INTRODUCTION TO MICROSCOPY. Urs Ziegler THE PROBLEM

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1 INTRODUCTION TO MICROSCOPY Urs Ziegler THE PROBLEM 1

2 ORGANISMS ARE LARGE LIGHT AND ELECTRONS: ELECTROMAGNETIC WAVES v = Wavelength ( ) Speed (v) Frequency ( ) Amplitude (A) Propagation direction Vibration direction Light and electrons as a probe of matter Electromagnetic wave Murphy and Davidson,

3 INTERACTION OFELECTROMAGNETIC WAVES WITHMATTER Murphy and Davidson, 2013 LIGHT (ELECTROMAGNETIC WAVES) INTERACTS WITH MATTER Stained Specimen Medium Amplitude object Light is absorbed by parts of the specimen and so changed in brightness and colour Phase object Phase shift depends on the refractive index and thickness of the object. 2 ( n 2 n1 ) t Typically Phase shift of livingcells is /4, not visible for human eyes courtesy Heiko Gäthje, Olympus Europe SE & Co. KG 3

4 ORGANISMS ARE LARGE SAMPLE PREPARATION CHOOSING THE RIGHT TOOL FOR IMAGING Typical Organisms in Life Science Solutions to allow imaging Thin samples (e.g. cells) or generate sections Sample preparation to allow processing of tissue without deterioration (e.g. fixation, freezing) Choice of imaging method depending on sample and resolution to be achieved Confocal laser scanning microscopy In vivo microscopy Selective plane illumination microscopy Superresolution techniques Transmission electron microscopy Scanning electron mciroscopy MICROSCOPY WITH LIGHT 4

5 FLUORESCENCE IN MICROSCOPY DNA Bax Mitochondria Cytochrome C DNA Bax Mitochondria Cytochrome C DNA Bax Mitochondria Cytochrome C WIDEFIELD MICROSCOPY 5

6 ESSENTIAL PARTS OF A MICROSCOPE Compound microscope Main parts of a microscope Illumination light source Focusing of light collector lenses and condensor Sample holder Objective Eyepiece Focus WIDEFIELD MICROSCOPY Problem Classical example of widefield imaging various points of an object are viewed simultaneously points of planes, other than the object plane, produce background illumination lowering the contrast stunningdepth field photographs/ Principle of widefield imaging Example from microscopy: histology 6

7 WIDEFIELD MICROSCOPY Problem Classical example of widefield imaging various points of an object are viewed simultaneously points of planes, other than the object plane, produce background illumination lowering the contrast stunningdepth field photographs/ Principle of widefield imaging Example from microscopy: histology FUNDAMENTALSETUP OF LIGHT MICROSCOPES 7

8 CONFOCAL LASERSCANNING MICROSCOPY CONFOCAL LASERSCANNING MICROSCOPY: CLSM Problem in widefield microscopy various points of an object are viewed simultaneously points of planes, other than the object plane, produce background illumination lowering the contrast Principle of widefield confocal imaging Solution 1. Illuminate a point in the object (using a focused laser which is scanned over the object hence the name laserscanning) 2. Introduce a pinhole in the image plane 3. The image plane is confocal to the focused object plane hence the name: confocal Motoneuronal endplate: widefield CLSM data data 8

9 TEMPORAL RESOLUTION NIPKOW DISK (SPINNING DISK TANDEM) SCANNING MICROSCOPY Problem Solution Speed in (single) point scanning confocal is limited! Scanning with multiple focused laser spots 1 to a few frames per seconds Schematics Illumination Detection campus.magnet.fsu.edu/tutorials MULTIPHOTON (LASERSCANNING) MICROSCOPY 9

10 MULTIPHOTON MICROSCOPY 3D sectioning without pinhole Schematics Excitation with one photon linearly depends on the amount of photons from the light source. Multiphoton excitation is proportional to the square of the intensity of light. Exponential drop in excitation out of focus in multiphoton excitation. No pinhole needed because no emitted light from out of focus. microsystems.com/science lab MULTIPHOTON MICROSCOPY Imaging in scattering tissue and deep into tissue Pulsed infrared laser ( nm) excites fluorochromes by multiphoton absorbtion Excitation in a small volume defined by the probability (densitiy of photons high) of a simultaneous multiphoton absorbtion All fluorescent photons provide useful signals. Helmchen and Denk, Nature Methods

