CN108844929A - It is divided the discrete fluorescence spectrum of pupil differential confocal and fluorescence lifetime detection method and device - Google Patents
It is divided the discrete fluorescence spectrum of pupil differential confocal and fluorescence lifetime detection method and device Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
- G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
- G01J3/02—Details
- G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
- G01J3/0216—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using light concentrators or collectors or condensers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
- G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
- G01J3/02—Details
- G01J3/0262—Constructional arrangements for removing stray light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
- G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
- G01J3/02—Details
- G01J3/0291—Housings; Spectrometer accessories; Spatial arrangement of elements, e.g. folded path arrangements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
- G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
- G01J3/28—Investigating the spectrum
- G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
- G01J3/4406—Fluorescence spectrometry
Abstract
The invention belongs to chemical substance detection technique fields, are designed using light splitting pupil, effectively shield interference of the optical element autofluorescence to result in excitation light path, improve the signal-to-noise ratio of system.In addition, differential confocal body surface location technology and discrete fluorescence spectrum and fluorescence lifetime measurement technology are blended；The high-acruracy survey of sample to be tested three-dimensional surface shape is realized using differential confocal technology, the fluorescence spectrum of sample to be tested surface each point and the highly sensitive detection of fluorescence lifetime are realized using discrete fluorescence spectrum and fluorescence lifetime Detection Techniques simultaneously, and then obtains the material composition distributed intelligence of three-dimensional high definition space.And in the fluorescence information measurement process of sample surfaces, present invention uses a variety of different discrete fluorescence detection means, user can be according to the chemical characteristic of test substance, and selecting use is fluorescence spectrum detection or fluorescence lifetime detection.The present invention is with a wide range of applications in biology, medicine, material science and clinical medicine diagnostic field.
Description
Technical field
The invention belongs to chemical substance detection technique fields, and highly sensitive identification can be not only carried out to the ingredient of substance,
The spatial distribution of material composition can also be detected, it will be in the research fields such as biology, medicine, material science and clinical doctor
Learning diagnosis aspect has important application.
Background technique
In ambits such as biology, physics, chemistry and materials, spectral detection and analysis are since it is with very high sensitive
Degree, molecular specificity and non-cpntact measurement characteristic have become a basic measurement means of basic research.Wherein, fluorescence light
Spectrum detection is that the characteristic for being generated fluorescence under ultraviolet light using substance and its intensity are carried out the qualitative and quantitative of substance and divided
The method of analysis.Especially for most of organic compounds, the fluorescence generated usually has very strong specificity, is directed toward
Property.Therefore fluorescence spectrum detection is especially suitable for applying to the constituent analysis of the organic compound strong to these fluorescent specifics.?
Industry, fluorescence spectrum detection can be used for the composition detection, raw material of industry composition detection, petroleum crude oil of various pollutants into sorting
Survey etc..In field of food safety, fluorescence spectrum detection can be used for the progress of the information such as the mildew bacterium to food, persticide residue
Detection and monitoring.In medical domain, fluorescence spectroscopy technique and Induced Fluorescence Microscopy be tumour and cancer real time imagery and
Detection provides new direction, and is expected to develop into the medical technology suitable for clinical diagnosis.It can be seen that fluorescence spectrum
Detection technique is a kind of very useful and promising optical detection means.
In general, fluorescence measurement techniques include fluorescence spectral measuring and fluorescence lifetime measurement two ways.Fluorescence spectral measuring
Technology is by realizing to the spatial distribution detection for issuing fluorescence from measuring samples, i.e., fixed excitation wavelength detects sample
Transmitting light intensity and lambda1-wavelength relation curve.Fluorescence spectroscopy technique is often combined with fluorescence probe, is applied and is surveyed in DNA
The fields such as sequence, polymer material science, bioluminescence imaging.In contrast, fluorescence lifetime detection be light-pulse generator excitation after from
Time required for the 1/e of fluorescence maximum intensity when the fluorescence intensity inspired in measuring samples drops to excitation.Fluorescent material
Fluorescence lifetime it is directly related with the conditions such as the structure of itself, polarity, the viscosity of locating microenvironment, therefore inspired from sample
Carrying out the life value of fluorescence is absolute, the not influence of the factors such as stimulated luminescence intensity, the concentration of fluorogen and photobleaching, and not by
The restriction of other limitation ionization meter factors.It can also be to the micro-loop locating for sample to be tested by carrying out fluorescence lifetime measurement to sample
Many biochemical parameters in border such as pH value, ion concentration, temperature equal distribution carry out quantitative measurment.
However, during the surface excitation fluorescence to sample to be tested is scanned imaging, when sample to be tested surface is deposited
When uneven, the spot size that not can guarantee excitation beam in sample to be tested surface different location is consistent, and then causes to examine
Resolution ratio cannot keep unanimously examining system at different locations.Particularly with the sample of some big rise and falls, in addition exist due to
Systematic survey object lens operating distance very little causes a possibility that object lens collide sample to be tested during Sample Scan, finally not only
It cannot get the fluorescence distribution imaging of sample surfaces, and the surface of system object lens caused to be contaminated.In addition to this, micro- in detection
When hypofluorescence signal, on excitation light path, strong light can not only excite the fluorescence of sample, can also excite the spontaneous glimmering of optical element
Light generates interference to experimental result, reduces the signal-to-noise ratio of system.
It is divided pupil design, helps to shield the autofluorescence on excitation light path, improves the signal-to-noise ratio of system, realize highly sensitive
Detection.Focusing technology altogether, filters out the scattering light outside focus using pin hole, relative to conventional microscope laterally resolution with higher
Rate, and there is unique axial chromatography ability.And differential confocal technology, then it is to be examined on the basis of total focusing technology
It surveys in optical path and is added about symmetrical two pin holes of optical axis, the corresponding light intensity sensor of each pin hole penetrates needle by detection
The difference of the two-way light intensity in hole, i.e. the differential confocal response of system come reflect object deviate focal plane distance.Differential confocal
The range of linearity and axial resolution of response curve are twice of the total focusing technology of tradition；Differential fashion can effectively inhibit light source
Drift and detector electronics drift and measured object surface reflectivity fluctuation etc. generations common-mode noise, improve system
Signal-to-noise ratio and stability are widely used in non-contact measurement field.
If the differential confocal technology with accurate axial resolution and light splitting pupil design can be applied to fluorescent scanning at
As in system, can not only guarantee that fluoroscopic imaging systems guarantee identical laterally resolution in each position of sample to be tested surface
Rate, can also effectively shield light at a possibility that effectively avoiding the sample to be tested of object lens impact surface big rise and fall in scanning process
The autofluorescence for learning element, improves the signal-to-noise ratio of system.Meanwhile entire scanning survey process completion after can simultaneously obtain to
The high-resolution object dimensional topographical information of sample, and the fluorescence spectrum distribution measured can carry out accurately with three-dimensional appearance
It is corresponding.This will be of great significance to the spatial compositional distribution for accurately analyzing sample to be tested comprehensively, can be in biology, material
The research fields such as, medicine are used widely.
