iris filter (32) substitution for generating vector beam.
5. a kind of postposition is divided pupil confocal laser mass spectrum microscopic imaging device, it is characterised in that: the light including generating excitation beam
Source system (1), Amici prism (5), the dichroscope A (6) being sequentially placed along light source exit direction, it is anti-with dichroscope A (6)
The identical measurement object lens (7) in direction, precision three-dimensional workbench (10) are penetrated, the postposition light splitting pupil of Amici prism (5) reflection direction swashs
Light confocal measuring system (14), mass spectrograph (29) and computer (30) processing system.
6. postposition according to claim 5 is divided pupil confocal laser mass spectrum microscopic imaging device, it is characterised in that: postposition point
Pupil confocal laser detecting module (14) is made of relaying amplifying lens (15), pin hole (16) and light intensity detector (17), wherein
Pin hole (16) is located in the image planes of relaying amplifying lens (15).
7. postposition according to claim 5 is divided pupil confocal laser mass spectrum microscopic imaging device, it is characterised in that: postposition point
Pupil confocal laser detection system (14) is made of relaying amplifying lens (15) and ccd detector (35), wherein search coverage position
Image plane center in ccd detector (35).
8. postposition according to claim 5 is divided pupil confocal laser mass spectrum microscopic imaging device, it is characterised in that: light source system
System (1) is substituted simultaneously by the Optic transmission fiber (38) of pulse laser (36), collector lens (37), collector lens (37) focal point,
Outgoing beam attenuator (39) are introduced in laser focusing system, introduce detection in postposition light splitting pupil confocal laser detection system
Beam attenuator (40)；Light intensity regulating system is constituted by outgoing beam attenuator (39) and detection beam attenuator (40), is used for
The spot intensity of decaying focal beam spot and light intensity detector (17) detection, to adapt to the light intensity when positioning of sample (8) surface
Demand.
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