CN109187720A - Postposition is divided pupil laser differential confocal mass spectrum micro imaging method and device - Google Patents
Postposition is divided pupil laser differential confocal 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差动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差动共焦显微成像技术与质谱探测技术结合，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差动共焦探测结构融合实现样品微区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、矿产、微纳制造等领域形态成像及物质组分探测提供一条全新的有效技术途径。
The invention discloses a post-pupil laser differential confocal mass spectrometry microscopic imaging method and device, and belongs to the technical field of confocal microscopic imaging and mass spectrometry imaging measurement. The invention combines the post-pupil laser differential confocal microscopy imaging technology with the mass spectrometry detection technology, uses the mass spectrometry detection system to detect the charged molecules, atoms, etc. The fusion of detection structures realizes high spatial resolution and high sensitivity imaging and detection of sample micro-area component information and morphological parameters. The invention can provide a brand-new and effective technical approach for morphological imaging and material component detection in the fields of biomedicine, material science, minerals, micro-nano manufacturing and the like.
Description
Technical field
The invention belongs to confocal microscopic imaging technologies and mass spectrum imaging technical field, and postposition is divided pupil laser differential confocal
Micro-imaging technique is combined with mass spectrum imaging technology, is related to a kind of postposition light splitting pupil laser differential confocal 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 model
Enclose the various fields for expanding to life science, especially mass spectrum protein, nucleic acid, in terms of answer
With not only providing new tool for life science, but also also promote 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 science is ground in the accurate acquisition of mineral products, " microcell " pattern of space substance and biological sample and component information
Study carefully and produce detection and is all extremely important.In fact, how to detect micro-area composition information with sensitivity is current mine
Produce the important technological problems that the fields such as analysis, biochemistry detection are urgently studied.
Postposition is divided pupil laser differential confocal technology and is detected using illumination and the non-line structure altogether of detection optical path, is not only shown
The azimuthal resolution and Focus accuracy for improving optical path are write, 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 laser differential confocal mass spectrum micro imaging method and device,
Innovation is: for the first time will be with the postposition of high-space resolution ability light splitting pupil laser differential confocal microtechnic and mass spectrometry detection skill
Art blends imaging and detection, it can be achieved that sample microcell high-space resolution and highly sensitive pattern and component.
A kind of postposition light splitting pupil laser differential confocal mass spectrum micro imaging method of the present invention and device can be biomedical, material
Expect that the pattern component imaging detection in the fields such as science, physical chemistry, mineral products, minute manufacturing provides a completely new effective technology way
Diameter.
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 laser differential confocal mass spectrum micro imaging method and device, to obtain simultaneously
Obtain sample 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 laser differential confocal mass spectrum micro imaging method, utilizes high-space resolution postposition laser
The focal beam spot of differential confocal microscopic system carries out axial fixed-focus and imaging to sample, using mass spectrometry detection system to laser differential
Confocal microscope system focal beam spot desorption ionization sample and charged molecule, atom for generating etc. carry out microcell mass spectrum imaging, then
Sample microcell high-space resolution and highly sensitive form are then realized with analysis is compared by the fusion of detection data information again
With the imaging and detection of component, comprising the following steps:
Step 1: reflecting collimated light beam simultaneously by compression focal beam spot system, through Amici prism transmission, dichroscope A
It is focused on sample by measurement object lens；
Step 2: computer control precise three-dimensional working platform is made to drive sample attached in measurement object focal point along measuring surface
It closely moves up and down, the light through sample reflection reflects to obtain Returning beam, Returning beam by Amici prism by dichroscope A
After reflection, postposition light splitting pupil measuring beam is formed after the collection pupil filtering in postposition pupil, postposition is divided pupil measuring beam
It converges on dual-quadrant detector by detection object lens, relaying amplifying lens and after relaying amplifying lens, is put about relaying
The first symmetrically placed detection quadrant of big lens axis and the second detection quadrant are split detection to amplification Airy, are ended
In the first microcell of spot the off-axis confocal axial strength curve of strength characteristics first, the strength characteristics of the second microcell of Airy is bent
The off-axis confocal axial strength curve of line second；
It is handled Step 3: the first off-axis confocal axial strength curve and the second off-axis confocal axial strength curve are subtracted each other
It is divided pupil laser differential confocal axial strength curve to postposition, is divided pupil laser differential confocal axial strength curve energy using postposition
Enough it is accurately positioned this of sample axial height information；
Step 4: computer controls precision according to the dead-center position of postposition light splitting pupil laser differential confocal 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 particle detection, and particle detection, 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 sample that computer measures postposition light splitting pupil laser differential confocal detection system
The Information in Mass Spectra that the laser that product elevation information, mass spectrometry detection system measure focuses microcell carries out fusion treatment, is then focused
The height and Information in Mass Spectra of hot spot microcell；
Step 8: 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 7, obtain the height and Information in Mass Spectra of next focal zone to be measured；
Measured Step 9: repeating step 8 until all tested points on sample, then using computer into
Row processing obtains sample shape information and complete component information.