Summary of the invention
In order to solve the surface fluorescence spectrum with three-dimensional profiles sample to be tested and fluorescence lifetime measurement problem, and
Optical element autofluorescence is to the interference problem of result in excitation light path, while obtaining sample to be tested with high axial resolution
The fluorescence spectrum information and fluorescence lifetime information of three-dimensional appearance information and its corresponding high s/n ratio of each position point, the present invention propose
" the discrete fluorescence spectrum of light splitting pupil differential confocal and fluorescence lifetime detection method and device ".
Concrete thought of the invention is：It is designed using light splitting pupil, effectively shields optical element autofluorescence in excitation light path
Interference to result improves the signal-to-noise ratio of system.In addition, by differential confocal body surface location technology and discrete fluorescence spectrum and
Fluorescence lifetime measurement technology blends；The high-acruracy survey of sample to be tested three-dimensional surface shape is realized using differential confocal technology,
Fluorescence spectrum and the fluorescence longevity of sample to be tested surface each point are realized using discrete fluorescence spectrum and fluorescence lifetime Detection Techniques simultaneously
The highly sensitive detection of life, and then obtain the material composition distributed intelligence of three-dimensional high definition space.
On the one hand, the present invention provides a kind of discrete fluorescence spectrum of light splitting pupil differential confocal and fluorescence lifetime detection device, packet
Pulsed laser light source and continuous laser source are included, the first spectroscope, beam expanding lens, illumination iris, object lens, collection pupil, No.1 are anti-
Penetrate mirror, No.1 dichroic beamsplitter, light splitting pupil differential confocal detection system, discrete fluorescence spectrum and fluorescence lifetime detection system,
D translation platform, signal picker and computer；
Wherein, illumination iris is placed on the pupil plane of object lens and collects pupil；First spectroscope issues pulsed laser light source
Pulse laser and continuous laser source issue continuous laser merge, formed synthesis light beam；Beam expanding lens, illumination iris,
Object lens are sequentially located in the exit direction of the synthesis light beam, and beam expanding lens is expanded light beam is synthesized, after illumination iris,
Object lens will synthesize formation detection light beam after light beam is assembled and be radiated on sample to be tested；What is ejected from sample to be tested is backward scattered
After light and fluorescence are penetrated by object lens collection, by collecting pupil, then reflected by No.1 reflecting mirror；It is anti-from No.1 reflecting mirror
Light beam after penetrating is divided by No.1 dichroic beamsplitter, is all the way wavelength intrinsic light beam identical with detection light beam wavelength, is entered
It is divided pupil differential confocal detection system, another way is the fluorescent light beam that wavelength is different from detection light beam wavelength, into discrete fluorescence
Spectrum and fluorescence lifetime detection system；
Sample to be tested is placed on D translation platform, is controlled D translation platform by computer and is driven sample to be tested along three, space
Direction is scanned movement；Signal picker becomes what light splitting pupil differential confocal detection system detected with sample to be tested height
It is transferred to computer after the Intensity response signal conversion of change, by obtaining differential confocal response curve after computer disposal, passes through difference
The dead-center position for moving confocal response curve accurately determines the height value of sample to be tested surface each point；When detection light beam focus on it is to be measured
When each sampling point position of sample surfaces, signal picker detects discrete fluorescence spectrum and fluorescence lifetime detection system
The fluorescent intensity information changed over time under different wave length by conversion after be transferred to computer, by obtained after computer disposal from
Fluorescence lifetime and fluorescence spectrum under the different wave length that the sampled point ejects；And then the three of sample surfaces are obtained by scanning
Tie up pattern and surface each point fluorescence lifetime at different wavelengths and fluorescence spectrum.
On the other hand, the present invention also provides a kind of discrete fluorescence spectrum of light splitting pupil differential confocal and fluorescence lifetime detection sides
Method, operating procedure are as follows：
(a) continuous laser that the pulse laser and continuous laser source issued pulsed laser light source by the first spectroscope issues
It merges, forms synthesis light beam, the pulse laser is identical with continuous laser wavelength；The synthesis light beam expands by beam expanding lens
Shu Houjing over-illumination pupil, assembled by object lens formed detection light beam be radiated on sample to be tested；Definition is perpendicular to the object lens light
Two orthogonal directions of axis are respectivelyxWithyDirection, the direction along objective lens optical axis arezDirection；
(b) rear orientation light of light beam irradiating sample generation and the fluorescence ejected from sample to be tested are collected by object lens together
Afterwards by collecting pupil, then reflected by No.1 reflecting mirror, the light beam of No.1 reflecting mirror reflection passes through No.1 dichroic beamsplitter
After be divided into two-way, be all the way wavelength and the identical intrinsic light beam of detection light beam wavelength, another way is that wavelength is different from detection light beam
The fluorescent light beam of wavelength；The intrinsic light beam enters light splitting pupil differential confocal detection system, and the fluorescent light beam enters discrete glimmering
Light spectrum and fluorescence lifetime detection system；
(c) continuous laser source, edge are openedxWithyThe mobile sample to be tested in direction to transversal scanning initial position (x 1, y 1), then
Edge in the positionzSample to be tested is scanned in direction, measures the difference changed with scan position using light splitting pupil differential confocal detection system
Confocal response curve is moved, and then accurately determines that detection light beam is focused on to test sample according to the dead-center position of differential confocal response curve
The surface location of product；
(d) close continuous laser source, according to the mobile sample to be tested of step (c) measurement result so that detection light beam focus on to
Sample surface, control pulsed laser light source issue pulse laser, inspire fluorescence on sample to be tested surface by pulse laser,
By discrete fluorescence spectrum and fluorescence lifetime detection system, the fluorescent intensity information changed over time under different wave length is obtained；And
The fluorescence lifetime under different wave length is obtained after carrying out data analysis to the information；
(e) edgexWithySample to be tested is scanned in direction, repeats the above steps, each scanning element (x i , y i ) at position using poor
The dead-center position for moving confocal response curve determines the surface information of sample to be tested at this location, and using discrete fluorescence spectrum and
The fluorescence lifetime of the fluorescence that the measurement of fluorescence lifetime detection system ejects from the position at different wavelengths；
(f) by obtained sample to be tested each scanning element (x i , y i ) position surface position information and the corresponding fluorescence longevity
Life information is reconstructed, while obtaining the fluorescence longevity of the three-dimensional appearance profile and its surface each point of sample at different wavelengths
Life.
