CN103940796A - Novel multi-angle and multi-mode quick switching circular optical illumination microscopic imaging system - Google Patents

Abstract

The invention discloses a novel multi-angle and multi-mode quick switching circular optical illumination microscopic imaging system which comprises a laser, a single mode fiber, a collimating lens, a scanning galvanometer, a scanning lens, a first field lens, a focus lens, a dichroic mirror, a total internal reflection fluorescence (TIRF) microscope objective, a sample platform, a filter, a second field lens and a detector, wherein the single mode fiber, the collimating lens and the scanning galvanometer are sequentially located on an optical axis of an outgoing beam of the laser; the scanning lens, the first field lens, the focus lens and the dichroic mirror are sequentially located on an optical axis of an outgoing beam of the scanning galvanometer; the TIRF microscope objective and the sample platform are sequentially located on an optical axis of a reflected beam of the dichroic mirror; the filter, the second field lens and the detector are sequentially arranged on an optical axis of a transmission beam of the dichroic mirror; the sample platform is arranged on a focus plane of the TIRF microscope objective; a collecting hole of the detector is formed in a focus plane of the second field lens. On the basis of TIRF, the microscopic imaging system disclosed by the invention realizes microscopic experimental observation which is convenient and rapid, high in resolution and low in noise.

Description

Novel multiple angle multi-mode is switched ring-type illumination optical micro imaging system fast

Technical field

The invention belongs to novel micro-mode field, relate in particular to a kind of Novel multiple angle multi-mode and switch fast ring-type illumination optical micro imaging system.

Background technology

Utilizing total internal reflection fluorescence microscope (total internal reflection fluorescence microscope, TIRFM), utilize the characteristic that produces evanescent wave after light total reflection at medium another side, fluorescence excitation molecule is to observe the region as thin as a wafer of fluorescence calibration sample, and the dynamic range of observation is conventionally below 200nm.Because exciting light is the characteristic of exponential damping, only have the sample areas of extremely close fully reflecting surface can produce fluorescent reflection, greatly reduce background noise disturbance-observer sample, can help researcher to obtain high-quality image quality and reliable observation data.Therefore technique is widely used in the dynamic observation of cell surface material.Incident light in biologic applications is the glass cover slide of laser and interface normally, the aqueous solution between cover glass and the film of attached cell.

Ring-type TIRF utilizes a ring-shaped light circle to form TIRF illumination imaging, and its advantage is to have reduced interference fringe, and multi-angle imaging has fast reduced the aberration of 3D imaging and single angle imaging generation, and the possibility of carrying out image 3D cross sectional reconstruction is provided.

EPI is rectilinear fluorescent microscopic imaging mode.

HILO (Highly inclined laminated optical) will produce when incident angle will enter TIRF, during imaging, noise proportional is very little, signal to noise ratio (S/N ratio) can reach the several times of EPI, very be applicable to observing the activity of living cells, but compare the degree of depth that is difficult to measure imaging with TIRF, cannot carry out quantitative test.

Business utilizing total internal reflection fluorescence microscope does not in the market have the free handoff angle of energy and TIRF encircles big or small function, cannot realize fluorescence imaging pattern conversion easily yet.

Summary of the invention

The object that the present invention carries is not enough for prior art, provides a kind of Novel multiple angle multi-mode to switch fast ring-type illumination optical micro imaging system.

The technical solution adopted for the present invention to solve the technical problems is as follows:

The present invention includes laser instrument, single-mode fiber, collimation lens, scanning galvanometer, scanning lens, the first field lens, focus lamp, dichroscope, TIRF microcobjective, sample stage, filter plate, the second field lens, detector;

Single-mode fiber, collimation lens, scanning galvanometer are positioned on the optical axis of laser emitting light beam successively, and scanning lens, the first field lens, focus lamp, dichroscope are positioned at successively on the optical axis of scanning galvanometer outgoing beam; TIRF microcobjective, sample stage are positioned on dichroscope folded light beam optical axis successively, and filter plate, the second field lens, detector are positioned on dichroscope transmitted light beam optical axis successively; Described sample stage is positioned at the place, focal plane of TIRF microcobjective, and the collection hole of described detector is positioned at the place, focal plane of the second field lens;

