CN106442445B - One kind being based on single pass polychrome super-resolution microscopic system and method - Google Patents
One kind being based on single pass polychrome super-resolution microscopic system and method Download PDFInfo
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Abstract
The invention discloses one kind to be based on single pass polychrome super-resolution microscopic system and method, including loss light source, the first collimation unit, phase board, the first excitation light source, the first dichroic mirror, the second collimation unit, half-wave plate, polarizing beam splitter mirror, the second excitation light source, the first detector, the second dichroic mirror, lens-pin hole-lens group, third dichroic mirror, scanning mirror, the second detector, third collimation unit, quarter-wave plate, microcobjective and sample stage.The present invention is micro- using the super-resolution that dichromatism (or polychrome) can be realized in a loss light source, and system architecture is simple, and cost is relatively low.The present invention can overcome in previous single channel polychrome super-resolution system energy loss greatly and the stronger ambient noise of crosstalk bring, operating accuracy height, as a result accurately.Therefore, the present invention can be improved the working efficiency and experimental precision of polychrome super-resolution microscopic system compared with original technology, and effectively reduce cost.
Description
Technical field
The present invention relates to optical instrument fields, biomedical micro-imaging field, and in particular to one kind is based on single pass
Polychrome super-resolution microscopic system and method.
Background technique
Higher and higher in requirement of the research of biomedicine field to resolution ratio at present, researcher is it should be understood that various micro-
The three-dimensional structure of small form substance, however conventional white light wide-field microscope and the spot size of laser confocal microscope can not reach
To such resolution ratio, and the appearance of super-resolution microscopic system ideally solves this problem.In super-resolution microtechnic,
In terms of current research hotspot is mostly focused on all kinds of fluorescence microscopies, such as stimulated emission depletion (STED) fluorescence microscopy skill
Art and unimolecule positioning fluorescence microscope (SMS) etc..Wherein, there are exciting light and loss two class light of light in STED system, exciting
The region of the superposition of hot spot and hollow loss hot spot, the energy of transmitting fluorescent photon is lost by the fluorescent particles being lost, and is remained
Under the phosphor region that emits be limited in less than in diffraction limit region, be less than shining for diffraction limit in being that of obtaining one
Point.Based on the principle, the loss light in STED system must be positioned in the spectral region of fluorescence could be to the hot spot of stimulated radiation
It is wiped.
For such super-resolution microscopic system, different cell or tissues are seen if to realize simultaneously
It surveys, cell must just be demarcated respectively using different fluorescent dyes, observation is realized by the imaging of double-colored or polychrome.For
Realization is double-colored or the imaging observation of polychrome, can introduce multiple loss light, inhibits the stimulated emission of different fluorescent dyes respectively
Light, the article " Two- delivered on periodical Biophysical Journal such as Gerald Donnert et al. in 2007
Color Far-Field Fluorescence Nanoscopy " in refer to, this method can preferably realize it is double-colored at
Picture, however the situation of multiple loss light is introduced, it will definitely increase the complexity and cost of manufacture of system.In order to simplify polychrome at
As system, JanEt al. the article " Two- that was delivered on periodical Biophysical Journal in 2011
Color STED Microscopy of Living Synapses Using A Single Laser-Beam Pair".Article
Middle proposition two kinds of fluorescent dyes of a branch of excitation, and disappeared with a branch of loss light to the fluorescence of both fluorescent dyes
Light.Due to the impaired limitation for depleting wavelength, the type that can be used for the fluorescent dye of polychrome calibration is very limited, therefore fluorescence contaminates
The stimulated emission wavelength of material is relatively.This kind of structure utilizes band pass filter to different fluorescent dyes in actual use
Stimulated light emission be filtered so that the stimulated light emission of different wave length is collected by different image device.On the one hand, due to
The stimulated emission wavelength of the optional fluorescent dye of STED relatively, in order to preferably filter, it will usually select narrow band filter, so
And narrow band filter will cause the biggish loss of luminous energy；On the other hand, different if the bandwidth of the band pass filter of selection is larger
Crosstalk will occur for the stimulated emission wavelength of fluorescent dye, and introduce ambient noise, increase burden for later data processing.Cause
This, existing double-colored or polychrome super-resolution microscopic system is not well positioned to meet actual demand.
