DOCSLIB.ORG

Total Internal Reflection Fluorescence Microscopy (TIRFM)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Total Internal Reflection Fluorescence Microscopy (TIRFM) Total Internal Reflection Fluorescence Microscopy (TIRFM) Physics 598 BP Spring 2015 1 Contents • Optical microscopy • TIRFM basics and principles • Optics – components and uses • Alignment tips and techniques 2 Optical Microscopy Bright Field Fluorescence … Epifluorescence TIRFM 3 Fluorescence Microscopy • Use of fluorophores – Absorb and emit at unique wavelengths • Laser used to excite fluorophores • Dichroic mirror reflects light at excitation wavelength while allowing emitted light to pass through • Emission filter allows emitted light to pass through while absorbing lights at other wavelengths 4 Fluorescence Microscopy Incoming Laser Beam Dichroic Mirror Emission Filter Microscope Emitted Light CCD Camera Source: Mindy Hoffman: Hoffman-Single Enzym. Meth. Chapter figures PowerPoint slides 5 TIRFM Basics and Principles • Make use of total internal reflection at the interface between glass coverslip and specimen buffer • Principles: evanescent wave formation • Advantages: – Low fluorescence background – Penetration depth: ~ 100 nm www2.bioch.ox.ac.uk • Two configurations: prism method and objective lens method http://www.microscopyu.com/articles/fluore6 scence/tirf/tirfintro.html Epifluorescence vs TIRFM Epifluorescence TIRFM Picture source: http://www.me.mtu.edu/~cchoi/practice.html 7 Alignment Goal • Beam needs to pass through the center of the front and back focal plane of the objective (before the TIR lens is added) http://www.microscopyu.com/articles/fluore scence/tirf/tirfintro.html 8 General Strategy for Alignment • Draw beam path • Add and align laser, shutter and neutral density filter • Add and align appropriate mirrors at respective corners • Add and align beam expander • Add and align TIR lens • Image in TIR 9 10 11 12 13 14 15 Optical Components: Posts, Post Holders and Bases 16 Irises • Two irises are used to make sure that the beam line is parallel to the optical table Source: Thorlabs • They are also used to make sure that the beam passes through the beam path 17 Irises • Alignment tips: – First adjust the height of the iris to the same height as the microscope laser input port – Place two irises as far Source: Thorlabs away from each other along the direction of the beam path – Adjust mirrors/ lasers so that the beam passes through both irises 18 Mirrors • Mirrors help direct beams • One mirror can direct a beam to any point • Two mirrors can direct a beam so that it is parallel and Source: Thorlabs coincident to any line • Mirrors are mounted on the kinematic mount • They are usually placed at corners of beam path 19 Source: Mindy Hoffman: Hoffman-Single Enzym. Meth. Chapter figures PowerPoint slides Mirrors • Alignment tips: – First align the post holders (Note: center of the post holder is offset from the mirror surface). – Once post holders are aligned and secured, place the mirrors such that the beam hits the center of the mirror 20 Beam Expander • Purpose: – Expand beam (5x,10x, 20x) – Collimate beam parallel output beam • Alignment tips: – Beam needs to pass through the center of expander Source: http://www.edmundoptics.com/technical-support/lasers/beam-expanders/ – First make sure the post holder and base are centered – Then add the beam expander and center it in x and y direction 21 TIR Lens • Focuses beam onto the back focal plane of the objective • Translation of the TIR lens will cause the beam to enter the objective at an angle, allowing total internal reflection to be achieved • Alignment tips: – Place the TIR lens onto a three axes translation stage – Position the lens (x and y direction) such that the beam passes through its center – Position the lens (z direction) such that the beam is converging through the objective – Fine adjust the translation stage such that the laser beam (through the objective) is projecting upward onto the ceiling and its image is circular in shape 22 Alignment Technique: Laser with Irises 23 Alignment Technique: Laser with Irises 24 Alignment Technique: Laser with Irises 25 Alignment Technique: Laser with Irises 26 Alignment Technique: Laser with Irises 27 Conclusion: Laser with Irises • To correctly align a laser, we need to adjust both the tilt and the position of the laser 28 Alignment Technique: Mirrors with Irises • Two mirrors can align the laser beam with the center line of the irises • Mirror 1 (further from the irises) is responsible for the position of the beam onto the second mirror. It is adjusted to center the beam on the first iris • Mirror 2 (closer to the irises) is responsible for the tilt of the beam towards the first iris. It is adjusted to center the beam on the second iris 29 Alignment Technique: Mirror with Irises 30 Alignment Technique: Mirror with Irises 31 Alignment Technique: Mirror with Irises 32 Alignment Technique: Mirror with Irises 33 Alignment Technique: Mirror with Irises 34 Alignment Technique: Alignment through Objective • The last alignment with mirrors is to align the beam through the objective • The strategy is the same as aligning mirrors through irises. In place of the irises, we now have the front and back focal plane of the objective • Use the center of the eyepiece and center of the crosshair to align through the front and back focal plane respectively • Use mirror 1 (further from objective) to align through crosshair (back) and mirror 2 (closer to objective) to align through the eyepiece (front) 35 http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html http://www.labx.com/v2/spiderdealer2/vistasearchdetails.cfm?LVid=6853317.
