CN107014788A - The caliberating device and scaling method of the incident depth of new utilizing total internal reflection fluorescence microscope - Google Patents

The caliberating device and scaling method of the incident depth of new utilizing total internal reflection fluorescence microscope Download PDF

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Publication number
CN107014788A
CN107014788A CN201710103652.1A CN201710103652A CN107014788A CN 107014788 A CN107014788 A CN 107014788A CN 201710103652 A CN201710103652 A CN 201710103652A CN 107014788 A CN107014788 A CN 107014788A
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internal reflection
total internal
fluorescence microscope
slide
images
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许迎科
金璐红
刘旭
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Zhejiang University ZJU
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Zhejiang University ZJU
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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
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N2021/0106—General arrangement of respective parts
    • G01N2021/0112—Apparatus in one mechanical, optical or electronic block

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  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Optics & Photonics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

The invention discloses a kind of caliberating device of the incident depth of utilizing total internal reflection fluorescence microscope, the device includes：Slide, square cover glass, sheet glass, the square cover glass are placed on the upper surface of the slide and right edge alignment；The sheet glass is placed on the slide and left side contact and alignment, and the lower surface of the sheet glass is supported on the direction cover glass；The lower surface of the sheet glass is coated uniformly with fluorescent dye.The invention also discloses the method demarcated using described caliberating device to the incident depth of utilizing total internal reflection fluorescence microscope, the apparatus structure is simple, cost is low, flexible to operation.