The collimated laser beam of laser instrument transmitting, is first imported into single-mode fiber, and the laser beam penetrating from single-mode fiber completes collimation through collimation lens; Light beam after collimation incides in surface sweeping galvanometer and carries out light path deviation, incides afterwards scanning lens and focuses on; After the light beam being penetrated by scanning lens expands through the first field lens, after parallel ejaculation difference line focus mirror and dichroiscopic focusing and reflection, light beam focuses on TIRF microcobjective and is projeced on the testing sample on sample stage; The flashlight that testing sample is launched is collected by TIRF microcobjective, first, by wave plate elimination parasitic light after filtration successively again after dichroscope transmission, secondly through the second field lens, focuses on, and is finally detected device collection, the detector record light intensity signal detecting now.

When described light beam focuses on TIRF microcobjective and is projeced on the testing sample of sample stage 10, if the incidence angle θ of incident beam is greater than critical angle θ _c, incident laser will be on sample surface of contact experiences total internal reflection, critical angle θ _ccomputing formula is as follows:

θ _c＝sin ^-1(n ₁/n ₃)

Wherein, n ₁and n ₃it is respectively the refraction coefficient that light is dredged material and the close material of light;

Now, at the opposite side of testing sample surface in contact, can produce evanescent wave, and along with being exponential damping from contact measured sample surfaces apart from Z; This field energy that dies of declining enough excites at the fluorophor of near surface and also can avoid the more fluorophor of depths to be excited simultaneously; The die measure equation of field strength of the declining of this unlimited angle pencil of ray is as follows:

I(Z)＝I(O)e ^-z/d (2)

Wherein,

$d=\frac{{\mathrm{\λ}}_0}{4\mathrm{\π}}{(n_3^2{\sin }^2\mathrm{\θ}-n_1^2)}^{-1/2}---\left(3\right)$

λ ₀it is incident beam wavelength in a vacuum; Depth d does not rely on incident polarisation of light and along with incidence angle θ increases and reduces; The fluorescence that fluorophor sent of the field excitation that died by declining detects through being just detected device after dichroscope, filter and lens.

Described microcobjective is TIRF type microcobjective, its numerical aperture NA=1.45, and detector is CCD camera, can carry out signals collecting.

Described can regulate and control institute's on-load voltage on it by controller for changing the scanning galvanometer of light beam deviation direction, changes the size of sample surfaces illuminating bundle incident angle, thereby realizes multi-angle TIRF; Also can realize by the regulation and control of controller ring-type TIRF throws light on; In ring-type TIRF situation, controller is controlled and is made incident light around lap time, just equal the time shutter of detector scanning galvanometer, thereby reach to survey with on the time of scanning, synchronizes.

Beneficial effect of the present invention is as follows:

(1) the present invention can be switched micro-imaging pattern between EPI, HILO and TIRF;

(2) realization of ring-type TIRF has improved image quality, and light source homogenization is beneficial to the micro-structure of observation of cell inside;

(3) image taking speed is fast, and ring-type TIRF formation time, in 10ms left and right, does not affect cell observation;

(4) device is simple, easy to operate.

To sum up, that the present invention realizes on the basis of TIRF is convenient and swift, high resolving power and low noise microscope experiment are observed, and the present invention has that image taking speed is fast, device is simple, resolution is high can well be for actual experiment.

Accompanying drawing explanation

Fig. 1 is schematic diagram of the present invention.

Fig. 2 is the switching schematic diagram between the different imaging patterns of EPI of the present invention, TIRF, HILO and ring-type TIRF;

Fig. 3 is the resolution schematic diagram under TIRF of the present invention and ring-type TIRF illumination;

In figure, laser instrument 1, single-mode fiber 2, collimation lens 3, scanning galvanometer 4, scanning lens 5, the first field lens 6, focus lamp 7, dichroscope 8, TIRF microcobjective 9, sample stage 10, filter plate 11, the second field lens 12, detector 13.

Embodiment

Below in conjunction with embodiment and accompanying drawing, describe the present invention in detail, but the present invention is not limited to this.