Summary of the invention
The present invention crosstalk or complex structure and other problems present in polychrome super-resolution microscopic system for before, propose one
Kind is novel based on single pass polychrome super-resolution microscopic system.The system can be realized using single loss light to polychrome sample into
Row observation, and capacity usage ratio is high, and ambient noise is small.
The present invention also provides a kind of novel based on single pass polychrome super-resolution microscopic method, party's Guttae Phacosylini is utilized
The observation to polychrome sample is realized with single loss light, and capacity usage ratio is high, practical.
One kind be based on single pass polychrome super-resolution microscopic system, including loss light source, the first collimation unit, phase board,
First excitation light source, the first dichroic mirror, the second collimation unit, half-wave plate, polarizing beam splitter mirror, the second excitation light source, the first detection
Device, the second dichroic mirror, lens-pin hole-lens group, third dichroic mirror, scanning mirror, the second detector, third collimation unit, four points
One of wave plate, microcobjective and sample stage；
The loss light source successively reaches an incidence of polarizing beam splitter mirror by first collimation unit, phase board
Face；The excitation beam that the excitation beam and the second excitation light source that first excitation light source issues issue closes at the first dichroic mirror
Beam after combined beam light passes sequentially through the second collimation unit, half-wave plate, reaches another plane of incidence of polarizing beam splitter mirror；The conjunction beam
Light and loss light beam, by third dichroic mirror surface, reach scanning mirror surface, then successively after polarizing beam splitter mirror closes beam
The sample surfaces on sample stage are converged to by third collimation unit, quarter-wave plate and microcobjective, the sample is by extremely
Few two kinds of fluorescent dyes are demarcated in advance；
The fluorescence generated by the sample surfaces passes sequentially through the microcobjective, the quarter-wave plate, described
After three collimation units and the scanning mirror, then third dichroic mirror is transmitted through then in turn through third dichroic mirror, lens-needle
Hole-lens group reaches the second dichroic mirror surface；It is incident to second dichroiscopic fluorescence a part and is transmitted through the second dichroic mirror,
It is received after being focused by the first detector, another part is connect after being focused by the fluorescence that the second dichroic mirror reflects by the second detector
It receives.
Preferably, first collimation unit, the second collimation unit, third collimation unit are lens group.That is, described
One collimation unit, the second collimation unit, third collimation unit are respectively the first lens group, the second lens group, the third lens group.Institute
It states the first lens group, the second lens group, the third lens group and plays beam-expanding collimation, expand rear lasing beam diameter and become larger, dissipate
Angle is smaller, and light distribution more evenly, closer to directional light, is conducive to light beam convergence in the cross section perpendicular to optical axis direction
Form smaller hot spot.
Preferably, further including the first reflecting mirror, the second reflecting mirror or third reflecting mirror；Polarization state is adjusted by phase board
Light beam afterwards is reflected into the plane of incidence of polarizing beam splitter mirror by the first reflecting mirror；Second excitation light source is reflected by the second reflecting mirror
To the first dichroic mirror；The fluorescence of described another part is first passed through and is received after third reflecting mirror is focused again by the second detector.
Preferably, two parts fluorescence is visited after passing through the first lens, the 4th lens focus respectively by the first detector, second
Device is surveyed to receive.
Preferably, the laser wavelength of the loss light source is 775nm.
Preferably, the laser wavelength of the first excitation light source is 650nm.
Preferably, the laser wavelength of the second excitation light source is 594nm.
In the present invention:
The half-wave plate is used to adjust the ratio of horizontal polarization light and orthogonal polarized light in received light beam；
The phase board is used to do phase coding to loss light beam, generates hollow focal beam spot.
The quarter-wave plate is used to linearly polarized light switching to circularly polarized light.
Lens-pin hole-the lens group is made of the second lens, pin hole and the third lens；The pin hole is miscellaneous for obstructing
Astigmatism reduces ambient noise.