Postposition of the invention is divided pupil laser differential confocal-mass spectrum micro imaging method, makes the collimated light beam shaping
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 laser differential confocal mass spectrum micro imaging method, and the pupil is D type postposition pupil
Or round postposition pupil；Collecting pupil is that D type collects pupil or circular collection pupil；It is total that D type postposition pupil and D type collect pupil
With use；Round postposition pupil and circular collection pupil are used in conjunction with.
Postposition of the invention is divided pupil laser differential confocal mass spectrum micro imaging method, and compression focal beam spot system generates
The vector beam generating system and iris filter of vector beam substitute.
Postposition of the invention is divided pupil laser differential confocal mass spectrum microscopic imaging device, the light source including generating excitation beam
System, the Amici prism being sequentially placed along light source exit direction, dichroscope A and dichroscope A reflection direction measurement object
The postposition of mirror, 3 D scanning system, Amici prism reflection direction is divided pupil laser differential confocal measuring system, mass spectrometry detection system
And computer processing system.
Postposition of the invention is divided pupil laser differential confocal mass spectrum microscopic imaging device, and postposition is divided pupil laser differential confocal
The optional following two mode of detecting module is realized:
Mode one, postposition are divided pupil laser differential confocal detecting module by relaying amplifying lens and dual-quadrant detector structure
At wherein the first detection quadrant on dual-quadrant detector test surface and the second detection quadrant are symmetrical about optical axis.
Mode two: postposition light splitting pupil laser differential confocal detecting module is made of relaying amplifying lens, ccd detector,
The first microcell of Airy and the second microcell of Airy that middle ccd detector detects are symmetrical about optical axis.
Postposition of the invention is divided pupil laser differential confocal mass spectrum microscopic imaging device, light-source system by pulse laser,
Collector lens, collector lens focal point Optic transmission fiber substitution simultaneously, in laser focusing system introduce outgoing beam attenuator,
Detection beam attenuator is introduced in postposition light splitting pupil laser differential confocal detection system.By outgoing beam attenuator and detection light
Beam attenuation device constitutes Light intensity regulating system, for the spot intensity of decay focal beam spot and dual-quadrant detector detection, to adapt to
Light intensity demand when sample surfaces position.
The utility model has the advantages that
1) " zero crossing " and high-acruracy survey object lens of pupil laser differential confocal axial response curve are divided by postposition
Focus accurately corresponds to this characteristic, realizes accurate fixed-focus to sample, be able to suppress existing mass spectrograph because long-time mass spectrum at
Drifting problem of the focal beam spot with respect to sample as in；
2) the preparatory fixed-focus of sample is carried out using " zero crossing " of postposition light splitting pupil laser differential confocal curve, makes minimum focusing
Hot spot focuses on sample surfaces, can be realized sample microcell high-space resolution mass spectrometry detection and microcell micro-imaging, effectively sends out
Wave the potential differentiated between postposition light splitting pupil laser differential confocal 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, it can be set on system detection focal plane by changing
The parameter of tiny area is to match the reflectivity of different samples, so as to extend its application field；Calculating can also only be passed through
The matching of the measurement object lens to different NA values is realized in the processing of machine system software, without carrying out any hardware adjustment to system again,
It is advantageously implemented the versatility of instrument.
Detailed description of the invention
Fig. 1 is that postposition of the present invention is divided pupil laser differential confocal mass spectrum micro imaging method schematic diagram；
Fig. 2 is that the postposition light splitting pupil laser differential confocal mass spectrum micro imaging method of the embodiment of the present invention 1 shows with device
It is intended to；
Fig. 3 is that the postposition light splitting pupil laser differential confocal mass spectrum micro imaging method of the embodiment of the present invention 2 shows with device
It is intended to；
Fig. 4 is light splitting pupil differential confocal 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- detects object lens, 14- postposition light splitting pupil laser differential confocal detection system, 15- relay amplifying lens,
It is micro- that 16- dual-quadrant detector, 17- first detect quadrant, 18- second detects quadrant, 19- amplifies Airy, 20- Airy first
Area, the second microcell of 21- Airy, the off-axis confocal axial strength curve of 22- first, the off-axis confocal axial strength curve of 23- second,
24- is divided pupil laser differential confocal axial strength curve, 25- modulates light beam, 26- particle detection, 27- Returning beam, 28- postposition
Be divided pupil measuring beam, 29- decaying light beam, 30- optical fiber incidence end, 31- fiber exit end, 32- ion suction pipe, 33- mass spectrograph,
34- computer, 35- vector optical generator, 36- iris filter, 37- circle postposition pupil, 38- circular collection pupil, 39-
Ccd detector, 40- pulse laser, 41- collector lens, 42- Optic transmission fiber, 43- outgoing beam attenuator, 44- detect light beam
Attenuator.