The present invention, which compares prior art, has following innovative point：
1. differential confocal technology is combined with fluorescence spectrum and service life Detection Techniques, the height to material composition both may be implemented
Sensitive detection, at the same again can the information that detects, and provide of the three-dimensional spatial distribution information to material composition it is more abundant；
2. being designed using light splitting pupil, the effectively shielding secondary fluorescence signal that optical element generates from excitation light path is to experimental result
Interference, greatly improve the signal-to-noise ratio of system；
3. detecting the service life of fluorescence under different wave length simultaneously using multiple light intensity sensors, and sample to be tested chemistry is identified based on this
Ingredient, identification speed with higher and identification accuracy；
4. the relative fluorescence that the present invention can detect between sample to be tested fluorescence lifetime at different wavelengths and different wave length is strong
Information is spent, is contained much information.
The present invention, which compares prior art, has following remarkable advantage：
1. the space topography measurement of three-dimensional sample to be tested and the fluorescence spectrum and fluorescence of its spatial surface each point can be achieved at the same time
Lifetime measurement provides information more abundant for the three dimensional chemical constituent analysis of sample；
2. differential confocal detection has preferable signal-to-noise ratio and stability, facilitate the accurate positioning being distributed to material space；
3. the present invention can be used for the high-sensitivity measurement of autofluorescence, detected using the autofluorescence of sample to be tested its chemistry at
Point, in the detection process without using fluorescent marker, therefore detection process is very convenient.
Detailed description of the invention
Fig. 1 is the schematic diagram of the discrete fluorescence spectrum of present invention light splitting pupil differential confocal and fluorescence lifetime detection method；
Fig. 2 is the schematic diagram of the discrete fluorescence spectrum of present invention light splitting pupil differential confocal and fluorescence lifetime detection device；
Fig. 3 is the schematic diagram of present invention light splitting pupil differential confocal detection system；
Fig. 4 is that the present invention utilizes dichroic beamsplitter, the discrete fluorescence spectrum and glimmering of narrow band filter and light intensity sensor composition
Light service life detection system；
Fig. 5 is the present invention dividing using dichroic beamsplitter, narrow band filter, convergent lens, pin hole and light intensity sensor composition
Vertical fluorescence spectrum and fluorescence lifetime detection system；
Fig. 6 is to be substituted in the discrete fluorescence spectrum of the present invention and fluorescence lifetime detection system with more light intensity sensor groupsNA light intensity passes
The schematic diagram of sensor；
Fig. 7 is that the present invention is passed using dichroic beamsplitter, narrow band filter, fiber focus lens, fiber delay line and light intensity
The discrete fluorescence spectrum and fluorescence lifetime detection system of sensor composition；
Fig. 8 is that the discrete fluorescence spectrum that the present invention is formed using optical filter runner and light intensity sensor and fluorescence lifetime detect system
System；
Fig. 9 is the schematic diagram that the discrete fluorescence spectrum of present invention light splitting pupil differential confocal and fluorescence lifetime detect embodiment；
Figure 10 be the present invention light splitting pupil differential confocal answer curve FES (z) schematic diagram；
Wherein：1- pulsed laser light source, 2- continuous laser source, the first spectroscope of 3-, 4- beam expanding lens, 5- illumination iris, 6- are received
Collect pupil, 7- object lens, 8- sample to be tested, 9- D translation platform, 10- No.1 reflecting mirror, 11- No.1 dichroic beamsplitter, 12- point
Pupil differential confocal detection system, the discrete fluorescence spectrum of 13- and fluorescence lifetime detection system, 14- signal picker, 15- are calculated
No. bis- machine, the confocal convergent lens of 16- difference, 17- diplopore pin hole, 18- No.1 differential confocal light intensity sensor, 19- differential confocals
Light intensity sensor, No. bis- dichroic beamsplitters of 20-, 21- No. tri- dichroic beamsplitters, 22-NNumber dichroic beamsplitter, 23- No.1
Narrow band filter, 24- No. bis- narrow band filters, 25-（N-1）Number narrow band filter, 26-NNumber narrow band filter, 27- No.1 light
Strong sensor, 28- No. bis- light intensity sensors, 29-（N-1）Number light intensity sensor, 30-NNumber light intensity sensor, 31- No.1 are assembled
Lens, 32- No. bis- convergent lenses, 33-（N-1）Number convergent lens, 34-NNumber No. bis- convergent lens, 35- No.1 pin hole, 36- needles
Hole, 37-（N-1）Number pin hole, 38-NNumber pin hole, No. bis- reflecting mirrors of 39-, the more light intensity sensor groups of 40-, the first optical fiber of 41- focus
Lens, the second fiber focus lens of 42-, 43-（N-1）Fiber focus lens, 44-NFiber focus lens, the first light of 45-
Fine delay line, the second fibre delay line of 46-, 47-（N-1）Fibre delay line, 48-NFibre delay line, 49-（N+1）Light intensity
Sensor, 50- optical filter runner, 51-（N+2）No. tri- light intensity sensor, No. tetra- dichroic beamsplitters of 52-, 53- narrow-band-filters
Piece, No. tetra- narrow band filters of 54-, first photomultiplier tube of 55-, the second photomultiplier tube of 56-, 57- third photomultiplier tube,
The 4th photomultiplier tube of 58-.
Specific embodiment
Present invention will be further explained below with reference to the attached drawings and examples.
Basic thought of the present invention is effectively to shield the autofluorescence of excitation light path optical element using the design of light splitting pupil,
The signal-to-noise ratio of raising system；By the confocal body surface location technology and discrete fluorescence spectrum with accurate axial resolution and glimmering
Light lifetime measurement technology blends, and the high-acruracy survey of sample to be tested three-dimensional surface shape is solved using differential confocal technology, together
The discrete fluorescence spectrum of Shi Liyong and fluorescence lifetime Detection Techniques solve the fluorescence spectrum and fluorescence lifetime of sample to be tested surface each point
Highly sensitive detection, and then obtain the material composition distributed intelligence of three-dimensional high definition space.And believe in the fluorescence of sample surfaces
It ceases in measurement process, present invention uses a variety of different discrete fluorescence detection means, user can be according to specific application
Demand selects.Meanwhile user can also be according to the chemical characteristic of test substance, selecting use is fluorescence spectrum detection, also
It is that fluorescence lifetime detection or the two combine to identify its material composition.