Beam Propagation process of the present invention are as follows:
The excitation beam that second excitation light source issues is after the reflection of the second reflecting mirror, the deflection of the excitation beam direction of propagation
45 °, the excitation beam issued with the first excitation light source closes beam at the first dichroic mirror.Excitation beam after closing beam is saturating by second
It is collimated after microscope group, after adjusting polarization state by half-wave plate later, is incident on a plane of incidence of polarizing beam splitter mirror.
The loss light of light source sending is lost after the first lens group collimation, passes through phase board and adjusts polarization state and exciting light
After beam polarization state is vertical, loss light is incident on the first mirror surface, is reflected into the another of polarizing beam splitter mirror by the first reflecting mirror
A plane of incidence.On polarizing beam splitter mirror, exciting light and loss light are incident on the three or two after the cemented surface of polarizing beam splitter mirror closes beam
Look mirror surface is reflected into scanning mirror surface by third dichroic mirror.The light of scanning mirror surface outgoing, collimates by the third lens group
Afterwards, by quarter-wave plate, after the bit phase delay for generating π/4, sample surfaces are converged to via microcobjective.Sample is by two kinds
(or two or more) fluorescent dye is calibrated in advance, thus two kinds of fluorescent dyes by exciting light irradiate after, can generate two kinds it is glimmering
Light, i.e. fluorescence spectrum one and fluorescence spectrum two.Both fluorescence spectrum opposite directions are by microcobjective, again by a quarter
Wave plate generates the bit phase delay of π/4, the i.e. bit phase delay of pi/2 in total again, so that polarization state is hung down with polarization state before
Directly, third dichroic mirror can be transmitted through after the third lens group, scanning mirror.It is transmitted through the dichroiscopic light beam warp of third
Cross the third lens post-concentration, convergent beam passes through after pin hole the beam-expanding collimation after by the second lens, to the second dichroic mirror surface.Two
Kind fluorescence spectrum a part is transmitted through the second dichroic mirror, after the first lens focus, is received by the first detector.Another part
Fluorescence third mirror surface is reflected by the second dichroic mirror after, then reflected, into the 4th lens, visited after focusing by second
Device is surveyed to receive.
It include two excited respectively kind fluorescence spectrum of two kinds of dyestuffs excitation, respectively one He of fluorescence spectrum in the fluorescence
Fluorescence spectrum two；Two filter plate of one filter plate of installation passage and channel, channel are distinguished on the first detector and the second detector
One filter plate filters out other spectrum and only retains the part not being superimposed with fluorescence spectrum two in fluorescence spectrum one, and channel two is filtered
Wave plate filters out the part that fluorescence spectrum medium wavelength is greater than exciting light, only retains the portion that fluorescence spectrum one is superimposed with fluorescence spectrum two
Point.
It adds up to the number of photons in channel one, obtains the number of photons in channel one.Due to the curve of fluorescence spectrum one
It is determined by its corresponding fluorescent dye, so known to the integrated curved of the spectrum.According to the number of photons in channel one, channel one
The curve of spectrum and fluorescence spectrum one integrated curved, can calculate in proportion, obtain channel two in by one tribute of fluorescence spectrum
The number of photons offered.The number of photons contributed in channel two by fluorescence spectrum one is subtracted with the number of photons in channel two, channel can be obtained
The number of photons contributed in two by fluorescence spectrum two.One He of fluorescence spectrum will be come from above-mentioned calculated channel one and channel two
The number of photons of fluorescence spectrum two is sent into subsequent calculation processing system, that is, realizes that single pass polychrome super-resolution is micro-.