As shown in Figure 1, Novel multiple angle multi-mode is switched ring-type illumination optical micro imaging system fast, comprises laser instrument 1, single-mode fiber 2, collimation lens 3, scanning galvanometer 4, scanning lens 5, the first field lens 6, focus lamp 7, dichroscope 8, TIRF microcobjective 9, sample stage 10, filter plate 11, the second field lens 12, detector 13.

Single-mode fiber 2, collimation lens 3, scanning galvanometer 4 are positioned on the optical axis of laser instrument 1 outgoing beam successively, and scanning lens 5, the first field lens 6, focus lamp 7, dichroscope 8 are positioned at successively on the optical axis of scanning galvanometer 4 outgoing beams.TIRF microcobjective 9, sample stage 10 are positioned on dichroscope 8 folded light beam optical axises successively, and filter plate 11, the second field lens 12, detector 13 are positioned on dichroscope 8 transmitted light beam optical axises successively; Described sample stage 10 is positioned at the place, focal plane of TIRF microcobjective 9, and the collection hole of described detector 13 is positioned at the place, focal plane of the second field lens 12.

The collimated laser beam of laser instrument 1 transmitting, is first imported into single-mode fiber 2, and the laser beam penetrating from single-mode fiber 2, completes collimation through collimation lens 3; Light beam after collimation incides in surface sweeping galvanometer 4 and carries out light path deviation, incides afterwards scanning lens 5 and focuses on.After the light beam being penetrated by scanning lens 5 expands through the first field lens 6, after parallel ejaculation line focus mirror 7 and dichroscope 8 focusing and reflection, light beam focuses on TIRF microcobjective 9 and is projeced on the testing sample on sample stage 10.The flashlight that testing sample is launched is collected by TIRF microcobjective 9, first by wave plate 11 elimination parasitic lights after filtration successively again after dichroscope 8 transmissions, secondly through the second field lens 12, focus on, be finally detected device 13 and gather, the detector 13 records light intensity signal detecting now.

When described light beam focuses on TIRF microcobjective 9 and is projeced on the testing sample on sample stage 10, the incidence angle θ of incident laser is greater than critical angle θ _c, incident laser will be on sample surface of contact experiences total internal reflection (TIR), θ _ccomputing formula is as follows:

θ _c＝sin ^-1(n ₁/n ₃)

(the n here ₁and n ₃respectively the refraction coefficient that light is dredged material and the close material of light)

At this moment the surface in contact opposite side at medium can produce evanescent wave, and along with being exponential damping from contact medium surface distance Z.This region can excite at the raw fluorophor of near surface also can avoid the more fluorophor of depths to be excited simultaneously.For declining of this unlimited angle pencil of ray die field strength measurement (unit is used: energy/region/s) formula is:

I(Z)＝I(O)e ^-z/d (2)

Wherein,

$d=\frac{{\mathrm{\λ}}_0}{4\mathrm{\π}}{(n_3^2{\sin }^2\mathrm{\θ}-n_1^2)}^{-1/2}---\left(3\right)$

λ ₀it is incident ray wavelength in a vacuum.Depth d does not rely on incident polarisation of light and along with incidence angle θ increases and reduces.The fluorescence that fluorophor sent of the field excitation that at this moment died by declining detects through being just detected device after dichroscope, filter and lens.

Microcobjective 9 is TIRF type microcobjective, its numerical aperture NA=1.45, and detector 13 is CCD camera, can carry out signals collecting.

As shown in Figure 2, described can regulate and control institute's on-load voltage on it by controller for changing the scanning galvanometer 4 of light beam deviation direction, change the size of sample surfaces illuminating bundle incident angle, thereby realize multi-angle TIRF, thereby also can realize by the regulation and control of controller ring-type TIRF illumination and reach elimination interference fringe, improve the object of observed image resolution, referring specifically to Fig. 3.Wherein, in ring-type TIRF situation, controller is controlled and is made incident light around lap time, just equal the time shutter of detector scanning galvanometer, can reach like this to survey with on the time of scanning to synchronize.