Description

The caliberating device and scaling method of the incident depth of new utilizing total internal reflection fluorescence microscope
Technical field
The invention belongs to fluorescence microscope imaging field, and in particular to a kind of new utilizing total internal reflection fluorescence microscope is incident deep The caliberating device and scaling method of degree.
Background technology
Utilizing total internal reflection fluorescence microscope (total internal reflection fluorescence microscopy, TIRFM it is) a kind of optical image technology emerging in recent years, the evanescent wave produced when the technology is using total internal reflection is glimmering to excite Optical molecule, to observe fluorescence labeling sample in the very thin region close to interface.Because there is energy in the axial direction in evanescent wave The characteristic exponentially decayed so that the dynamic range in observable region has efficiently controlled generally in below 200nm and excited body Product, greatly reduces background noise interference, therefore with the incomparable high s/n ratio of other optical image technologies and contrast Degree.Therefore technique is widely used to the dynamic observation of cell membrane surface material at present.
It can be seen from the characteristic of evanescent wave, when incidence angle is bigger, the incident depth of the evanescent wave of generation is more shallow.Therefore Every utilizing total internal reflection fluorescence microscope is shot in obtained two-dimensional projection image all containing abundant three-dimensional information.Accordingly, it is determined that Go out the corresponding incident depth of each incidence angle to be just particularly important.
The incidence angle and TIRF that business utilizing total internal reflection fluorescence microscope in the market can not provide exciting light are incident deep The corresponding informance of degree.Although utilizing total internal reflection fluorescence microscope has a theoretical exponential decay model, during this is ideal situation Optical model, is not particularly suited for actual conditions.Further, since the difference of experiment condition, different microscopical axial attenuation models Also it is not quite similar.So, it is proposed that a kind of system calibrating method of the incident depth of conveniently utilizing total internal reflection fluorescence microscope is just It is particularly important.
At present, many researchers paid energy to study the incident depth of demarcation utilizing total internal reflection fluorescence microscope, than More common two methods are the sub- method of Fluorescent Beads and the big pearl method of fluorescence.Schematic diagram such as Fig. 1 institutes of the sub- standardization of Fluorescent Beads Show, its principle is to choose a, the several points such as b place Fluorescent Beads, per the two apart from 25nm.Sent out with certain galvanometer voltage V Light is penetrated, measures and obtains a, the light intensity of b etc. fluorescence is I (a) and I (b).Light intensity will be measured with the sub- position of Fluorescent Beads to join Vertical matched curve, obtains I (z)-z curves, penetration depth d can be obtained according to formula (1).
Its difficult point is the special instrument of needs to ensure the distance between Fluorescent Beads for 25nm and need to make work Fluorescent Beads of skill complexity and diameter very little.
The schematic diagram of the big pearl standardization of fluorescence is as shown in Fig. 2 its principle is the excitation under certain incidence angle Evanescent wave depth of field is certain, when fluorescent bead diameter is sufficiently large, can be extrapolated using the perspective view clapped real incident deep Degree.
Although both approaches can obtain preferable calibration result, special fluorescent bead or experimental tool is needed, And the later stage also needs to carry out the processes such as complicated image processing data fitting, application method is inconvenient.
Ring-type utilizing total internal reflection fluorescence microscope resides in reduced using a ring-shaped light circle formation TIRF image, its advantage Interference fringe, quick multi-angle imaging reduces the aberration that 3D imagings and single angle imaging are produced.
EPI refers to rectilinear fluorescence microscope.Exciting light is incident in the form of near vertical interface so that enter sample The fluorescence intensity of one's respective area is still very strong, can excite the whole fluorescent dyes marked on sample.
The content of the invention
In view of above-mentioned, the invention provides a kind of caliberating device of the incident depth of utilizing total internal reflection fluorescence microscope, the device Simple in construction, cost is low, flexible to operation.
A kind of caliberating device of the incident depth of utilizing total internal reflection fluorescence microscope, including：
Slide；
Square cover glass, the square cover glass is placed on the upper surface of the slide and right edge alignment；
Sheet glass, the sheet glass is placed on the slide and left side contact and alignment, the sheet glass Lower surface is supported on the direction cover glass；
The lower surface of the sheet glass is coated uniformly with fluorescent dye.
Described sheet glass is identical with the size of slide.