Load more

Recommended publications
  • 502-13 Magnifiers and Telescopes
    13-1 I and Instrumentation Design Optical OPTI-502 © Copyright 2019 John E. Greivenkamp E. John 2019 © Copyright Section 13 Magnifiers and Telescopes 13-2 I and Instrumentation Design Optical OPTI-502 Visual Magnification Greivenkamp E. John 2019 © Copyright All optical systems that are used with the eye are characterized by a visual magnification or a visual magnifying power. While the details of the definitions of this quantity differ from instrument to instrument and for different applications, the underlying principle remains the same: How much bigger does an object appear to be when viewed through the instrument? The size change is measured as the change in angular subtense of the image produced by the instrument compared to the angular subtense of the object. The angular subtense of the object is measured when the object is placed at the optimum viewing condition. 13-3 I and Instrumentation Design Optical OPTI-502 Magnifiers Greivenkamp E. John 2019 © Copyright As an object is brought closer to the eye, the size of the image on the retina increases and the object appears larger. The largest image magnification possible with the unaided eye occurs when the object is placed at the near point of the eye, by convention 250 mm or 10 in from the eye. A magnifier is a single lens that provides an enlarged erect virtual image of a nearby object for visual observation. The object must be placed inside the front focal point of the magnifier. f h uM h F z z s The magnifying power MP is defined as (stop at the eye): Angular size of the image (with lens) MP Angular size of the object at the near point uM MP d NP 250 mm uU 13-4 I and Instrumentation Design Optical OPTI-502 Magnifiers – Magnifying Power Greivenkamp E.
    [Show full text]
  • Depth of Focus (DOF)
    Erect Image Depth of Focus (DOF) unit: mm Also known as ‘depth of field’, this is the distance (measured in the An image in which the orientations of left, right, top, bottom and direction of the optical axis) between the two planes which define the moving directions are the same as those of a workpiece on the limits of acceptable image sharpness when the microscope is focused workstage. PG on an object. As the numerical aperture (NA) increases, the depth of 46 focus becomes shallower, as shown by the expression below: λ DOF = λ = 0.55µm is often used as the reference wavelength 2·(NA)2 Field number (FN), real field of view, and monitor display magnification unit: mm Example: For an M Plan Apo 100X lens (NA = 0.7) The depth of focus of this objective is The observation range of the sample surface is determined by the diameter of the eyepiece’s field stop. The value of this diameter in 0.55µm = 0.6µm 2 x 0.72 millimeters is called the field number (FN). In contrast, the real field of view is the range on the workpiece surface when actually magnified and observed with the objective lens. Bright-field Illumination and Dark-field Illumination The real field of view can be calculated with the following formula: In brightfield illumination a full cone of light is focused by the objective on the specimen surface. This is the normal mode of viewing with an (1) The range of the workpiece that can be observed with the optical microscope. With darkfield illumination, the inner area of the microscope (diameter) light cone is blocked so that the surface is only illuminated by light FN of eyepiece Real field of view = from an oblique angle.
    [Show full text]
  • The Microscope Parts And
    The Microscope ­ Parts and Use Name:_______________________ Period:______ Historians credit the invention of the compound microscope to the Dutch spectacle maker, Zacharias Janssen, around the year 1590. The compound microscope uses lenses and light to enlarge the image and is also called an optical or light microscope (vs./ an electron microscope). The simplest optical microscope is the magnifying glass and is good to about ten times (10X) magnification. The compound microscope has two systems of lenses for greater magnification, 1) the ocular, or eyepiece lens that one looks into and 2) the objective lens, or the lens closest to the object. Before purchasing or using a microscope, it is important to know the functions of each part. Eyepiece Lens: the lens at the top that you look through. They are usually 10X or 15X power. Tube: Connects the eyepiece to the objective lenses Arm: Supports the tube and connects it to the base. It is used along with the base to carry the microscope Base: The bottom of the microscope, used for support Illuminator: A steady light source (110 volts) used in place of a mirror. Stage: The flat platform where you place your slides. Stage clips hold the slides in place. Revolving Nosepiece or Turret: This is the part that holds two or more objective lenses and can be rotated to easily change power. Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope. They almost always consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common) eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X.