Embodiment 1
Current embodiment require that solving the problems, such as to be the three-dimensional appearance for scanning sample to be tested simultaneously and analysis tumor tissues to test sample
Space distribution situation in product, and the boundary information of tumor tissues is judged accordingly.The present embodiment is visited using light splitting pupil differential confocal
Examining system realizes the measurement of three-dimensional appearance, using 400 nm ejected from sample by 355 nm wavelength pulsed lasers,
The fluorescence lifetime of this four wavelength of 450 nm, 530 nm and 580 nm judges whether each scanning element is tumour cell.Due to sample
The fluorescence signal of product is very faint, therefore the present embodiment has used photomultiplier tube as the light intensity sensor of fluorescence detection to mention
The fluorescent intensity detectivity of high system.Fig. 9, which is the present embodiment, is realizing the confocal discrete fluorescence spectrum of light splitting pupil difference and glimmering
Specific implementation device when the light service life detects, including pulsed laser light source 1, continuous laser source 2, the first spectroscope 3, beam expanding lens
4, illumination iris 5, collection pupil 6, object lens 7, D translation platform 9, No.1 reflecting mirror 10, No.1 dichroic beamsplitter 11, light splitting
Pupil differential confocal detection system 12, discrete fluorescence spectrum and fluorescence lifetime detection system 13, signal picker 14, computer 15.
Wherein, pulsed laser light source 1 and the wavelength of continuous laser source 2 are 355 nm, and the pulse width of pulsed laser light source 1 is 2
ns.The laser that pulsed laser light source 1 and continuous laser source 2 issue successively passes through beam expanding lens after closing beam by the first spectroscope 3
4, detection light beam is formed after illumination iris 5, object lens 7 to be radiated on sample to be tested 8.Sample to be tested 8 is placed in D translation platform 9
On, it is scanned by D translation platform 9.Light beam irradiates the rear orientation light that sample to be tested 8 generates and from sample to be tested 8
After the fluorescence ejected passes through the collection of object lens 7 together, after collecting pupil 6, is reflected by No.1 reflecting mirror 10, then passed through
It is divided into two-way after No.1 dichroic beamsplitter 11, is all the way wavelength intrinsic light beam identical with detection light beam wavelength, into light splitting
Pupil differential confocal detection system 12, another way is the fluorescent light beam that wavelength is different from detection light beam wavelength, into discrete fluorescence light
Spectrum and fluorescence lifetime detection system 13.
In the present system, light splitting pupil differential confocal detection system 12 include differential confocal convergent lens 16, diplopore pin hole 17,
No.1 differential confocal light intensity sensor 18, No. two differential confocal light intensity sensors 19.Wherein, two pin holes of diplopore pin hole 17,
It is put on the focal plane of differential confocal convergent lens 16, and about the optical axis of differential confocal convergent lens 16 bilateral symmetry
It sets.After No.1 differential confocal light intensity sensor 18 and No. two differential confocal light intensity sensors 19 are displaced the hole of diplopore pin hole respectively
Face.The light beam for entering light splitting pupil differential confocal detection system 12 is focused by differential confocal convergent lens 16, edgezAxis scans sample
Product, light beam passes through two pin holes of diplopore pin hole 17 respectively, by No.1 differential confocal light intensity sensor 18 and No. two differential confocals
Light intensity sensor 19 separately detects.
Discrete fluorescence spectrum and fluorescence lifetime detection system 13 include No. two dichroic beamsplitters 20, No.1 narrow band filter
23, the first photomultiplier tube 55, No. three dichroic beamsplitters 21, No. two narrow band filters 24, the second photomultiplier tube 56, No. four
Dichroic beamsplitter 52, No. three narrow band filters 53, third photomultiplier tube 57, No. four narrow band filters 54, the 4th photoelectricity times
Increase pipe 58.20, No. three dichroic beamsplitters 21 of No. two dichroic beamsplitters and No. four dichroic beamsplitters 52 will enter discrete glimmering
Light spectrum and the light beam of fluorescence lifetime detection system 13 carry out 3 light splitting, obtain the fluorescent light beam of 4 road different wave length bands.This 4 tunnel
The fluorescent light beam of different wave length band is respectively through the narrow band filter 53 of narrow band filter 24, three of No.1 narrow band filter 23, two
With after No. four narrow band filters 54 by the first photomultiplier tube 55, the second photomultiplier tube 56, third photomultiplier tube 57 and
The detection of four photomultiplier tubes 58 receives.The corresponding central wavelength of each narrow band filter is 400 nm respectively, 450 nm, 530 nm and
580 nm.Signal picker 14 is used to acquire light splitting pupil differential confocal detection system 12 and discrete fluorescence spectrum and fluorescence lifetime is visited
The intensity signal that each light intensity sensor collects in examining system 13, and computer 15 is transferred to after being converted.Computer 15
The fluorescent intensity changed over time under confocal response curve and each wavelength is obtained after analyzing the intensity signal collected
Information.It defines and is respectively perpendicular to two orthogonal directions of 7 optical axis of object lensxWithyDirection, the direction along 7 optical axis of object lens arezDirection.
The step of it identifies sample to be tested ingredient is as follows.
(a) continuous laser source 2, edge are openedxWithyThe mobile sample to be tested 8 in direction to transversal scanning initial position (x 1 , y 1), then edge in the positionzScan sample to be tested 8 in direction.Using light splitting pupil differential confocal detection system 12, measure with scanning
Change in location differential confocal response curve FES as shown in Fig. 10 (z), and then according to differential confocal response curve FES (z)
Zero point response point accurately determine that detection light beam focuses on the surface location of sample to be tested, record the scan position (x 1 , y 1) at
The surface location height of sample 8 isz 1。
(b) continuous laser source is closed, according to the mobile sample to be tested 8 of step (a) measurement result, so that detection light beam is poly-
Coke controls pulsed laser light source 1 and issues pulse laser on 8 surface of sample to be tested, is swashed on 8 surface of sample to be tested by pulse laser
Fluorescence is issued, after the fluorescence ejected is collected by object lens 7, by collecting pupil 6, is reflected by No.1 reflecting mirror 10, then
Enter discrete fluorescence spectrum and fluorescence lifetime detection system 13 through No.1 dichroic beamsplitter 11.Into discrete fluorescence spectrum and
The fluorescence of fluorescence lifetime detection system 13 is divided into two-way after No. two dichroic beamsplitters 20, wherein the wavelength model of the reflected beams
It encloses for the nm of 360 nm ~ 430, the wave-length coverage through light beam is the nm of 430 nm ~ 700；It is penetrated from No. two dichroic beamsplitters 20
Light beam after No. three dichroic beamsplitters 21 again be divided into two-way, wherein the wave-length coverage of the reflected beams be 430 nm ~ 480
Nm, the wave-length coverage through light beam are the nm of 480 nm ~ 700；From No. three dichroic beamsplitters penetrate light beam by No. four two to
It is divided into two-way again after color spectroscope 54, wherein the wave-length coverage of the reflected beams is the nm of 480 nm ~ 550, through the wavelength model of light beam
It encloses for the nm of 550 nm ~ 700.The light beam reflected by No. two dichroic beamsplitters 20 is radiated at after penetrating No.1 narrow band filter 23
On first photomultiplier tube 57.The central wavelength of No.1 narrow band filter 23 is 400 nm, and bandpass width is 10 nm.Therefore by
The central wavelength of the received fluorescence of first photomultiplier tube 55 is 400 nm.The light beam reflected by No. three dichroic beamsplitters 21 is saturating
It is radiated on the second photomultiplier tube 56 after crossing No. two narrow band filters 24.The central wavelength of No. two narrow band filters 24 is 450
Nm, bandpass width are 10 nm.It therefore is 450 nm by the central wavelength of the received fluorescence of the second photomultiplier tube 56.By No. four
The light beam that dichroic beamsplitter 52 reflects is radiated on third photomultiplier tube 57 after penetrating No. three narrow band filters 53；It is No. three narrow
Central wavelength with optical filter 54 is 530 nm, and bandpass width is 10 nm.Therefore by the received fluorescence of third photomultiplier tube 57
Central wavelength be 530 nm.The light beam penetrated by No. four dichroic beamsplitters 52 is radiated at after penetrating No. four narrow band filters 54
On 4th photomultiplier tube 58；The central wavelength of No. four narrow band filters 54 is 580 nm, and bandpass width is 10 nm；Therefore by
The central wavelength of the received fluorescence of 4th photomultiplier tube 58 is 580 nm.