The present invention also provides one kind to be based on single pass polychrome super-resolution microscopic method, using any of the above-described technical solution
It is described based on single pass polychrome super-resolution microscopic system；It wherein include two kinds of spectrum, respectively fluorescence light in the fluorescence
Spectrum one and fluorescence spectrum two, one filter plate of channel filter out other spectrum and only retain no and fluorescence spectrum in fluorescence spectrum one
The part of two superpositions, two filter plate of channel filter out the part that fluorescence spectrum medium wavelength is greater than exciting light, only retain fluorescence spectrum
One part being superimposed with fluorescence spectrum two；
Further include the steps that calculating the number of photons from fluorescence spectrum one and fluorescence spectrum two in channel one and channel two:
(1) it adds up to the number of photons in channel one, obtains the number of photons in channel one；According to the photon in channel one
Number, the curve of spectrum integral area in channel one and the integrated curved integral area of fluorescence spectrum one obtain in channel two by glimmering
The number of photons that light spectrum one is contributed；
(2) number of photons contributed in channel two by fluorescence spectrum one is subtracted with the number of photons in channel two, channel can be obtained
The number of photons contributed in two by fluorescence spectrum two；
(3) number of photons from fluorescence spectrum one and fluorescence spectrum two in above-mentioned calculated channel one and channel two is sent
Enter subsequent calculation processing system, that is, realizes that single pass polychrome super-resolution is micro-.
It should be noted that in case of no particular description, in the present invention, " first ", " second ", " third " " the
Four " etc. only for distinguishing each element, to the unlimited effect such as the installation order or structure of element, function.
Therefore, existing double-colored or polychrome super-resolution microscopic system is not well positioned to meet actual demand.With it is existing
Technology is compared, the beneficial effects of the present invention are embodied in:
1, the super-resolution that dichromatism (or polychrome) can be realized using a loss light source in the present invention is micro-, system architecture letter
Single, cost is relatively low.
2, the present invention can overcome in previous single channel polychrome super-resolution system that energy loss is big and crosstalk bring is stronger
Ambient noise, operating accuracy is high, as a result accurately.
Therefore, the technical solution is compared with original technology, can be improved polychrome super-resolution microscopic system working efficiency and
Experimental precision, and effectively reduce cost.
Detailed description of the invention
Fig. 1 is the index path of one embodiment of the present of invention；
Wherein: 1, light source is lost；2, the first lens group；3, phase board；4, the first reflecting mirror；5, the first excitation light source；6,
First dichroic mirror；7, the second lens group；8, half-wave plate；9, polarizing beam splitter mirror；10, the second excitation light source；11, the second reflecting mirror；
12, the first detector；13, the first lens；14, the second dichroic mirror；15, the second lens；16, pin hole；17, the third lens；18,
Three dichroic mirrors；19, scanning mirror；20, the second detector；21, the 4th lens；22, third reflecting mirror；23, the third lens group；24,
Quarter-wave plate；25, microcobjective；26, sample；
Fig. 2 is a stimulated emission spectrogram of the invention, including two kinds of dyestuffs of fluorescent dye A and fluorescent dye B by
The spectrum of stimulated emission light, in which: 27 be the spectrum in channel one；28 be the spectrum in channel two；
Fig. 3 is the stimulated emission spectrogram of the embodiment of the present invention, including the glimmering of fluorescent dye ATTO594 and ATTO647N
Light spectrum, in which: 29 be the spectrum of 545~590nm of channel；30 be the spectrum in channel 591~778.
Specific embodiment
Illustrate the present invention with reference to the accompanying drawing, but the present invention is not limited thereto.
It is the single channel polychrome super-resolution microscopic system embodiment of one embodiment of the invention as shown in Figure 1.The embodiment
Dual-beam optical optical tweezers system include:
Light source 1 is lost；First lens group 2；Phase board 3；First reflecting mirror 4；First excitation light source 5；First dichroic mirror 6；
Second lens group 7；Half-wave plate 8；Polarizing beam splitter mirror 9；Second excitation light source 10；Second reflecting mirror 11；First detector 12；First
Lens 13；Second dichroic mirror 14；Second lens 15；Pin hole 16；The third lens 17；Third dichroic mirror 18；Scanning mirror 19；Second visits
Survey device 20；4th lens 21；Third reflecting mirror 22；The third lens group 23；Quarter-wave plate 24；Microcobjective 25；Sample stage
With sample 26；
Wherein, loss light source 1 is preferably the Katana -08HP of OneFive company.
First excitation light source 5 is preferably the LDH-TA-595 of PicoQuant company.
Second excitation light source 10 is preferably the LDH-P-C-650B of PicoQuant company.
First detector 12 is preferably the SPCM-AQRH-13FC of Excelitas company.