Preferably, the size of described sheet glass is 25mm × 75mm, in order to overcome the point diffusion effect of fluorescence imaging, The thickness of fluorescent dye on sheet glass is 8~12nm.
Preferably, the specification that described slide is the production of Electron Microscopy Sciences companies is 25mm × 75mm slide.
Preferably, the specification that described square cover glass is the production of FisherBrand companies is 18mm × 18mm thickness For 0.17mm square cover glass.
The incident depth of utilizing total internal reflection fluorescence microscope is carried out using above-mentioned caliberating device present invention also offers a kind of The method of demarcation, is concretely comprised the following steps：
(1) by caliberating device be placed in can free switching incidence angle utilizing total internal reflection fluorescence microscope objective table on；
(2) by microscope can field of view border B points are set in the relevant position for the sheet glass for being evenly coated with fluorescent dye, B points are set to z apart from the distance of slide0；
(3) it is any as the fluorescent target point A demarcated that selection microscope can be in the range of field of view, then fluorescence mesh Punctuate A actual grade ZAIt is represented by：
zA=z0+xtanα
Wherein, x be fluorescent target point A with can the distance between field of view border, α is the glass for being evenly coated with fluorescent dye Angle between glass piece and slide；
(4) fixation can field of view, the incidence angle θ of change microscope exciting lighti, obtain the TIRFM figures under the incidence angle Picture, and then obtain intensity Is of the fluorescent target point A in TIRFM imagesA(TIRFM)(θi)；
(5) change microscope exciting light vertically to inject, obtain EPI images, and then obtain fluorescent target point A in EPI images Intensity IA(EPI), then the logarithm value of strength ratios of the fluorescent target point A in TIRFM images and EPI images be expressed as：
Wherein, IA(0,θi) represent exciting light incidence angle be θiWhen evanescent wave at the interface of two kinds of refractive indexes Light intensity, dp(θi) represent the incidence angle of exciting light for θiWhen evanescent wave penetration depth, e is about 2.718,For incidence angle θiIt is a constant.
(6) the different fluorescent target point of a row is chosen, each fluorescence mesh is obtained by the method for step (4) and step (5) The logarithm value of strength ratio of the punctuate in intensity TIRFM images and EPI images, then to all fluorescent target points in intensity TIRFM The logarithm value of image and the strength ratio in EPI images carries out linear fit, and the negative inverse for obtaining the slope of fitting a straight line is incidence Angle θiCorresponding penetration depth dp(θi)。
The present invention provides one kind newly for the corresponding relation that utilizing total internal reflection fluorescence microscope demarcates incidence angle and penetration depth Thinking, can effectively determine the incident depth under utilizing total internal reflection fluorescence microscope different incidence angles.The present invention has easy to operate, dress Put the characteristics such as simple and demarcation efficiency height.To experiment condition without particular/special requirement, it is adaptable to which various types of total internal reflection fluorescents show The demarcation of micro mirror.
Brief description of the drawings
Fig. 1 is the sub- scaling method schematic diagram of Fluorescent Beads in background technology；
Fig. 2 is the big pearl scaling method schematic diagram of fluorescence in background technology；
Fig. 3 is the caliberating device side view of the incident depth of utilizing total internal reflection fluorescence microscope of the present invention；
Fig. 4 is the scaling method schematic diagram of the incident depth of utilizing total internal reflection fluorescence microscope of the present invention.
Embodiment
In order to more specifically describe the present invention, below in conjunction with the accompanying drawings and embodiment is to technical scheme It is described in detail.
The caliberating device of the incident depth of utilizing total internal reflection fluorescence microscope as shown in Figure 1 is set up, the master of caliberating device is built The equipment is wanted to be：Size is sheet glass, the Electron that 25mm × 75mm and one side are evenly coated with the thick fluorescent dyes of 10nm The specification of Microscopy Sciences companies production is 25mm × 75mm slide and the production of FisherBrand companies Specification be square cover glass that 18mm × 18mm thickness is 0.17mm.Square cover glass is stacked on the right side of slide, makes two pieces The right hand edge alignment of slide.Then sheet glass is up stacked, and ensures that the left hand edge of sheet glass aligns with slide left hand edge, And the left end frame of sheet glass is on square cover glass.So as to ensure to scribble the slide of dyestuff and bottom carry inclined angle alpha between glass= 0.18°。
Schematic diagram such as Fig. 4 institutes that the incident depth of utilizing total internal reflection fluorescence microscope is demarcated are carried out using above-mentioned caliberating device Show, 1. represent the liquid environment residing for sample, in this calibration experiment, the liquid in the region is water, and refractive index is 1.2. represent The slide on microscope carrier is positioned over, its refractive index is 1.52.3. represent be microscope ocular can field of view, It is the oil immersion that refractive index and slide match between eyepiece and slide (i.e. oil immersion refractive index is also 1.52).