(c) the first photomultiplier tube 55 is detected to the fluorescence information changed over time under obtained 400 nm of central wavelength,
The fluorescence information changed over time under 450 nm of central wavelength that the detection of second photomultiplier tube 56 obtains, third photomultiplier tube
The fluorescence information and the detection of the 4th photomultiplier tube 58 changed over time under 530 nm of central wavelength that 57 detections obtain obtains
580 nm of central wavelength under the fluorescence information that changes over time while passing through after signal picker 14 acquires and be transferred to computer
15.Computer 15 obtains each wavelength pair by handling the fluorescence signal changed over time under these different central wavelengths
The fluorescence lifetime answered.Wherein, the corresponding fluorescence lifetime of 400 nm of central wavelength is 10.5 ns, and 450 nm of central wavelength is corresponding
Fluorescence lifetime is 7.3 ns, and the corresponding fluorescence lifetime of 530 nm of central wavelength is 13.3 ns, and 580 nm of central wavelength is corresponding glimmering
The light service life is 6.7 ns.
(d) according to the fluorescence lifetime of normal tissue fluorescence under each wavelength：The corresponding fluorescence lifetime of 400 nm of wavelength is 9
Between the ns of ns ~ 13, the corresponding fluorescence lifetime of 450 nm of wavelength is between the ns of 10 ns ~ 14,530 nm of the wavelength corresponding fluorescence longevity
Life is between the ns of 12 ns ~ 15, and the corresponding fluorescence lifetime of 580 nm of wavelength is between the ns of 11 ns ~ 14；Tumor tissues are in each wave
The fluorescence lifetime of long lower fluorescence：For the corresponding fluorescence lifetime of 400 nm of wavelength between the ns of 8 ns ~ 11,450 nm of wavelength is corresponding
Fluorescence lifetime is between the ns of 6 ns ~ 8, and the corresponding fluorescence lifetime of 530 nm of wavelength is between the ns of 13 ns ~ 16,580 nm of wavelength
Corresponding fluorescence lifetime is between the ns of 5 ns ~ 7；It is available from 7 surface of sample to be tested（x 1, y 1 , z 1）What place ejected
Fluorescence lifetime information and the fluorescence lifetime of tumor tissues exactly match, thus may determine that（x 1 , y 1 , z 1）Place is tumor group
It knits.
(e) edgexWithySample to be tested is scanned in direction, repeats the above steps, each scanning element (x i , y i ) benefit at position
The surface information of sample to be tested 8 at this location is determined with light splitting pupil differential confocal detection system 12z i , utilize discrete fluorescence light
Spectrum and fluorescence lifetime detection system 13 measure the fluorescence lifetime of the fluorescence ejected from the position at different wavelengths, and base
Judge at this in this for normal tissue or tumor tissues.
(f) sample to be tested for obtaining above- mentioned information each scanning element (x i , y i ) position surface location letter
Breathz i It is reconstructed with corresponding fluorescence lifetime information, while obtaining the three-dimensional appearance profile and its surface each point of sample 8
Fluorescence lifetime at different wavelengths, and obtain the judging result that surface each point is tumor tissues or normal tissue.And then root
According to these information, the boundary of space distribution situation and tumor tissues of the available tumor tissues in sample to be tested 8 is believed
Breath.
Embodiment 2
Unlike the first embodiment, this example judged using discrete fluorescence spectrum 8 surface each point of sample to be tested be tumor tissues also
It is normal tissue.Equipment therefor and sample are identical with embodiment 1.For the stability for improving fluorescence spectral measuring, the present embodiment is adopted
Sample to be tested 8 is excited to generate fluorescence with the light beam that continuous laser source 2 issues, measuring process is as follows.
