Introduction to light microscopy

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1 Center for Microscopy and Image Anaylsis Introduction to light microscopy (an overview)

2 Microscopy with light

3 Components of a light microscope 1. Light source 2. Objective 3. Sample or specimen holder 4. Focusing mechanism 5. Lens for focusing light on specimen 6. Eyepiece / Camera

4 Interaction of light with matter Samples tissue Visible! mouse Visible! Visible! cells Not visible!

$Diffraction by matter Introduce optical elements to detect only diffracted light (dark$

5 IMAGING IN LIGHT MICROSCOPY Interaction of light with matter Absorbtion by matter Introduce dyes which absorb light (Histology) Phase shift by matter Introduce optical elements measuring the phase shift and transfer it to an absorbtion (phase contrast) Diffraction by matter Introduce optical elements to detect only diffracted light (dark field)

6 IMAGING IN LIGHT MICROSCOPY Interaction of light with matter Advantages: Very high contrast resulting in high sensitivity Tagging of specific entities possible Excitation / emission allows for various variants of microscopy techniques Absorbtion by matter Introduce dyes which absorb light -> Histology Absorbtion by matter Introduce dyes which absorb light and subsequently emit light -> Fluorescence

7 Resolution limits d xy = 0.61 λ NA d z = n λ NA 2 These formula are used for the calculation of resolution in widefield microscopy. In other techniques like confocal laser scanning, multiphoton microscopy, etc other formula are used.

8 Fluorescence in microscopy DNA Bax Mitochondria Cytochrome C DNA Bax Mitochondria Cytochrome C DNA Bax Mitochondria Cytochrome C

10 CONFOCAL LASERSCANNING MICROSCOPY: TRUE 3D MICROSCOPY Regular widefield fluorescence Problem: out of focus light Reduced contrast from out of focus light

12 CONFOCAL LASER SCANNING MICROSCOPY Excitation of fluorescence in sample: Notes: Sample is excited by a laser focused to a point

13 CONFOCAL LASER SCANNING MICROSCOPY Emission of fluorescence from sample: Notes: Sample is excited by a laser focused to a Point Emitted fluorescent from focus is focused to a point and then reaches a detector measuring the incoming fluorescent light. A computer records the amount of emitted light and computes an image point by point over time

CONFOCAL LASER SCANNING MICROSCOPY Excluding out of focus light: Notes: Sample is excited by a laser focused to a Point Emitted fluorescent from focus is focused to a point and then reaches a

14 CONFOCAL LASER SCANNING MICROSCOPY Excluding out of focus light: Notes: Sample is excited by a laser focused to a Point Emitted fluorescent from focus is focused to a point and then reaches a detector measuring the incoming fluorescent light. A computer records the amount of emitted light and computes an image point by point over time Emitted fluorescent from out-of-focus is also out-of- focus at pinhole and largely excluded from detector by the presence of the pinhole

15 CONFOCAL LASER SCANNING MICROSCOPY Excluding out of focus light: Notes: Sample is excited by a laser focused to a Point Emitted fluorescent from focus is focused to a point and then reaches a detector measuring the incoming fluorescent light. A computer records the amount of emitted light and computes an image point by point over time Emitted fluorescent from out-of-focus is also out-of- focus at pinhole and largely excluded from detector by the presence of the pinhole

Comparison of widefield and confocal microscopy d z = n λ

microscopy has a very high signal to noise ratio

well resolved 3D imaging (without any image processing) dz

16 Comparison of widefield and confocal microscopy d z = n λ NA 2 Image acquired with a widefield microscope Confocal microscopy has a very high signal to noise ratio (prominent in thick samples) Confocal microscopy allows well resolved 3D imaging (without any image processing) dz n n NA 2 em 2 2 n PH NA 2 Image acquired with a confocal microscope

17 Spinning disk microscopy Increase acquisition speed

18 Temporal resolution Nipkow disk (spinning disk tandem) scanning microscopy

19 Multiphoton microscopy Imaging deep into tissue

20 Multiphoton microscopy Imaging in scattering tissue All fluorescent photons provide useful signals. Helmchen and Denk, Nature Methods 2005

21 Multiphoton microscopy Deep tissue two-photon microscopy Helmchen and Denk, Nature Methods 2005

23 Light sheet microscopy Huisken J, Stainier D Y R Development 2009;136:

24 Light sheet microscopy Development, September 1, 2012vol. 139 no

25 Superresolution microscopy Structured illumination microscopy José María Mateos

$Super resolution microscopy Beyond the diffraction limit The common feature: switching fluorophores on and off$

26 Super resolution microscopy Beyond the diffraction limit The common feature: switching fluorophores on and off sequentially in time and space so that the signals can be recorded consecutively beneath the diffraction limit

27 SUPER RESOLUTION MICROSCOPY

28 SUMMARY Light (and electrons) interact weakly with biological matter Diffraction, phase differences, absorbtions contribute all to the image formation Widefield microscopy: a large volume and the whole plane imaged is illuminated Laserscanning microscopy: a spot of the sample is imaged. Images are formed by sequentially illuminating a whole plane Lightsheet microscopy: a plane is illumated orthogonal to the detection direction Superresolution microscopy: microscopy techniques surpassing the classical lightmi roscopy resolution, common them: switching fluorescent dyes / tags off and having only a subset in the bright state.

29 Literatur Thank you Fundamentals of light microscopy and electronic imaging, Douglas B. Murphy; Wiley-Liss, 2001 ISBN X (Sehr verständliches Buch mit allem nötigen Grundlagenwissen zu Lichtmikroskopie) Light Microscopy in Biology A practical approach, A. J. Lacey; Oxford University Press, 2004 (Einfache Beschreibung der Lichtmikroskopie mit praktischen Übungen und Anleitungen) Light and Electron Microscopy, E. M. Slayter, H. S. Slayter; Cambridge University Press, 1992 (Detailierte und oft mathematische Beschreibung der Licht und Elektronenmikroskopie. Gutes Referenzwerk) (Ausführliche und vorzügliche Beschreibung der Lichtmikroskopie mit Demonstrationen, sehr empfehlenswert)

Introduction to light microscopy

Introduction to light microscopy Center for Microscopy and Image Anaylsis Introduction to light Basic concepts of imaging with light Urs Ziegler ziegler@zmb.uzh.ch Microscopy with light 1 Light interacting with matter Absorbtion Refraction