11 MULTIPHOTON MICROSCOPY Kidney Brain Living mouse: kidney (Hoechst, 10kD dextran FITC, 150kD dextran Texas Red Helmchen, F., and W. Denk Deep tissue two photon microscopy. Nature methods. 2: LIGHTSHEET (SELECTIVE PLANE ILLUMINATION) MICROSCOPY 11

12 SELECTIVE PLANE ILLUMINATION MICROSCOPY LIGHTSHEET MICROSCOPY 3D Imaging with low phototoxicity and high speed Excitation of focal plane only Detection of whole plane (CCD parallel) Light sheet imaging technique Better signal to noise ratio Low phototoxicity 4D imaging Huisken J, Stainier D Y R Development 2009;136: SELECTIVE PLANE ILLUMINATION MICROSCOPY LIGHTSHEET MICROSCOPY Excitation of focal plane only Detection of whole plane (CCD parallel) Light sheet imaging technique Better signal to noise ratio Low phototoxicity 4D imaging Huisken J, Stainier D Y R Development 2009;136:

13 SUPERRESOLUTION MICROSCOPY SUPERRESOLUTION IMAGING Why superresolution imaging? Is there a limit in resolution that cannot be overcome? Why do we want to overcome the limit in resolution? 13

14 RESOLUTION LIMITS _ = (0.61 λ)/ _ = ( λ)/ ^2 These formula are used for the calculation of resolution in widefield microscopy. In other techniques like confocal laser scanning, multiphoton microscopy, etc slightly other formulas are used. SUPERRESOLUTION MICROSCOPY: STATISTICAL MICROSCOPY LIKE PALM, STORM, GSD PALM: PhotoActivated LightMicroscopy STORM: Stochastic Optical Reconstruction Microscopy GSD: Ground State Depletion microscopy stochastic photoswitching of fluorescent proteins where most of the molecules remain dark 14

15 STIMULATED EMISSION DEPLETION MICROSCOPY : STED In STED, an initial excitation pulse is focused on a spot. The spot is narrowed by a second, donut shaped pulse that prompts all excited fluorophores in the body of the donut to emit (this is the emission depletion part of STED). This leaves only the hole of the donut in an excited state, and only this narrow hole is detected as an emitted fluorescence. ELECTRON MICROSCOPY 15

16 THE TYPES OF ELECTRON MICROSCOPES Transmission electron microscope (TEM) Scanning electron microscope (SEM) The types of electron microscopes Transmission electron microscope (TEM) Electron beam Scanning electron microscope (SEM) Electron beam Specimen ~100 nm Projection Specimen Surface Hela Cells 16

17 Transmission electron microscope vs. Widefield light microscope Transmission electron microscope Widefield light microscope Illumination Condenser lens Specimen Objective lens Projector lens Final image Examples TEM Mouse cerebellum Microtubule Dendrite Synapse Ribosomes Golgi Mitochondrium Nucleus 500 nm Specimen courtesy of B. Sobottka, Institute of Experimental Immunology, University of Zurich 17

18 Examples TEM Mouse cerebellum Mitochondrium Lipid bilayer Golgi Nucleus 100 nm Scanning electron microscope vs. Confocal laser scanning microscope Scanning electron microscope Confocal laser scanning microscope Illumination Detector Lens system Beam scanner Lens system Specimen 18

19 Examples SEM Mouse kidney 500 µm Examples SEM Mouse kidney (glomerulus) 10 µm 19

20 LITERATURE AND ACKNOWLEDGMENTS Literature Fundamentals of light microscopy and electronic imaging, Douglas B. Murphy; Wiley Liss, 2001 ISBN X Light Microscopy in Biology A practical approach, A. J. Lacey; Oxford University Press, 2004 Light and Electron Microscopy, E. M. Slayter, H. S. Slayter; Cambridge University Press, Acknowledgments 20