Center for Microscopy and Image Anaylsis

Introduction to light

microscopy

(an overview)

Microscopy with light

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

tissue

cells

Interaction of light with

matter

Samples

mouse

Visible!Visible!

Visible!

Not

visible!

IMAGING IN LIGHT MICROSCOPY

Interaction of light with matter

Absorbtion by matter

Introduce dyes whichabsorb light (Histology)

Phase shift by matter

Introduce optical elementsmeasuring the phase shiftand transfer it to an absorbtion (phase contrast)

Diffraction by matter

Introduce optical elementsto detect only diffractedlight (dark field)

IMAGING IN LIGHT MICROSCOPY

Interaction of light with matter

Absorbtion by matter

Introduce dyes whichabsorb light-> Histology

Absorbtion by matter

Introduce dyes whichabsorb light andsubsequently emit light-> Fluorescence

Advantages:Very high contrast resulting in high sensitivityTagging of specific entities possibleExcitation / emission allows for various variants of microscopy techniques

Resolution limits

𝑑𝑥𝑦 =0.61 × λ

𝑁𝐴

𝑑𝑧 =𝑛 × λ

𝑁𝐴2

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.

Fluorescence in

microscopy

DNA

Bax

Mitochondria

Cytochrome C

DNA

Bax

Mitochondria

Cytochrome C

DNA

Bax

Mitochondria

Cytochrome C

Regular widefield fluorescence Problem: out of focus light

CONFOCAL LASERSCANNING MICROSCOPY: TRUE 3D MICROSCOPY

Reduced contrast from out of focus light

http://smokingdesigners.com/34-stunning-depth-field-photographs/

CONFOCAL LASER SCANNING MICROSCOPY

Excitation of fluorescence in sample: Notes:

Sample is excited by a laser focused to a

point

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 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

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

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)Image acquired with a

widefield microscope

Image acquired with a

confocal microscope

𝑑𝑧 =𝑛 × λ

𝑁𝐴2

NA

PHn

NAnn

em 288.0dz

2

22

2

Spinning disk microscopy

Increase acquisition speed

Temporal resolution –

Nipkow disk (spinning disk

– tandem) scanning

microscopy

http://zeiss-campus.magnet.fsu.edu/tutorials

Multiphoton microscopy

Imaging deep into tissue

Multiphoton microscopy

Imaging in scattering tissue

All fluorescent photonsprovide useful signals.

Helmchen and Denk, Nature Methods2005

Multiphoton microscopy

Deep tissue two-photon

microscopy

Helmchen and Denk, NatureMethods 2005

Light sheet microscopyHuisken J , Stainier D Y R Development 2009;136:1963-1975

Light sheet microscopy

Development, September 1, 2012vol. 139 no. 17 3242-3247

José María Mateos

Superresolutionmicroscopy

Structured illuminationmicroscopy

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

SUPER RESOLUTION MICROSCOPY

SUMMARY

• Light (and electrons) interact weakly with biological matter

• Diffraction, phase differences, absorbtions contribute all tothe 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 thedetection direction

• Superresolution microscopy: microscopy techniquessurpassing the classical lightmi roscopy resolution, commonthem: switching fluorescent dyes / tags off and having only a subset in the bright state.

Thank you

Literatur

Fundamentals of light microscopy and electronic imaging, Douglas B.

Murphy; Wiley-Liss, 2001

ISBN 0-471-25391-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)

http://microscopy.fsu.edu/primer/index.html (Ausführliche und vorzügliche

Beschreibung der Lichtmikroskopie mit Demonstrationen, sehr empfehlenswert)