    [Show full text]
  • How Do the Lenses in a Microscope Work?
    Student Name: _____________________________ Date: _________________ How do the lenses in a microscope work? Compound Light Microscope: A compound light microscope uses light to ​ ​ transmit an image to your eye. Compound ​ deals with the microscope having more than one lens. Microscope is the ​ ​ combination of two words; "micro" meaning small and "scope" meaning view. Early microscopes, like Leeuwenhoek's, were called simple because they only had one lens. Simple scopes work like magnifying glasses that you have seen and/or used. These early microscopes had limitations to the amount of magnification no matter how they were constructed. The creation of the compound microscope by the Janssens helped to advance the field of microbiology light years ahead of where it had been only just a few years earlier. The Janssens added a second lens to magnify the image of the primary (or first) lens. Simple light microscopes of the past could magnify an object to 266X as in the case of Leeuwenhoek's microscope. Modern compound light microscopes, under optimal conditions, can magnify an object from 1000X to 2000X (times) the specimens original diameter. "The Compound Light Microscope." The Compound Light Microscope. Web. 16 Feb. 2017. ​ ​ http://www.cas.miamioh.edu/mbi-ws/microscopes/compoundscope.html Text is available under the Creative Commons Attribution-NonCommercial 4.0 ​ International (CC BY-NC 4.0) license. - 1 – Student Name: _____________________________ Date: _________________ Now we will describe how a microscope works in somewhat more detail. The first lens of a microscope is the one closest to the object being examined and, for this reason, is called the objective.
    [Show full text]
  • Objective (Optics) 1 Objective (Optics)
    Objective (optics) 1 Objective (optics) In an optical instrument, the objective is the optical element that gathers light from the object being observed and focuses the light rays to produce a real image. Objectives can be single lenses or mirrors, or combinations of several optical elements. They are used in microscopes, telescopes, cameras, slide projectors, CD players and many other optical instruments. Objectives are also called object lenses, object glasses, or objective glasses. Microscope objectives are typically designed to be parfocal, which means that when one changes from one lens to another on a Several objective lenses on a microscope. microscope, the sample stays in focus. Microscope objectives are characterized by two parameters, namely, magnification and numerical aperture. The former typically ranges from 5× to 100× while the latter ranges from 0.14 to 0.7, corresponding to focal lengths of about 40 to 2 mm, respectively. For high magnification applications, an oil-immersion objective or water-immersion objective has to be used. The objective is specially designed and refractive index matching oil or water must fill the air gap between the front element and the object to allow the numerical aperture to exceed 1, and hence give greater resolution at high magnification. Numerical apertures as high as 1.6 can be achieved with oil immersion.[1] To find the total magnification of a microscope, one multiplies the A photographic objective, focal length 50 mm, magnification of the objective lenses by that of the eyepiece. aperture 1:1.4 See also • List of telescope parts and construction Diastar projection objective from a 35 mm movie projector, (focal length 400 mm) References [1] Kenneth, Spring; Keller, H.
    [Show full text]
  • 6. Imaging: Lenses & Curved Mirrors
    6. Imaging: Lenses & Curved Mirrors The basic laws of lenses allow considerable versatility in optical instrument design. The focal length of a lens, f, is the distance at which collimated (parallel) rays converge to a single point after passing through the lens. This is illustrated in Fig. 6.1. A collimated laser beam can thus be brought to a focus at a known position simply by selecting a lens of the desired focal length. Likewise, placing a lens at a distance f can collimate light emanating from a single spot. This simple law is widely used in experimental spectroscopy. For example, this configuration allows collection of the greatest amount of light from a spot, an important consideration in maximizing sensitivity in fluorescence or Raman measurements. The collection Figure 6.1: Parallel beams focused by a lens efficiency of a lens is the ratio Ω/4π, where Ω is the solid angle of the light collected and 4π is the solid angle over all space. The collection efficiency is related to the F-number of the lens, also abbreviated as F/# or f/n. F/# is defined as the ratio of the distance of the object from the lens to the lens diameter (or limiting aperture diameter). In Fig. 6.1 above, the f- number of lens is f/D - ! 1 = 2 . 4" $#4(F / #)&% The collection efficiency increases with decreased focal length and increased lens diameter. Collection efficiency is typically low in optical spectroscopy, such as fluorescence or Raman. On the other hand, two-dimensional imaging (as opposed to light collection) is limited when the object is at a distance of f from the lens because light from only one point in space is collected in this configuration.