Second detector 20 is preferably the SPCM-AQRH-13FC of Excelitas company
For the excitation beam that second excitation light source 10 issues after the reflection of the second reflecting mirror 11, the excitation beam direction of propagation is inclined
Turn 90 °, the excitation beam issued with the first excitation light source 5 closes beam at the first dichroic mirror 6.Excitation beam after closing beam passes through the
It is collimated after two lens groups 7, after adjusting polarization state by half-wave plate 8 later, is incident on a plane of incidence of polarizing beam splitter mirror 9.
The loss light of the sending of light source 1 is lost after the first lens group 2 collimation, polarization state is adjusted by phase board 3 and is swashed
After hair light polarization is vertical, loss light is incident on 4 surface of the first reflecting mirror, is reflected into polarizing beam splitter mirror 9 by the first reflecting mirror 4
Another plane of incidence.
On polarizing beam splitter mirror 9, exciting light and loss light are incident on third after the cemented surface of polarizing beam splitter mirror 9 closes beam
18 surface of dichroic mirror is reflected into 19 surface of scanning mirror by third dichroic mirror 18.The light of 19 surface of scanning mirror outgoing, it is saturating by third
After microscope group 23 collimates, by quarter-wave plate 24, after the bit phase delay for generating π/4, sample is converged to via microcobjective 25
26 surfaces.Sample 26 is calibrated in advance by two kinds of (or two or more) fluorescent dyes, i.e. fluorescent dye A and fluorescent dye B, therefore
After two kinds of fluorescent dyes are irradiated by exciting light, two kinds of fluorescence spectrums can be generated, respectively fluorescence spectrum A (is swashed by fluorescent dye A
Hair generates) and fluorescence spectrum B (excited and generated by fluorescent dye B), as shown in Figure 2.Both fluorescence spectrum opposite directions pass through aobvious
Speck mirror 25 generates the bit phase delay of π/4 again by quarter-wave plate 24 again, i.e. the bit phase delay of pi/2 in total,
So that polarization state is vertical with polarization state before, the three or two can be transmitted through after the third lens group 23, scanning mirror 19
Look mirror 18.The light beam of third dichroic mirror 18 is transmitted through by 17 post-concentration of the third lens, convergent beam passes through after passing through pin hole 16
By beam-expanding collimation after the second lens 15,14 surface of the second dichroic mirror is reached.The fluorescence spectrum A and fluorescence spectrum B mono- that sample generates
Part is by the second dichroic mirror 14, after being focused by the first lens 13, receives 12 by the first detector.The fluorescence spectrum that sample generates
It after another part of A and fluorescence spectrum B are reflected into 22 surface of third reflecting mirror by the second dichroic mirror 14, then is reflected, into
Four lens 21 are received after focusing by the second detector 20.
The filter plate of installation passage one and channel two is distinguished on the first detector 12 and the second detector 20, channel one is filtered
Wave plate filters out other spectrum and only retains the part not being superimposed with fluorescence spectrum B in fluorescence spectrum A, i.e., logical as shown in Figure 2
The spectrum 27 in road one；Two filter plate of channel filters out the part for being greater than exciting light in spectrum and only retains fluorescence spectrum A and fluorescence
The part of spectrum B superposition, i.e., the spectrum 28 in channel two as shown in Figure 2.
It adds up to number of photons all in the spectrum 27 in channel one, obtains the number of photons N in channel one.Due to fluorescence
The curve of spectrum of spectrum A is determined by fluorescent dye, so known to the integrated curved of the spectrum.According to the number of photons in channel one
N, the integral area b of the integrated curved of the integral area a and fluorescence spectrum A of the curve of spectrum in channel one, can calculate in proportion,
Obtain the number of photons M, i.e. M=N/a (b-a) contributed in channel two by fluorescence spectrum A.
It adds up to number of photons all in the spectrum 28 in channel two, obtains the number of photons U in channel two.With channel two
Number of photons subtract the number of photons contributed in channel two by fluorescence spectrum A, can obtain in channel two and be contributed by fluorescence spectrum B
Number of photons Z=U-M.It will be above-mentioned calculated from the subsequent calculation processing of the number of photons of fluorescence spectrum A and fluorescence spectrum B feeding
System realizes that single pass polychrome super-resolution is micro-.