(a) continuous laser source 2, edge are openedxWithyThe mobile sample to be tested 8 in direction to transversal scanning initial position (x 1 , y 1), then edge in the positionzScan sample to be tested 8 in direction.Using light splitting pupil differential confocal detection system 12, measure with scanning
Change in location differential confocal response curve FES as shown in Fig. 10 (z), and then song is responded according to differential confocal response curve
Line FES (z) zero point response point accurately determine that detection light beam focuses on the surface location of sample to be tested, record the scan position
(x 1 , y 1) at the surface location height of sample 8 bez 1。
(b) according to the mobile sample to be tested 8 of step (a) measurement result, so that detection light beam focuses on 8 table of sample to be tested
Face is radiated on 8 surface of sample to be tested by the continuous laser that continuous laser source 2 issues and inspires fluorescence, ejected
Fluorescence by object lens 7 collect after, through collection pupil 6, by No.1 reflecting mirror reflection 10 after, through No.1 dichroic beamsplitter 9 into
Enter discrete fluorescence spectrum and fluorescence lifetime detection system 13.Into discrete fluorescence spectrum and the fluorescence of fluorescence lifetime detection system 13
It is divided into two-way after No. two dichroic beamsplitters 20, wherein the wave-length coverage of the reflected beams is the nm of 360 nm ~ 430, through light
The wave-length coverage of beam is the nm of 430 nm ~ 700；The light beam penetrated from No. two dichroic beamsplitters 20 is by No. three dichroic light splitting
It is divided into two-way again after mirror 21, wherein the wave-length coverage of the reflected beams is the nm of 430 nm ~ 480, and the wave-length coverage through light beam is
480 nm~700 nm；The light beam penetrated from No. three dichroic beamsplitters 23 is divided into two again after No. four dichroic beamsplitters 54
Road, wherein the wave-length coverage of the reflected beams is the nm of 480 nm ~ 550, and the wave-length coverage through light beam is the nm of 550 nm ~ 700.By
The light beam of No. two dichroic beamsplitters 20 reflection is radiated on the first photomultiplier tube 55 after penetrating No.1 narrow band filter 23.One
The central wavelength of number narrow band filter 23 is 400 nm, and bandpass width is 10 nm.Therefore received by the first photomultiplier tube 55
The central wavelength of fluorescence is 400 nm.The light beam reflected by No. three dichroic beamsplitters 21 shines after penetrating No. two narrow band filters 24
It penetrates on the second photomultiplier tube 56.The central wavelength of No. two narrow band filters 24 is 450 nm, and bandpass width is 10 nm.Cause
This is 450 nm by the central wavelength of the received fluorescence of the second photomultiplier tube 56.The light reflected by No. four dichroic beamsplitters 52
Beam is radiated on third photomultiplier tube 57 after penetrating No. three narrow band filters 53；The central wavelength of No. three narrow band filters 53 is
530 nm, bandpass width are 10 nm.It therefore is 530 nm by the central wavelength of the received fluorescence of third photomultiplier tube 57.By
The light beam that No. four dichroic beamsplitters 52 penetrate is radiated on the 4th photomultiplier tube 58 after penetrating No. four narrow band filters 54；Four
The central wavelength of number narrow band filter 54 is 580 nm, and bandpass width is 10 nm；Therefore received by the 4th photomultiplier tube 58
The central wavelength of fluorescence is 580 nm.Computer 15 is obtained by handling the fluorescence signal under these different central wavelengths
To the corresponding fluorescence intensity information of each wavelength.Wherein, the corresponding fluorescence intensity of 400 nm of central wavelength is 5.6 μ W；Central wavelength
The corresponding fluorescence intensity of 450 nm is 8.4 μ W；The corresponding fluorescence intensity of 530 nm of central wavelength is 4.5 μ W；Central wavelength 580
The corresponding fluorescence intensity of nm is 9.8 μ W.And then the available sample to be tested 8 is in 400 nm, 450 nm, 530 nm and 580
Relative intensity of fluorescence spectrum under nm is 0.57:0.86:0.46:1.
(c) according to normal tissue, peak fluorescence spectrum is 530 nm in this four wavelength fluorescent information, and tumor tissues exist
Peak fluorescence spectrum is 580 nm in this four wavelength fluorescent information, obtain sample to be tested 8 its surface point (x 1 ,y 1 ,z 1) at be swollen
Tumor tissue.
(d) edgexWithySample to be tested is scanned in direction, repeats the above steps, each scanning element (x i , y i ) benefit at position
The surface information of sample to be tested 8 at this location is determined with differential confocal detection system 12z i , utilize discrete fluorescence spectrum and glimmering
Light service life detection system 13 measures the fluorescence intensity of the fluorescence ejected from the position at different wavelengths, and is sentenced based on this
The tissue at this that breaks is normal tissue or tumor tissues.
(e) sample to be tested for obtaining above- mentioned information each scanning element (x i , y i ) position surface location letter
Breathz i It is reconstructed with corresponding fluorescence lifetime information, while obtaining the three-dimensional appearance profile and its surface each point of sample 8
Discrete fluorescence spectrum, and obtain the judging result that surface each point is tumor tissues or normal tissue.And then according to these letters
Breath, the boundary information of space distribution situation and tumor tissues of the available tumor tissues in sample to be tested 8.
Embodiment 3
It unlike the first embodiment, as shown in Fig. 5, is the resolution ratio for improving detection light beam, before all light intensity sensors all
Convergent lens and pin hole are added respectively.Pin hole is placed at the focal position of convergent lens, and convergent lens is by variant wavelength
Space filtering is carried out by pin hole after fluorescent light beam convergence.Therefore, the fluorescence signal that each light intensity sensor detects is filtering
Fluorescent intensity information afterwards, these filtered fluorescence accurately correspond to the fluorescence letter that detection light beam focusing focus ejects
Number, the fluorescence signal except focus has been subjected to effective shielding.
Embodiment 4
Unlike the first embodiment, as shown in Fig. 6, it is simplied system structure, reduces system cost, the present embodiment array
Photomultiplier tube detectors substitute four used the photomultiplier tube detectors as more light intensity sensor groups, with array light
Different probe units separately detects to obtain the fluorescence information of different wave length in electric multiplier-detector.
Embodiment 5
Unlike the first embodiment, as shown in Fig. 7, discrete fluorescence spectrum and fluorescence lifetime detection system include 3 dichroics
Spectroscope（In the embodimentN= 4）, 4 narrow band filters, 4 fiber focus lens, 4 light with the different delayed time time
Fine delay line and one（N+1）Number light intensity sensor 49.It will be ejected from sample to be tested by this 3 dichroic beamsplitter
Fluorescent light beam carries out 3 light splitting, obtains the fluorescent light beam of 4 road different wave length bands；The fluorescent light beam of this 4 road different wave length band is distinguished
4 fiber delay times with the different delayed time time are coupled by 4 fiber focus lens after 4 narrow band filters filter
Line.The 4 road fluorescence after fiber delay line is delayed are synthesized in fiber delay line end to be exported, by（N+1）Number light intensity sensor
51 detections receive.The fluorescence of different wave length will be reached in different moments at this time（N+1）Number light intensity sensor 51, therefore can basis
The different periods separates the fluorescence information under different wave length.This mode is conducive to simplied system structure, reduction system volume,
Reduce system cost.
Embodiment 6
Unlike the first embodiment, as shown in Fig. 8,50 He of optical filter runner is utilized（N+2）Number 51 groups of ingredients of light intensity sensor
Vertical fluorescence spectrum and fluorescence lifetime detection system 13.Optical filter runner 50 is by 4 narrow band filters with different central wavelengths
Composition（In the embodimentN= 4）, optical filter runner 50 is every to be rotated, and pulsed laser light source issues a pulse laser, from
The fluorescent light beam that sample to be tested 8 ejects penetrates the narrow band filter under corresponding central wavelength,（N+2）Number light intensity sensor 51
Measure the fluorescent intensity information changed over time under respective wavelength.Thus, available 4 after the rotation of optical filter runner 50 4 times
The fluorescent intensity information changed over time under different wave length.Utilize the method for rotation optical filter runner 50, it is possible to reduce use
The number of light intensity sensor, therefore significantly reduce system development cost.