Carrying out the method that the incident depth of utilizing total internal reflection fluorescence microscope demarcated using above-mentioned caliberating device is：
Step 1, eyepiece oil, the refractive index of eyepiece oil and slide are coated on the eyepiece of utilizing total internal reflection fluorescence microscope Match；
Step 2, by caliberating device be placed in can free switching incidence angle utilizing total internal reflection fluorescence microscope objective table on；
Step 3, by microscope can field of view border in the relevant position for the sheet glass for being evenly coated with fluorescent dye be set to B Point, is set to z by B points apart from the distance of slide0；
Step 4, it is any as the fluorescent target point A demarcated that selection microscope can be in the range of field of view, then glimmering Optical target point A actual grade ZAIt is represented by：
zA=z0+xtanα
Wherein, x be fluorescent target point A with can the distance between field of view border, α is the glass for being evenly coated with fluorescent dye Angle between glass piece and slide；
Step 5, fixation can field of view, the incidence angle θ of change microscope exciting lighti, obtain the TIRFM under the incidence angle Image, and then obtain intensity Is of the fluorescent target point A in TIRFM imagesA(TIRFM)(θi)；
Step 6, change microscope exciting light vertically to inject, obtain EPI images, and then obtain fluorescent target point A in EPI figures The intensity I of pictureA(EPI), then the logarithm value of strength ratios of the fluorescent target point A in TIRFM images and EPI images be expressed as：
Wherein, IA(0,θi) represent exciting light incidence angle be θiWhen evanescent wave at the interface of two kinds of refractive indexes Light intensity, dp(θi) represent the incidence angle of exciting light for θiWhen evanescent wave penetration depth, e is 2.178,For incidence angle θiIt is a constant.
Step 7, the different fluorescent target point of a row is chosen, each fluorescence is obtained by the method for step (4) and step (5) The logarithm value of strength ratio of the target point in intensity TIRFM images and EPI images, then to all fluorescent target points in intensity The logarithm value of TIRFM images and the strength ratio in EPI images carries out linear fit, and the negative inverse for obtaining the slope of fitting a straight line is Incidence angle θiCorresponding penetration depth dp(θi)。
Embodiment 1
The caliberating device of the incident depth of the utilizing total internal reflection fluorescence microscope of above-mentioned foundation is placed in sweeping for Thornlabs companies Retouch in galvanometer system, the laser that then laser is produced from Melles Griot companies is sent, and is imported by single-mode fiber The collimation lens of Thornlabs companies completes collimation；Light beam after collimation incides the scanning galvanometer of Thornlabs companies Carry out being focused in light path deviation, the scanning lens that Thornlabs companies are incided afterwards in system.Projected by scanning lens Light beam expanded by the first field lens after parallel focus lamp of the injection through Thornlabs companies and dichroic mirror focus on and reflect Afterwards, light beam is focused on the object lens of the utilizing total internal reflection fluorescence microscope of OLYMPUS companies production and is projeced on its sample stage.Treat The fiber object lens that the flashlight that test sample product are launched is accordingly totally internally reflected fluorescence microscope are collected, and are first passed through dichroic mirror and are sequentially passed through again Filter plate filters off veiling glare, is focused on by the second field lens, is finally gathered by the ccd detector of OLYMPUS companies, ccd detector Record the light intensity signal now detected.The voltage for changing control scanning galvanometer is that can obtain in this visual field under different incidence angles Utilizing total internal reflection fluorescence microscope image.Finally incident beam is vertically injected, shoot to obtain EPI images under same visual field. So far, the view data needed for demarcating prepares complete.
Any one target point in the utilizing total internal reflection fluorescence microscope image obtained for shooting, according to inclining for inclined plane Angle obtains its axial depth relative to slide finally.
For every utilizing total internal reflection fluorescence microscope image, a series of target point for taking axial depths different exists the point Corresponding intensity is taken the logarithm compared with the corresponding intensity in EPI images in utilizing total internal reflection fluorescence microscope image, you can obtained A series of linear relation, is fitted to linear relation, and the negative inverse for obtaining the slope of straight line is corresponding for incidence angle Penetration depth.
Technical scheme and beneficial effect are described in detail above-described embodiment, Ying Li Solution is to the foregoing is only presently most preferred embodiment of the invention, is not intended to limit the invention, all principle models in the present invention Interior done any modification, supplement and equivalent substitution etc. are enclosed, be should be included in the scope of the protection.