In the present embodiment, the UPLSAPO 100XS of Olympus company is can be selected in microcobjective 25.
Embodiment
Single pass polychrome super-resolution microscopic system proposed by the invention is carried out furtherly below with reference to embodiment
It is bright, but the present invention is not limited thereto.
The excitation beam that second excitation light source 10 issues, wavelength 594nm.After the reflection of the second reflecting mirror 11, excitation
Direction of beam propagation deflects 45 °, the excitation beam issued with the first excitation light source 5, wavelength 650nm.At the first dichroic mirror 6
Close beam.Excitation beam after closing beam is incident after adjusting polarization state by half-wave plate 8 later by being collimated after the second lens group 7
To a plane of incidence of polarizing beam splitter mirror 9.
The loss light that light source 1 issues is lost, wavelength 775nm is adjusted after the first lens group 2 collimation by phase board 3
After section polarization state is vertical with excitation beam polarization state, loss light is incident on 4 surface of the first reflecting mirror, is reflected by the first reflecting mirror 4
To another plane of incidence of polarizing beam splitter mirror 9.On polarizing beam splitter mirror 9, exciting light and loss light polarizing beam splitter mirror 9 gluing
After beam is closed in face, it is incident on 18 surface of third dichroic mirror, 19 surface of scanning mirror is reflected by third dichroic mirror 18.19 surface of scanning mirror
The light of outgoing, after the collimation of the third lens group 23, by quarter-wave plate 24, after the bit phase delay for generating π/4, via aobvious
Speck mirror 25 converges to sample surfaces.Sample 26 is calibrated in advance by ATTO594 and two kinds of fluorescent dyes of ATTO647N, therefore two
After kind fluorescent dye is by exciting light irradiation, corresponding fluorescence spectrum can be generated, as shown in Figure 3.Both fluorescence spectrums negative side
To by microcobjective 25, again by quarter-wave plate 24, the bit phase delay of π/4 is generated again, i.e. the position of pi/2 in total
Phase retardation can be transmitted through so that polarization state is vertical with polarization state before after the third lens group 23, scanning mirror 19
Third dichroic mirror 18.The light beam of third dichroic mirror 18 is transmitted through by 17 post-concentration of the third lens, convergent beam passes through pin hole
After 16 after by the second lens 15 beam-expanding collimation, to 14 surface of the second dichroic mirror.The 50% of fluorescent spectrum is transmitted through
Two dichroic mirrors 14 after being focused by the first lens 13, receive 12 by the first detector.Another the 50% of fluorescent spectrum is by the two or two
It after Look mirror 14 is reflected into 22 surface of third reflecting mirror, then is reflected, into the 4th lens 21, is connect after focusing by the second detector 20
It receives.
The bandpass filter that 545~590nm is had on the first detector 12, on the second detector 20 with 591~
The bandpass filter of 778nm, the bandpass filter of 545~590nm only retain in the spectrum of ATTO594 fluorescence not with
The part of the spectrum superposition of ATTO647N fluorescence, i.e., the spectrum 29 of 545~590nm of channel as shown in Figure 3；The band of 591~778nm
Pass filter filters out the part for being greater than 778nm in spectrum, and the part that the spectrum one for only retaining fluorescence is superimposed with spectrum two, i.e.,
The spectrum 30 of 591~778nm of channel as shown in Figure 2.
It adds up to number of photons all in the spectrum 29 of 545~590nm of channel, obtains the light of 545~590nm of channel
Number of photons N in spectrum 29.Since the curve of spectrum of the fluorescence of ATTO594 is determined by fluorescent dye, so the entirety of the spectrum
Known to curve.According to the number of photons N in channel, the integral area a of the curve of spectrum of the spectrum 29 of 545~590nm of channel and
The integral area b of the fluorescence integrated curved of ATTO594, can calculate in proportion, obtain 591~778nm of channel spectrum 30 in by
The number of photons M that the fluorescence of ATTO594 is contributed, i.e. M=N/a (b-a).