Claims (10)
1. being divided the discrete fluorescence spectrum of pupil differential confocal and fluorescence lifetime detection device, including pulsed laser light source and continuous laser
Light source, it is characterised in that：Including the first spectroscope, beam expanding lens, illumination iris, object lens, collect pupil, No.1 reflecting mirror, No.1
Dichroic beamsplitter, light splitting pupil differential confocal detection system, discrete fluorescence spectrum and fluorescence lifetime detection system, D translation
Platform, signal picker and computer；
Wherein, illumination iris is placed on the pupil plane of object lens and collects pupil；First spectroscope issues pulsed laser light source
Pulse laser and continuous laser source issue continuous laser merge, formed synthesis light beam；Beam expanding lens, illumination iris,
Object lens are sequentially located in the exit direction of the synthesis light beam, and beam expanding lens is expanded light beam is synthesized, after illumination iris,
Object lens will synthesize formation detection light beam after light beam is assembled and be radiated on sample to be tested；What is ejected from sample to be tested is backward scattered
After light and fluorescence are penetrated by object lens collection, by collecting pupil, then reflected by No.1 reflecting mirror；It is anti-from No.1 reflecting mirror
Light beam after penetrating is divided by No.1 dichroic beamsplitter, is all the way wavelength intrinsic light beam identical with detection light beam wavelength, is entered
It is divided pupil differential confocal detection system, another way is the fluorescent light beam that wavelength is different from detection light beam wavelength, into discrete fluorescence
Spectrum and fluorescence lifetime detection system；
Sample to be tested is placed on D translation platform, is controlled D translation platform by computer and is driven sample to be tested along three, space
Direction is scanned movement；Signal picker becomes what light splitting pupil differential confocal detection system detected with sample to be tested height
It is transferred to computer after the Intensity response signal conversion of change, by obtaining differential confocal response curve after computer disposal, passes through difference
The dead-center position for moving confocal response curve accurately determines the height value of sample to be tested surface each point；When detection light beam focus on it is to be measured
When each sampling point position of sample surfaces, signal picker detects discrete fluorescence spectrum and fluorescence lifetime detection system
The fluorescent intensity information changed over time under different wave length by conversion after be transferred to computer, by obtained after computer disposal from
Fluorescence lifetime and fluorescence spectrum under the different wave length that the sampled point ejects；And then the three of sample surfaces are obtained by scanning
Tie up pattern and surface each point fluorescence lifetime at different wavelengths and fluorescence spectrum.
2. the discrete fluorescence spectrum of light splitting pupil differential confocal according to claim 1 and fluorescence lifetime detection device, feature
It is：The light splitting pupil differential confocal detection system includes differential confocal convergent lens, diplopore pin hole, No.1 differential confocal light intensity
Sensor, No. two differential confocal light intensity sensors；Two pin holes of diplopore pin hole, positioned at the focal plane of differential confocal convergent lens
On, and placed about the optical axis of differential confocal convergent lens bilateral symmetry；No.1 differential confocal light intensity sensor and No. two differences
Confocal light intensity sensor is moved to be located at behind two pin holes of diplopore pin hole；No.1 differential confocal light intensity sensor detects
The light intensity signal that light intensity signal and No. two differential confocal light intensity sensors obtain subtracts each other, and differential confocal response curve can be obtained.
3. the discrete fluorescence spectrum of light splitting pupil differential confocal according to claim 1 and fluorescence lifetime detection device, described point
Vertical fluorescence spectrum and fluorescence lifetime detection system include（N-1）A dichroic beamsplitter,NA narrow band filter,NA light intensity sensing
Device：By described（N-1）A dichroic beamsplitter carries out the fluorescent light beam ejected from sample to be tested（N-1）Secondary light splitting, obtains
It arrivesNThe fluorescent light beam of road different wave length band；It is describedNThe fluorescent light beam of road different wave length band passes through respectivelyNA narrow band filter filters
Afterwards byNA light intensity sensor detection receives, and obtainsNThe fluorescent intensity information changed over time under a different wave length；Each narrowband filter
The corresponding central wavelength of mating plate isλ n , bandpass width δλ n , whereinn=1, 2, …, N。
4. the discrete fluorescence spectrum of light splitting pupil differential confocal according to claim 3 and fluorescence lifetime detection device, feature
It is：Described use is substituted with more light intensity sensor groupsNA light intensity sensor：More light intensity sensor groups byNA light intensity sensing
Device composition, is separately detected to obtain corresponding fluorescence intensity information with each light intensity sensor in more light intensity sensor groups.
5. the discrete fluorescence spectrum of light splitting pupil differential confocal according to claim 1 and fluorescence lifetime detection device, feature
It is：The discrete fluorescence spectrum and fluorescence lifetime detection system include（N-1）A dichroic beamsplitter,NA narrow band filter,NA fiber focus lens,NA fiber delay line with the different delayed time time and（N+1）Number light intensity sensor：By described
（N-1）A dichroic beamsplitter carries out the fluorescent light beam ejected from sample to be tested（N-1）Secondary light splitting, obtainsNRoad is different
The fluorescent light beam of wavelength band；It is describedNThe fluorescent light beam of road different wave length band passes through respectivelyNA narrow band filter filter after byNA light
Fine condenser lens is coupled intoNA fiber delay line with the different delayed time time；After fiber delay line is delayedNLu Ying
Light is synthesized in fiber delay line end and is exported, by（N+1）The detection of number light intensity sensor receives；The fluorescence of different wave length will be at this time
Different moments reach（N+1）Number light intensity sensor, therefore can be according to the different periods by the fluorescence information under different wave length point
It opens.
6. the discrete fluorescence spectrum of light splitting pupil differential confocal according to claim 1 and fluorescence lifetime detection device, feature
It is：The discrete fluorescence spectrum and fluorescence lifetime detection system include an optical filter runner and（N+2）Number light intensity sensor：
On the optical filter runner byNA narrow band filter composition with different central wavelengths, optical filter runner is every to be rotated, arteries and veins
Impulse radiant issues a pulse laser, and the fluorescent light beam ejected from sample to be tested is through narrow under corresponding central wavelength
Band optical filter,（N+2）Number light intensity sensor measures the fluorescent intensity information changed over time under respective wavelength；Optical filter runner turns
It is dynamicNIt is available after secondaryNThe fluorescent intensity information changed over time under a different wave length.
7. the discrete fluorescence spectrum of light splitting pupil differential confocal and fluorescence lifetime detection device according to claim 3 or 6, special
Sign is：Add convergent lens and pin hole respectively before each light intensity sensor：Pin hole is placed on the focus position of convergent lens
Place is set, convergent lens will carry out space filtering, each light intensity sensing by pin hole after the fluorescent light beam convergence of variant wavelength
Fluorescent intensity information after device detection filtering.