Specific embodiment
Invention is further described in detail with reference to the accompanying drawings and examples.
Embodiment 1
Pupil 11 is collected as shown in Figure 1, placing on detection 13 pupil plane of object lens.Light-source system 1 selects point light source, puts light
The excitation beam of source outgoing is by collimation lens 2, compression focal beam spot system 4, Amici prism 5, dichroscope A6 and measurement object
It after mirror 7, converges on sample 8, computer 34 controls precision three-dimensional workbench 10 and drives sample 8 in measurement object lens 7
Near focal point moves up and down, and the light through sample reflection passes through D type postposition by dichroscope A6 reflection, the reflection of Amici prism 5
D type in pupil 11 converges on dual-quadrant detector 16 after collecting pupil 12, detection object lens 13, relaying amplifying lens 14, closes
It is split detection in the first symmetrically placed detection quadrant 17 of acquisition optical axis and second detection 18 pairs of amplification Airy 19 of quadrant,
The strength characteristics for obtaining the first microcell of Airy 20 and the second microcell of Airy 21 are respectively that the first off-axis confocal axial direction is strong
It writes music line 22 and the second off-axis confocal axial strength curve 23；
First off-axis confocal axial strength curve 22 and the second off-axis confocal axial strength curve 23 are subtracted each other processing and obtained
Postposition is divided pupil laser differential confocal axial strength curve 24, is divided pupil laser differential confocal axial strength curve 24 using postposition
This axial height information of sample 8 can be accurately positioned；
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 32, original
Mass spectrum imaging is carried out in son and ion sucking mass spectrometry detection system 33, measures the Information in Mass Spectra in corresponding focal beam spot region；
The laser that postposition light splitting pupil laser differential confocal detection system measures is focused micro-raman spectra information, matter by computer 34
The Information in Mass Spectra that the laser that measures of spectrum detection system 33 focuses microcell carries out fusion treatment, obtain focal beam spot microcell height and
Information in Mass Spectra；
Computer 34, 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 34 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 in postposition light splitting pupil laser differential confocal mass spectrum microscopic imaging device
It being substituted by vector beam generating system 35, iris filter 36, D type postposition pupil 11 can be substituted by round postposition pupil 37, and two
Quadrant detector is substituted by ccd detector 39, and wherein the first microcell of Airy and Airy second of ccd detector detection are micro-
Area, it is symmetrical about optical axis.
Remaining imaging method and process are same as Example 1.
Embodiment, 3
As shown in figure 3, point light source 1 can be swashed by pulse in postposition light splitting pupil laser differential confocal mass spectrum microscopic imaging device
The substitution of Optic transmission fiber 42 of light device 40, collector lens 41,41 focal point of collector lens, the light beam that laser 40 is emitted pass through optically focused
Lens 41 are assembled, and the optical fiber incidence end 30 by being located at 41 focal point of collector lens receives, by light after the transmission of Optic transmission fiber 42
The fine sending of exit end 31 forms point light source；Meanwhile outgoing beam attenuator 43 is introduced in laser focusing system, it is divided in postposition
Detection beam attenuator 44 is introduced in pupil laser differential confocal detection system.By outgoing beam attenuator 43 and detection beam attenuation
Device 44 constitutes Light intensity regulating system, and illuminating bundle is modulated to decaying light beam 29 and is used to illuminate by irradiating light beam attenuator 43, for declining
Subtract the spot intensity that focal beam spot and dual-quadrant detector 16 detect, 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 laser differential confocal mass spectrum micro imaging method, it is characterised in that: swashed using high-space resolution postposition
The focal beam spot that the equation of light moves confocal microscope system carries out axial fixed-focus and imaging to sample (8), utilizes mass spectrometry detection system
Charged molecule, atom generated to laser differential confocal microscopic system focal beam spot desorption ionization sample (8) etc. carries out
Then microcell mass spectrum imaging then realizes that sample (8) microcell is high with analysis is compared by the fusion of detection data information again
The imaging and detection of spatial discrimination and highly sensitive form and component, comprising the following steps:
Step 1: collimated light beam (3) is made to transmit (5), dichroscope A by compression focal beam spot system (4), through Amici prism
(6) it reflects and is focused on sample (8) by measurement object lens (7)；
Step 2: computer (34) control precision three-dimensional workbench (10) is made to drive sample (8) along measuring surface in measurement object
Mirror (7) near focal point moves up and down, and the light through sample (8) reflection obtains Returning beam by dichroscope A (6) reflection
(27), Returning beam (27) forms postposition point after Amici prism (5) reflection after the collection pupil filtering in postposition pupil
Pupil measuring beam (28), postposition light splitting pupil measuring beam (28) are by detection object lens (13), relaying amplifying lens (15) and position