    [Show full text]
  • What's the Size of What You See? Determine the Field Diameter of a Compound Microscope
    4th NGSS STEM Conference MAKING SCIENCE COUNT Integrating Math into an NGSS Classroom January 20, 2017 | Pier 15, San Francisco, CA What's the Size of What You See? Determine the field diameter of a compound microscope. By measuring the field diameter of a microscope, you can calculate the real sizes of objects too small to see with the naked eye. Tools and Materials • Compound microscope • Pencil and notepaper to record results • Calculator • Clear plastic metric ruler with millimeter markings (copy this template onto a transparency to make your own) • Optional: microscopic objects to view and measure 4th NGSS STEM Conference MAKING SCIENCE COUNT Integrating Math into an NGSS Classroom January 20, 2017 | Pier 15, San Francisco, CA Assembly None needed. To Do and Notice Find the total magnification of your microscope. First, read the power inscribed on the eyepiece. You’ll find it marked as a number followed by an X, which stands for “times.” Record the eyepiece power. Find the three barrel-shaped objective lenses near the microscope stage. Each will have a different power, which should be marked on the side of the lens. Record the power for each objective. Find the total magnification for each objective lens by multiplying the power of the eyepiece by the power of the objective. Lowest Magnification: Eyepiece x Lowest power objective = ________X Medium Magnification: Eyepiece x Medium power objective = ________X Highest Magnification: Eyepiece x Highest power objective = ________X Set the microscope to its lowest magnification. Slide the plastic metric ruler onto the stage and focus the microscope on the millimeter divisions.
    [Show full text]
  • Basic Microscopy Microscopy: Historical Perspective Introduction
    Basic Microscopy Microscopy: Historical Perspective Introduction Throughout history, the purpose of the optical or light microscope has not always been for research and the production of beautiful, accurate images. For many centuries, the construction of microscopes and the underpinning optical systems was entirely an issue of exterior cosmetic craftsmanship, with the design of optical components lagging seriously behind advances in the fabrication of microscope bodies and frames. Greater trouble and effort was invested in the outer appearance of these early instruments, while their performance depended on the experience of the lens grinders, and the (good or bad) results obtained were often sheer coincidence. In many cases, microscopes were simply toys or show pieces intended to increase the prestige of their owners. The dichotomy between appearance and quality of the images such instruments provided was, by today's standards, striking (see Figure 1). Nevertheless, microscopy became a beloved pastime of the wealthy in the 18th century and many nights were spent observing local specimens, an affair that often involved entire families. For example, Elizabeth Charlotte of the Palatinate, who became famous as the sister-in-law of Louis XIV, owned a valuable microscope and used it regularly. She correctly predicted that this instrument would be of inestimable value for medical applications in the future. Her foresight was almost visionary at that time. While working in a store where magnifying glasses were used to count the number of threads in cloth, Anton van Leeuwenhoek of Holland, who is often referred to as the father of microscopy, taught himself new methods for grinding and polishing small, curved lenses that magnified up to 270 diameters.
    [Show full text]
  • Chapter 8 the Telescope
    Chapter 8 The Telescope 8.1 Purpose In this lab, you will measure the focal lengths of two lenses and use them to construct a simple telescope which inverts the image like the one developed by Johannes Kepler. Because one lens has a large focal length and the other lens has a small focal length, you will use different methods of determining the focal lengths than was used in ’Optics of Thin Lenses’ lab. 8.2 Introduction 8.2.1 A Brief History of the Early Telescope Although eyeglass-makers had been experimenting with lenses well before 1600, the first mention of a telescope appears in a letter written in 1608 by Hans Lippershey, a Dutch spectacle maker, seeking a patent for a telescope. The patent was denied because of easy telescope duplication and difficulty in patent enforcement. The instrument spread rapidly. Galileo heard of it in the early 1600s and quickly made improvements in lens grinding that increased the magnification from a relatively low value of 2 to as much as 30. With these more powerful telescopes, he observed the Milky Way, the mountains on the Moon, the phases of Venus, and the moons of Jupiter. These early telescopes were a type of ‘opera glass,’ producing erect or ‘right side up’ images but having limited magnification. When Johannes Kepler, a German mathematician and astronomer working in Prague under Tycho Brahe, heard of Galileo’s discoveries, he perfected a different form of telescope. Although Kepler’s design inverts the image, it is much more powerful than the Galilean type. This lab we will use the lenses supplied with the telescope kit.