Claims (4)

1. a kind of caliberating device of the incident depth of utilizing total internal reflection fluorescence microscope, it is characterised in that：
Slide；
Square cover glass, the square cover glass is placed on the upper surface of the slide and right edge alignment；
Sheet glass, the sheet glass is placed on the slide and left side contact and alignment, the following table of the sheet glass Face is supported on the direction cover glass；
The lower surface of the sheet glass is coated uniformly with fluorescent dye.
2. the caliberating device of the incident depth of utilizing total internal reflection fluorescence microscope according to claim 1, it is characterised in that：Described The thickness of fluorescent dye is 8~12nm.
3. the caliberating device of the incident depth of utilizing total internal reflection fluorescence microscope according to claim 2, it is characterised in that：Described Sheet glass is identical with the size of slide.
4. a kind of carried out using any described caliberating device of claims 1 to 33 to the incident depth of utilizing total internal reflection fluorescence microscope The method of demarcation, is concretely comprised the following steps：
(1) by caliberating device be placed in can free switching incidence angle utilizing total internal reflection fluorescence microscope objective table on；
(2) by microscope can field of view border B points are set in the relevant position for the sheet glass for being evenly coated with fluorescent dye, by B Point is set to z apart from the distance of slide0；
(3) it is any as the fluorescent target point A demarcated that selection microscope can be in the range of field of view, then fluorescent target point A actual grade ZAIt is represented by：
zA=z0+x tanα
Wherein, x be fluorescent target point A with can the distance between field of view border, α is the sheet glass for being evenly coated with fluorescent dye Angle between slide；
(4) fixation can field of view, the incidence angle θ of change microscope exciting lighti, the TIRFM images under the incidence angle are obtained, are entered And obtain intensity Is of the fluorescent target point A in TIRFM imagesA(TIRFM)(θi)；
(5) change microscope exciting light vertically to inject, obtain EPI images, and then obtain fluorescent target point A in the strong of EPI images Spend IA(EPI), then the logarithm value of strength ratios of the fluorescent target point A in TIRFM images and EPI images be expressed as：
Wherein, IA(0,θi) represent exciting light incidence angle be θiWhen light of the evanescent wave at the interface of two kinds of refractive indexes By force, dp(θi) represent the incidence angle of exciting light for θiWhen evanescent wave penetration depth, e is about 2.718；
(6) the different fluorescent target point of a row is chosen, each fluorescent target point is obtained by the method for step (4) and step (5) The logarithm value of strength ratio in intensity TIRFM images and EPI images, then to all fluorescent target points in intensity TIRFM images Linear fit is carried out with the logarithm value of the strength ratio in EPI images, the negative inverse for obtaining the slope of fitting a straight line is incidence angle θi Corresponding penetration depth dp(θi)。
CN201710103652.1A 2017-02-24 2017-02-24 The caliberating device and scaling method of the incident depth of new utilizing total internal reflection fluorescence microscope Pending CN107014788A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101949849A (en) * 2010-09-08 2011-01-19 华中科技大学 Light-activated positioning microscopic imaging system based on optical fiber evanescent field illuminator
CN102183499A (en) * 2011-02-24 2011-09-14 中国科学院植物研究所 Method for measuring concentration of calcium ions in plant cells by utilizing total internal reflection fluorescence microscope
CN103940796A (en) * 2014-04-22 2014-07-23 浙江大学 Novel multi-angle and multi-mode quick switching circular optical illumination microscopic imaging system
CN106226895A (en) * 2016-08-25 2016-12-14 浙江大学 The rotation total internal reflection microscopic method of a kind of band feedback and device

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101949849A (en) * 2010-09-08 2011-01-19 华中科技大学 Light-activated positioning microscopic imaging system based on optical fiber evanescent field illuminator
CN102183499A (en) * 2011-02-24 2011-09-14 中国科学院植物研究所 Method for measuring concentration of calcium ions in plant cells by utilizing total internal reflection fluorescence microscope
CN103940796A (en) * 2014-04-22 2014-07-23 浙江大学 Novel multi-angle and multi-mode quick switching circular optical illumination microscopic imaging system
CN106226895A (en) * 2016-08-25 2016-12-14 浙江大学 The rotation total internal reflection microscopic method of a kind of band feedback and device

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JIAN WU 等: "A New Method for Axial Decay Function Calibration of Evanescent Field in Multi-Angle Total Internal Reflection Fluorescence Microscopy", 《JOURNAL OF PHYSICS: CONFERENCE SERIES》 *

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Application publication date: 20170804