It adds up to number of photons all in the spectrum 30 of 591~778nm of channel, obtains the light of 591~778nm of channel
Number of photons U in spectrum 30.It is subtracted in the spectrum 30 of 591~778nm of channel with the number of photons of the spectrum 30 of 591~778nm of channel
The number of photons contributed by ATTO594 fluorescence can obtain the fluorescence institute in the spectrum 30 of 591~778nm of channel by ATTO647N
The number of photons Z=U-M of contribution.It will be above-mentioned calculated from the subsequent calculating of the number of photons of ATTO594 fluorescence and ATTO647 feeding
Processing system realizes that single pass polychrome super-resolution is micro-.
Finally, it should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting.Such as, except real
It applies outside two kinds of fluorescent dyes of ATTO594 described in example and ATTO647N, matches with exciting light described in embodiment and loss light
Fluorescent dye further include: ATTO590, SiR, Alexa635P, KK140 etc..But according to the difference of selected fluorescent dye, accordingly
The selection and parameter of optical device, such as the passband of optical filter, still should rationally be finely tuned.
It in addition, using system and method for the invention, can also be achieved micro- greater than the super-resolution of the polychrome of two colors, need at this time
To increase corresponding probe and filter plate as needed, while configure dichroic mirror appropriate, according to above-mentioned similar method, one by one
It is calculated.It is micro- for polychrome, in filter plate design, for example, sample will issue S for S (S > 2) color system
A fluorescence spectrum, needs to configure S probe, and each probe needs to configure a filter plate；For i-th of filter plate: when i=1,
Filtering condition is same as above, i.e., only retains the 1st fluorescence spectrum not part with the 2nd Overlapping of fluorescence spectra；For the i-th of i ≠ 1
A filter plate only retains the part of (i-1)-th and the superposition of i-th of fluorescence spectrum, successively calculates when calculating.
Although the invention is described in detail with reference to an embodiment, those skilled in the art should understand that, it is right
Technical solution of the present invention is modified or replaced equivalently, without departure from the spirit and scope of technical solution of the present invention,
It should cover in the spirit and scope of the present invention, be intended to be within the scope of the claims of the invention.
Claims (9)
1. one kind is based on single pass polychrome super-resolution microscopic system, which is characterized in that single including loss light source, the first collimation
Member, phase board, the first excitation light source, the first dichroic mirror, the second collimation unit, half-wave plate, polarizing beam splitter mirror, the second exciting light
Source, the first detector, the second dichroic mirror, lens-pin hole-lens group, third dichroic mirror, scanning mirror, the second detector, third are quasi-
Straight unit, quarter-wave plate, microcobjective and sample stage；
The loss light source successively reaches a plane of incidence of polarizing beam splitter mirror by first collimation unit, phase board；Institute
The excitation beam that the excitation beam and the second excitation light source for stating the sending of the first excitation light source issue closes beam at the first dichroic mirror, closes
After Shu Guang passes sequentially through the second collimation unit, half-wave plate, another plane of incidence of polarizing beam splitter mirror is reached；The combined beam light and damage
Beam is depleted after polarizing beam splitter mirror closes beam, by third dichroic mirror surface, reaches scanning mirror surface, then in turn through the
Three collimation units, quarter-wave plate and microcobjective converge to the sample surfaces on sample stage, and the sample is by two kinds of fluorescence
Dyestuff is demarcated in advance；
It is quasi- to pass sequentially through the microcobjective, the quarter-wave plate, the third for the fluorescence generated by the sample surfaces
After straight unit and the scanning mirror, then third dichroic mirror is transmitted through then in turn through lens-pin hole-lens group, reach the
Two dichroic mirror surfaces；It is incident to second dichroiscopic fluorescence a part and is transmitted through the second dichroic mirror, visited after being focused by first
It surveys device to receive, another part is received after the fluorescence that the second dichroic mirror reflects is focused by the second detector；
Two filter plate of one filter plate of channel and channel is separately installed on first detector and the second detector；
It include two kinds of spectrum, respectively fluorescence spectrum one and fluorescence spectrum two in the fluorescence；One filter plate of the channel filtering
Fall other spectrum and only retains the part not being superimposed with fluorescence spectrum two in fluorescence spectrum one, two filter plate of the channel filtering
Fall the part that fluorescence spectrum medium wavelength is greater than exciting light, only retains the part that fluorescence spectrum one is superimposed with fluorescence spectrum two；
Two kinds in ATTO594, ATTO647N, ATTO590, SiR, Alexa635P, KK140 of described two fluorescent dyes.