Embodiment:

First laser sends from Melles Griot company production laser instrument, and the collimation lens that imports Thornlabs company through single-mode fiber completes collimation; Light beam after collimation incides in the scanning galvanometer system of Thornlabs company and carries out light path deviation, incides afterwards in the scanning lens of Thornlabs company and focuses on.The light beam being penetrated by scanning lens through parallel ejaculation after the first field lens expands through the focus lamp of Thornlabs company and dichroscope focuses on and reflection after, light beam focuses on the microscopical object lens of TIRF that OLYMPUS company produces and is projeced on its sample stage.The flashlight that testing sample is launched is collected by TIRF microcobjective, first by dichroscope wave plate elimination parasitic light after filtration successively again, through the second field lens, focus on, finally by the EM-CCD detector of Andor company, gathered, the light intensity signal that EM-CCD detector record now detects.

When light beam enters optically thinner medium from optically denser medium, according to snell law part light, can reflect, and a part of light can reflect, when incident angle constantly increases, refraction angle also can constantly increase, and when refraction angle just reaches 90 while spending, total reflection phenomenon has just occurred.After total reflection occurs, refract light has a critical conditions to propagate along plane of refraction, and we are referred to as evanescent wave this part light, and its energy presents exponential damping on Z axis.

TIRF has the super-resolution (between 30nm～100nm) in Z-direction, when experiences total internal reflection (TIR), surface in contact opposite side at medium can produce evanescent wave, for example, between the surface of contact of glass and water, the incident angle angle (this angle θ is called incident angle) of measuring from normal to both sides must enough allow greatly light beam that TIR can occur rather than at interface, occur refraction situation, will surpass alinternal reflection angle critical angle in this case.TIR can produce an evanscent field with the same frequency of incident ray in fluid, and along with being exponential damping from contact medium surface distance Z.This region can excite at the raw fluorophor of near surface also can avoid the more fluorophor of depths to be excited simultaneously.

The simplest a kind of TIR situation is exactly that a branch of straight incident light has incided on single surface.For example, when a transparent optical for example, enters into an optically thinner medium 1 (water) from optically denser medium 3 (glass), when being greater than the cirtical angle of total reflection, the incident angle from normal to interface orientation measurement will there is total reflection.This critical angle θ _ccomputing formula is as follows:

θ _c＝sin ^-1(n ₁/n ₃) (1)

The n here ₁and n ₃it is respectively the refraction coefficient that light is dredged material and the close material of light.N ₁and n ₃ratio must be less than 1 total reflection could occur.As θ < θ _ctime, by the known most light of Snell formula, all through a refraction angle, by interface transmission, go out (be equally also by normal to interface orientation measurement).As θ > θ _ctime, all light all can be reflected back in optically denser medium.But some projectile energies can be penetrated through surface of contact and along the surface that is parallel to incident aspect and propagate.This is called ' field that dies of declining ' (or ' ripple ') in optically thinner medium, and such field just can excite near fluorescence molecule surface in contact.

For declining of a unlimited angle pencil of ray die field strength measurement (unit is used: energy/region/s) be accompanied by the surperficial vertical range Z of distance and be exponential damping:

I(Z)＝I(O)e ^-z/d (2)

Here

$d=\frac{{\mathrm{\&lambda;}}_0}{4\mathrm{\&pi;}}{(n_3^2{\sin }^2\mathrm{\&theta;}-n_1^2)}^{-1/2}---\left(3\right)$

λ ₀it is incident ray wavelength in a vacuum.Depth d does not rely on incident polarisation of light and along with incidence angle θ increases and reduces.Except when θ → θ _c(d → ∞ here) d approximates λ ₀or smaller.

So, when in TIRF state, change incident angle size its decline the degree of depth dying can be different, constantly change angular dimension, just can obtain the image relevant for cell surface information of a series of different depths, then these different depth images are analyzed near the three-dimensional information that just can obtain cell surface.And can significantly eliminate when replace traditional single-point illumination with ring-shaped lighting in TIRF situation the interference fringe that point light source of single illumination produces.

EPI is standard fluorescence microscope mode of operation, excitation line is vertically injected through sample fluorophor is excited from object lens direction, in TIRF microscope, when incident ray is EPI pattern when object lens central vertical is injected, now the very wide observation degree of depth of field range is dark.

HILO is that (incident angle between zero degree and the angle of total reflection time), on contact interface, two kinds of situations of refraction and reflection can occur simultaneously when incident angle is in subcritical angular region, adjust different incident angles and reflect with deflecting light beams angulation and energy and change thereupon, can obtain like this having the image of the low backscatter of depth illumination ability.The signal to noise ratio (S/N ratio) of HILO is higher 4 to 8 times than EPI.