    [Show full text]
  • Total Internal Reflection Fluorescence (TIRF) Microscopy
    Application Note: TIRF Microscopy Total Internal Reflection Fluorescence (TIRF) Microscopy Introduction Fluorescence microscopy is a fundamental set of techniques in the life sciences for visualizing structures in living systems. Typically, a fluorescent molecule, either synthetic or biological, is associated with a structure of interest in a biological sample. When the sample is illuminated with light of an appropriate wavelength to excite the fluorophore, photons are emitted by the molecule which allows the molecule of interest to be visualized. The resolution at which fluorescent molecules can be visualized depends greatly on the preparation of the sample and the objectives used, but this can be limited by out of focus light being collected in a focal plane. Total internal reflection fluorescence microscopy (TIRF) makes use of specific optics to produce illumination light only at the 50-100 nm range at the interface of the slide, massively reducing out of focus light and improving the ability to detect fluorescent molecules. Because of its low light intensity and high spatial resolution, it is a key technique in live-cell imaging. Epifluorescence vs TIRF microscopy In traditional epifluorescence microscopy, illumination light is focused through the objective, exciting fluorescent molecules in the sample. The resultant emitted photons are collected by the same objective and focused onto a scientific camera for detection. This exposes the sample to excess and out of focus light which increases the effective light dose to the cells being imaged. Light itself, as well as radicals given off in the process of fluorescence, are toxic to cells and contribute to sample degradation. Further, fluorescence outside of the focal plane contributes to noise in the image, reducing overall signal to noise and spatial resolution.
    [Show full text]
  • The F-Word in Optics
    The F-word in Optics F-number, was first found for fixing photographic exposure. But F/number is a far more flexible phenomenon for finding facts about optics. Douglas Goodman finds fertile field for fixes for frequent confusion for F/#. The F-word of optics is the F-number,* also known as F/number, F/#, etc. The F- number is not a natural physical quantity, it is not defined and used consistently, and it is often used in ways that are both wrong and confusing. Moreover, there is no need to use the F-number, since what it purports to describe can be specified rigorously and unambiguously with direction cosines. The F-number is usually defined as follows: focal length F-number= [1] entrance pupil diameter A further identification is usually made with ray angle. Referring to Figure 1, a ray from an object at infinity that is parallel to the axis in object space intersects the front principal plane at height Y and presumably leaves the rear principal plane at the same height. If f is the rear focal length, then the angle of the ray in image space θ′' is given by y tan θ'= [2] f In this equation, as in the others here, a sign convention for angles heights and magnifications is not rigorously enforced. Combining these equations gives: 1 F−= number [3] 2tanθ ' For an object not at infinity, by analogy, the F-number is often taken to be the half of the inverse of the tangent of the marginal ray angle. However, Eq. 2 is wrong.
    [Show full text]
  • Total Internal Reflection Fluorescence Microscopy
    Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on April 25, 2017 - Published by Cold Spring Harbor Laboratory Press Topic Introduction Total Internal Reflection Fluorescence Microscopy Ahmet Yildiz and Ronald D. Vale The goal in fluorescence microscopy is to detect the signal of fluorescently labeled molecules with great sensitivity and minimal background noise. In epifluorescence microscopy, it is difficult to observe weak signals along the optical axis, owing to the overpowering signal from the out-of-focus particles. Confocal microscopy uses a small pinhole to produce thin optical sections (500 nm), but the pinhole rejects some of the in-focus photons as well. Total internal reflection fluorescence microscopy (TIRFM) is a wide-field illumination technique that illuminates only the molecules near the glass coverslip. It has become widely used in biological imaging because it has a significantly reduced background and high temporal resolution capability. TIRFM has been used to study proteins in vitro as well as signaling cascades by hormones and neurotransmitters, intracellular cargo transport, actin dynamics near the plasma membrane, and focal adhesions in living cells. Because TIRF illumi- nation is restricted to the glass–water interface and does not penetrate the specimen, it is well suited for studying the interaction of molecules within or near the cell membrane in living cells. INTRODUCTION Principles of Total Internal Reflection Total internal reflection fluorescence microscopy (TIRFM) relies on the refraction and reflection of light from a planar surface. When the excitation beam travels from a high index of refraction medium (n1 = 1.518 for glass) to a low index of refraction medium (n2 = 1.33 for water), the angle of the beam changes in accordance with Snell’s law.
    [Show full text]