2. according to claim 1 be based on single pass polychrome super-resolution microscopic system, which is characterized in that described first is quasi-
Straight unit, the second collimation unit, third collimation unit are lens group.
3. according to claim 1 be based on single pass polychrome super-resolution microscopic system, which is characterized in that further include first
Reflecting mirror, the second reflecting mirror or third reflecting mirror；Light beam after phase board adjusts polarization state is reflected by the first reflecting mirror
To the plane of incidence of polarizing beam splitter mirror；Second excitation light source reflexes to the first dichroic mirror by the second reflecting mirror；Described another part
Fluorescence first pass through after third reflecting mirror is focused again by the second detector receive.
4. according to claim 1 be based on single pass polychrome super-resolution microscopic system, which is characterized in that the loss light
The laser wavelength in source is 775nm.
5. according to claim 1 be based on single pass polychrome super-resolution microscopic system, which is characterized in that the first exciting light
The laser wavelength in source is 650nm.
6. according to claim 1 be based on single pass polychrome super-resolution microscopic system, which is characterized in that the second exciting light
The laser wavelength in source is 594nm.
7. according to claim 1 be based on single pass polychrome super-resolution microscopic system, which is characterized in that two parts fluorescence
Respectively by being received after the first lens, the 4th lens focus by the first detector, the second detector.
8. one kind is based on single pass polychrome super-resolution microscopic method, it is characterised in that: using described in claim 1 based on single
The polychrome super-resolution microscopic system in channel；It wherein include two kinds of spectrum, respectively fluorescence spectrum one and fluorescence light in the fluorescence
Spectrum two, one filter plate of channel filters out other spectrum and only retains the portion not being superimposed with fluorescence spectrum two in fluorescence spectrum one
Point, two filter plate of channel filters out the part that fluorescence spectrum medium wavelength is greater than exciting light, only retains fluorescence spectrum one and fluorescence light
The part of two superposition of spectrum；
Further include the steps that calculating the number of photons from fluorescence spectrum one and fluorescence spectrum two in channel one and channel two:
(1) it adds up to the number of photons in channel one, obtains the number of photons in channel one；According to the number of photons in channel one,
The curve of spectrum integral area in channel one and the integrated curved integral area of fluorescence spectrum one obtain in channel two by fluorescence light
The number of photons that spectrum one is contributed；
(2) number of photons contributed in channel two by fluorescence spectrum one is subtracted with the number of photons in channel two, can obtained in channel two
The number of photons contributed by fluorescence spectrum two；
(3) by above-mentioned calculated channel one and channel two from the number of photons of fluorescence spectrum one and fluorescence spectrum two feeding after
Continuous calculation processing system, that is, realize that single pass polychrome super-resolution is micro-.
9. according to claim 8 be based on single pass polychrome super-resolution microscopic method, which is characterized in that the loss light
The laser wavelength in source is 775nm；The laser wavelength of first excitation light source is 650nm；The laser wave of second excitation light source
A length of 594nm.
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| CN108107034B (en) * | 2017-12-27 | 2020-11-13 | 中国科学院长春应用化学研究所 | Raman super-resolution microscopic imaging system and method based on structured light illumination |
| CN108414445A (en) * | 2018-03-12 | 2018-08-17 | 清华大学深圳研究生院 | A kind of liquid phase biomolecule multi detection method and device |
| CN111912796B (en) * | 2019-12-12 | 2023-03-10 | 南开大学 | Super-resolution imaging system and method based on visible absorption spectrum |
| CN113063766A (en) * | 2021-03-29 | 2021-07-02 | 新羿制造科技（北京）有限公司 | Micro-droplet fluorescence signal detection device containing biconvex lens |
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