5/4/2015 INTRODUCTION TO LIGHT MICROSCOPY. Urs Ziegler MICROSCOPY WITH LIGHT. Image formation in a nutshell. Overview of techniques

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1 INTRODUCTION TO LIGHT MICROSCOPY Urs Ziegler MICROSCOPY WITH LIGHT INTRODUCTION TO LIGHT MICROSCOPY Image formation in a nutshell Overview of techniques Widefield microscopy Resolution limits Light emission from molecules and fluorescent imaging Confocal laser scanning microscopy Fluorescence energy transfer Fluorescence recovery after photobleaching In vivo microscopy Selective plane illumination microscopy Superresolution techniques Correlative techniques light and electron microscopy 1

2 WIDEFIELD MICROSCOPY 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 Principle of widefield imaging Example from microscopy: histology 2

3 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 Principle of widefield imaging Example: fluoresc. histology motoneuronal of kidney section endplate FUNDAMENTAL SETUP OF LIGHT MICROSCOPES FLUORESCENCE IN MICROSCOPY DNA Bax Mitochondria Cytochrome C DNA Bax Mitochondria Cytochrome C DNA Bax Mitochondria Cytochrome C 3

4 FLUORESCENCE IN MICROSCOPY Advantages Jablonski scheme Very high contrast resulting in high sensitivity Tagging of specific entities possible Excitation / emission allows for various variants of microscopy techniques 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 laserwhich is scannedover the object hence the name laserscanning) 2. Introduce a pinhole in the image plane 3. The image plane is confocalto the focused object plane hence the name: confocal Motoneuronal endplate: widefield CLSM data data 4

5 FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING(FRAP) Principle of FRAP FRAP Movie and Measurement Image sample using confocal microscopy Bleach defined region using intense illumination Measure fluorescence intensity over time in the photobleached region Time for recovery of fluorescence is an indication for: Diffusion Mobility Binding TEMPORAL RESOLUTION NIPKOW DISK(SPINNING DISK TANDEM) SCANNING MICROSCOPY Problem Solution Speed in (single) point scanning confocal is limited! 1 to a few frames per seconds Scanning with multiple focused laser spots Schematics Illumination - Detection MULTIPHOTON (LASERSCANNING) MICROSCOPY 5

6 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 squareofthe intensityof light. Exponential drop in excitation out of focus in multiphoton excitation. No pinhole needed because no emitted light fromout of focus. 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 (densitiyof photons high) of a simultaneous multiphoton absorbtion All fluorescent photons provide useful signals. Helmchen and Denk, Nature Methods 2005 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:

7 LIGHTSHEET (SELECTIVE PLANE ILLUMINATION) MICROSCOPY 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:

8 5/4/2015 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? SOME CONCEPTS ABOUT IMAGE FORMATION 8

9 5/4/2015 SPATIAL RESOLUTION IN X,Y AND Z Theory 0.1 µm bead Implications: focal plane Objects smaller than the resolution limit of the chosen objective will always be 1Airy disk Reality Objects larger than the resolution limit of the chosen objective will always be the size of the object convolved with the optical transfer function Note: the optical transfer function is a function describing how the imaging is occurring in the microscope Crossection 1 µm RESOLUTION AND RAYLEIGH CRITERION Resolving power of microscope: = a) Single diffraction pattern b) Two Airy disks with maximum of one overlapping first minimum of the other objects just resolved c) Two Airy disks with maximum of one overlapping the second minimum objects well resolved 0.61 λ Concept: an image of an extended object consists of a pattern of overlapping diffraction spots Resolution: the larger the NA of the objective, the smaller the diffraction spots (airy disks). 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. 9

10 5/4/2015 RESOLUTION AND SIZE OF AIRY DISK SUPERRESOLUTION IMAGING Concept: an image of an extended object consists of a pattern of overlapping diffraction spots Resolution: the larger the NA of the objective, the smaller the diffraction spots (airy disks). Note: this theme of diffraction limited spots and their separation in space and time will again be used and taken up in superresolution microscopy. 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 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. 10

11 OUTLOOK: ELECTRONMICROSCOPY AND SUPERRESOLUTION NOT COVERED Technique TIRF: Total Internal Reflection Microscopy Structures around 100nm from the coverslip can be visualized (see image at bottom right) FRET: Fluorescence Resonance Energy Transfer Proximity of proteins can be accessed SIM: Structured illumination microscopy Superresolution technique achieving 2x higher resolution compared to widefield microscopy 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 andelectronmicroscopy, E. M. Slayter, H. S. Slayter; Cambridge University Press, 1992 Acknowledgments Jana Doehner Therese Bruggmann Gery Barmettler Andres Kaech Txema José María Mateos Melero Claudia Dumrese Dominik Haenni Bruno Guhl 11