Claims (4)

1. Novel multiple angle multi-mode is switched ring-type illumination optical micro imaging system fast, it is characterized in that comprising laser instrument, single-mode fiber, collimation lens, scanning galvanometer, scanning lens, the first field lens, focus lamp, dichroscope, TIRF microcobjective, sample stage, filter plate, the second field lens, detector;

Single-mode fiber, collimation lens, scanning galvanometer are positioned on the optical axis of laser emitting light beam successively, and scanning lens, the first field lens, focus lamp, dichroscope are positioned at successively on the optical axis of scanning galvanometer outgoing beam; TIRF microcobjective, sample stage are positioned on dichroscope folded light beam optical axis successively, and filter plate, the second field lens, detector are positioned on dichroscope transmitted light beam optical axis successively; Described sample stage is positioned at the place, focal plane of TIRF microcobjective, and the collection hole of described detector is positioned at the place, focal plane of the second field lens;

The collimated laser beam of laser instrument transmitting, is first imported into single-mode fiber, and the laser beam penetrating from single-mode fiber completes collimation through collimation lens; Light beam after collimation incides in scanning galvanometer and carries out light path deviation, incides afterwards scanning lens and focuses on; After the light beam being penetrated by scanning lens expands through the first field lens, after parallel ejaculation difference line focus mirror and dichroiscopic focusing and reflection, light beam focuses on TIRF microcobjective and is projeced on the testing sample on sample stage; The flashlight that testing sample is launched is collected by TIRF microcobjective, first, by wave plate elimination parasitic light after filtration successively again after dichroscope transmission, secondly through the second field lens, focuses on, and is finally detected device collection, the detector record light intensity signal detecting now.

2. Novel multiple angle multi-mode as claimed in claim 1 is switched ring-type illumination optical micro imaging system fast, it is characterized in that:

When described light beam focuses on TIRF microcobjective and is projeced on the testing sample of sample stage 10, if the incidence angle θ of incident beam is greater than critical angle θ _c, incident laser will be on sample surface of contact experiences total internal reflection, critical angle θ _ccomputing formula is as follows:

θ _c＝sin ^-1(n ₁/n ₃)

Wherein, n ₁and n ₃it is respectively the refraction coefficient that light is dredged material and the close material of light;

Now, at the opposite side of testing sample surface in contact, can produce evanescent wave, and along with being exponential damping from contact measured sample surfaces apart from Z; This field energy that dies of declining enough excites at the fluorophor of near surface and also can avoid the more fluorophor of depths to be excited simultaneously; The die measure equation of field strength of the declining of this unlimited angle pencil of ray is as follows:

I(Z)＝I(O)e ^-z/d (2)

Wherein,

$d=\frac{{\mathrm{\&lambda;}}_0}{4\mathrm{\&pi;}}{(n_3^2{\sin }^2\mathrm{\&theta;}-n_1^2)}^{-1/2}---\left(3\right)$

λ ₀it is incident beam wavelength in a vacuum; Depth d does not rely on incident polarisation of light and along with incidence angle θ increases and reduces; The fluorescence that fluorophor sent of the field excitation that died by declining detects through being just detected device after dichroscope, filter and lens.

3. Novel multiple angle multi-mode as claimed in claim 1 is switched ring-type illumination optical micro imaging system fast, it is characterized in that:

Described microcobjective is TIRF type microcobjective, its numerical aperture NA=1.45, and detector is CCD camera, can carry out signals collecting.

4. Novel multiple angle multi-mode as claimed in claim 1 is switched ring-type illumination optical micro imaging system fast, it is characterized in that:

Described can regulate and control institute's on-load voltage on it by controller for changing the scanning galvanometer of light beam deviation direction, changes the size of sample surfaces illuminating bundle incident angle, thereby realizes multi-angle TIRF; Also can realize by the regulation and control of controller ring-type TIRF throws light on; In ring-type TIRF situation, controller is controlled and is made incident light around lap time, just equal the time shutter of detector scanning galvanometer, thereby reach to survey with on the time of scanning, synchronizes.