It is converged on dual-quadrant detector (16) after relaying amplifying lens (15), it is symmetrically placed about relaying amplifying lens (15) optical axis
The first detection quadrant (17) and the second detection quadrant (18) be split detection to Airy (19) are amplified, obtain Airy the
The off-axis confocal axial strength curve (22) of the strength characteristics first of one microcell (20), the intensity of the second microcell of Airy (22)
The off-axis confocal axial strength curve (23) of characteristic curve second；
Step 3: the first off-axis confocal axial strength curve (22) and the second off-axis confocal axial strength curve (23) are subtracted each other place
Reason obtains postposition light splitting pupil laser differential confocal axial strength curve (24), axially strong using postposition light splitting pupil laser differential confocal
Line (24) of writing music can be accurately positioned sample (8) this axial height information；
Step 4: dead-center position control of the computer (34) according to postposition light splitting pupil laser differential confocal axial strength curve (24)
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 (32)
Molecule, atom and ion suck to be formed particle detection (26), particle detection (26) 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 position that computer (34) measures postposition light splitting pupil laser differential confocal detection system (14)
Set sample (8) elevation information, the Information in Mass Spectra progress fusion treatment for the laser focusing microcell that mass spectrometry detection system (33) measures, after
And obtain the height and Information in Mass Spectra of focal beam spot microcell；
Step 8: computer (34) 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 7, and the height and matter of next focal zone to be measured are obtained
Spectrum information；
Step 9: repetition step 8 is measured until all tested points on sample (8), then computer (34) are utilized
It is handled and obtains sample (8) shape information and complete component information.
2. postposition according to claim 1 is divided pupil laser differential confocal mass spectrum micro imaging method, it is characterised in that: make
The collimated light beam (3) is shaped as annular beam, and the annular beam is anti-through Amici prism (5) transmission, dichroscope A (6) again
It penetrates, measure object lens (7) and focus on sample (8) desorption ionization and generate plasma plume (9).
3. postposition according to claim 1 is divided pupil laser differential confocal mass spectrum micro imaging method, it is characterised in that: institute
Stating postposition pupil is D type postposition pupil (11) or round postposition pupil (37)；Collecting pupil is that D type collects pupil (12) or round
It collects pupil (38)；D type postposition pupil (11) and D type are collected pupil (12) and are used in conjunction with；Round postposition pupil (37) and circle
Pupil (38) are collected to be used in conjunction with.
4. postposition according to claim 1 is divided pupil laser differential confocal mass spectrum micro imaging method, it is characterised in that: pressure
Polycondensation coke spot system (4) vector beam generating system (35) and iris filter (36) substitution for generating vector beam, is used
Light beam (25) are modulated in generating.
5. postposition is divided pupil laser differential confocal 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, is divided pupil with the postposition of Amici prism (5) reflection direction
Laser differential confocal measuring system (14), mass spectrometry detection system (33) and computer (34) processing system.
6. postposition according to claim 5 is divided pupil laser differential confocal mass spectrum microscopic imaging device, it is characterised in that: after
Set light splitting pupil laser differential detecting module (14) be made of relaying enlarging objective (15) and dual-quadrant detector (16), wherein two as
The first detection quadrant (17) and the second detection quadrant (18) on limit detector (16) test surface is symmetrical about optical axis.
7. postposition according to claim 5 is divided pupil laser differential confocal mass spectrum microscopic imaging device, it is characterised in that: after
It sets light splitting pupil laser differential confocal detecting module (14) to be made of relaying amplifying lens (15), ccd detector (39), wherein CCD
The first microcell of Airy (20) and the second microcell of Airy (21) that detector (39) detects are visited about the symmetrical two quadrant of optical axis
Surveying device (16) can be substituted with a ccd detector (36), wherein the first microcell of Airy and Airy of ccd detector detection
Second microcell, it is symmetrical about optical axis.
8. postposition according to claim 5 is divided pupil laser differential confocal mass spectrum microscopic imaging device, it is characterised in that: light
Source system (1) is substituted same by the Optic transmission fiber (42) of pulse laser (40), collector lens (41), collector lens (41) focal point
When, outgoing beam attenuator (43) are introduced in laser focusing system, in postposition light splitting pupil laser differential confocal detection system
Introduce detection beam attenuator (44)；Light intensity regulating system is constituted by outgoing beam attenuator (43) and detection beam attenuator (44)
System, for the spot intensity of decay focal beam spot and dual-quadrant detector (16) detection, to adapt to when sample (8) surface positions
Light intensity demand.
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