introduction to light microscopy - zmb uzh to light microscopy.pdf · introductiontolight...

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INTRODUCTION TO LIGHT MICROSCOPY

Urs Ziegler

[email protected]

MICROSCOPY WITH LIGHT

Image formation in a nutshell

Resolution limits

Light emission from molecules and

fluorescent imaging

Overview of techniques

Widefield microscopy

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

INTRODUCTION TO LIGHT MICROSCOPY

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WIDEFIELD MICROSCOPY

Compound microscope Main parts of a microscope

● Illumination - light source

● Focusing of light – collector lenses and

condensor

● Sample holder

● Objective

● Eyepiece

● Focus

ESSENTIAL PARTS OF A MICROSCOPE


Principle of widefield imaging

Problem Classical example of widefield imaging

Example from microscopy: histology

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various points of an object are viewed

simultaneously

points of planes, other than the object

plane, produce background illumination

lowering the contrast

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Example: fluoresc. motoneuronal endplate



Problem Classical example of widefield imaging

Example: histology of kidney section

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simultaneously




FUNDAMENTAL SETUP OF LIGHT MICROSCOPES

FLUORESCENCE IN MICROSCOPY

DNA

Bax

Mitochondria

Cytochrome C

DNA

Bax

Mitochondria

Cytochrome C

DNA

Bax

Mitochondria

Cytochrome C

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Advantages

Very high contrast resulting in high

sensitivity

Tagging of specific entities possible

Excitation / emission allows for various

variants of microscopy techniques

Jablonski scheme

FLUORESCENCE IN MICROSCOPY

CONFOCAL LASERSCANNING MICROSCOPY

CONFOCAL LASERSCANNING MICROSCOPY: CLSM


Problem in widefield microscopy Solution

Motoneuronal endplate: widefield data


simultaneously




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

Principle of confocal imaging Motoneuronal endplate: CLSM data

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Principle of FRAP

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

FRAP Movie and Measurement

FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING (FRAP)

TEMPORAL RESOLUTION – NIPKOW DISK (SPINNING DISK – TANDEM) SCANNING MICROSCOPY

Schematics

Problem Solution

Illumination

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

Speed in (single) point scanning confocal is

limited!

→ 1 to a few frames per seconds

Scanning with multiple focused laser spots

Illumination - Detection

MULTIPHOTON (LASERSCANNING) MICROSCOPY

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3D sectioning without pinhole

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.

Schematics

MULTIPHOTON MICROSCOPY

http://www.leica-microsystems.com/science-lab


Imaging in scattering tissue and deep into tissue

Pulsed infrared laser (700-1500nm) 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

2005

Kidney Brain


Helmchen, F., and W. Denk. 2005. Deep tissue two-photon microscopy.

Nature methods. 2:932-40.Living mouse: kidney (Hoechst, 10kD dextran FITC,

150kD dextran Texas Red

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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;136:1963-1975

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;136:1963- 1975

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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

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SPATIAL RESOLUTION IN X,Y AND Z

Implications:

Objects smaller than the resolution

limit of the chosen objective will

always be 1Airy disk

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

1 µm

Crossection

focal plane

0.1 µm bead

Reality

Theory

1 µm

Crossection

RESOLUTION AND RAYLEIGH CRITERION

Resolving power of microscope:

� = 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).

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

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, etcslightly other formulas are used.

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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.

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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

Fundamentals of light microscopy and

electronic imaging, Douglas B. Murphy; Wiley-

Liss, 2001

ISBN 0-471-25391-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, 1992

http://microscopy.fsu.edu/primer/index.html

Acknowledgments

Jana Doehner

Therese Bruggmann

Gery Barmettler

Andres Kaech

‚Txema‘ José María Mateos Melero

Claudia Dumrese

Dominik Haenni

Bruno Guhl

LITERATURE AND ACKNOWLEDGMENTS

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