8. being divided the discrete fluorescence spectrum of pupil differential confocal and fluorescence lifetime detection method, it is characterised in that：
(a) continuous laser that the pulse laser and continuous laser source issued pulsed laser light source by the first spectroscope issues
It merges, forms synthesis light beam, the pulse laser is identical with continuous laser wavelength；The synthesis light beam expands by beam expanding lens
Shu Houjing over-illumination pupil, assembled by object lens formed detection light beam be radiated on sample to be tested；Definition is perpendicular to the object lens light
Two orthogonal directions of axis are respectivelyxWithyDirection, the direction along objective lens optical axis arezDirection；
(b) rear orientation light of light beam irradiating sample generation and the fluorescence ejected from sample to be tested are collected by object lens together
Afterwards by collecting pupil, then reflected by No.1 reflecting mirror, the light beam of No.1 reflecting mirror reflection passes through No.1 dichroic beamsplitter
After be divided into two-way, be all the way wavelength and the identical intrinsic light beam of detection light beam wavelength, another way is that wavelength is different from detection light beam
The fluorescent light beam of wavelength；The intrinsic light beam enters light splitting pupil differential confocal detection system, and the fluorescent light beam enters discrete glimmering
Light spectrum and fluorescence lifetime detection system；
(c) continuous laser source, edge are openedxWithyThe mobile sample to be tested in direction to transversal scanning initial position (x 1, y 1), then
Edge in the positionzSample to be tested is scanned in direction, measures the difference changed with scan position using light splitting pupil differential confocal detection system
Confocal response curve is moved, and then accurately determines that detection light beam is focused on to test sample according to the dead-center position of differential confocal response curve
The surface location of product；
(d) close continuous laser source, according to the mobile sample to be tested of step (c) measurement result so that detection light beam focus on to
Sample surface, control pulsed laser light source issue pulse laser, inspire fluorescence on sample to be tested surface by pulse laser,
By discrete fluorescence spectrum and fluorescence lifetime detection system, the fluorescent intensity information changed over time under different wave length is obtained；And
The fluorescence lifetime under different wave length is obtained after carrying out data analysis to the information；
(e) edgexWithySample to be tested is scanned in direction, repeats the above steps, each scanning element (x i , y i ) at position using poor
The dead-center position for moving confocal response curve determines the surface information of sample to be tested at this location, and using discrete fluorescence spectrum and
The fluorescence lifetime of the fluorescence that the measurement of fluorescence lifetime detection system ejects from the position at different wavelengths；
(f) by obtained sample to be tested each scanning element (x i , y i ) position surface position information and the corresponding fluorescence longevity
Life information is reconstructed, while obtaining the fluorescence longevity of the three-dimensional appearance profile and its surface each point of sample at different wavelengths
Life.
9. the discrete fluorescence spectrum of light splitting pupil differential confocal according to claim 1 and fluorescence lifetime detection method, feature
It is：In the dead-center position of the differential confocal response curve of light splitting pupil differential confocal system, detection beam and focus vernier focusing exists
Sample surfaces are detected, focused spot size is minimum herein, and search coverage is minimum；The other positions of differential confocal response curve are corresponding
Detection light beam focuses at the position of departure surface, and the positive and negative of differential confocal response curve is able to reflect out object lens deviation or close
The direction of measured object；When measuring the fluorescence lifetime and relative intensity of fluorescence spectrum information at certain surface location under different wave length, root
Detection light beam is controlled in the surface spot size of sample to be tested according to differential confocal response curve, and then according to actual measurement demand control
The size of focal beam spot processed is realized to the controllable of sample search coverage size.
10. being divided the discrete fluorescence spectrum of pupil differential confocal and fluorescence lifetime detection method, it is characterised in that：
(a) continuous laser that the pulse laser and continuous laser source issued pulsed laser light source by the first spectroscope issues
It merges, forms synthesis light beam, the pulse laser is identical with continuous laser wavelength；The synthesis light beam expands by beam expanding lens
Shu Houjing over-illumination pupil, assembled by object lens formed detection light beam be radiated on sample to be tested；Definition is perpendicular to the object lens light
Two orthogonal directions of axis are respectivelyxWithyDirection, the direction along objective lens optical axis arezDirection；
(b) rear orientation light of light beam irradiating sample generation and the fluorescence ejected from sample to be tested are collected by object lens together
Afterwards, it by collecting pupil, is reflected by No.1 reflecting mirror；The light beam reflected by No.1 reflecting mirror is after No.1 dichroic beamsplitter
It is divided into two-way, is all the way wavelength intrinsic light beam identical with detection light beam wavelength, another way is that wavelength is different from detection light beam wave
Long fluorescent light beam；The intrinsic light beam enters light splitting pupil differential confocal detection system, and the fluorescent light beam enters discrete fluorescence
Spectrum and fluorescence lifetime detection system；
(c) continuous laser source, edge are openedxWithyThe mobile sample to be tested in direction to transversal scanning initial position (x 1, y 1), then
Edge in the positionzSample to be tested is scanned in direction, measures the difference changed with scan position using light splitting pupil differential confocal detection system
Confocal response curve is moved, and then accurately determines that detection light beam is focused on to test sample according to the dead-center position of differential confocal response curve
The surface location of product；
(d) according to the mobile sample to be tested of step (c) measurement result, so that detection light beam focuses on sample to be tested surface, by continuous
Laser inspires fluorescence on sample to be tested surface, by discrete fluorescence spectrum and fluorescence lifetime detection system, obtains different waves
Long corresponding fluorescent intensity information, and analyze it to obtain discrete fluorescence spectrum information；
(e) edgexWithySample to be tested is scanned in direction, repeats the above steps, each scanning element (x i, y i ) at position using confocal
Detection system determines the surface information of sample to be tested at this location, and utilizes discrete fluorescence spectrum and fluorescence lifetime detection system
Measure the discrete fluorescence spectrum information ejected from the position；
(f) by obtained sample to be tested each scanning element (x i , y i ) surface position information of position and corresponding discrete glimmering
Light spectral information is reconstructed, while obtaining the three-dimensional appearance profile of sample and its discrete fluorescence spectrum of surface each point.
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| CN204731160U (en) * | 2015-06-01 | 2015-10-28 | 复旦大学 | A kind of autofluorescence life-span imaging and fluorescence spectrum combine the device being used for early diagnosis of cancer |
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| CN113108697A (en) * | 2021-04-06 | 2021-07-13 | 合肥埃科光电科技有限公司 | Differential spectrum confocal sensor |
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