Advanced Fluorescence &

Confocal Microscopy

08/2007

Lecture by Dr. Dirk Lang

Dept. of Human BiologyUCT Medical School

Room 6.10.1Phone: 406-6419E-Mail: DIRK.LANG@UCT.AC.ZA

FluorescenceStokes Shift

– is the energy difference between the peak of the absorbence and the peak of the emission spectrum

495 nm 520 nm

Stokes Shift is 25 nmFluoresceinmolecule

Flu

ores

cnec

e In

tens

ity

Wavelength

Fluorescent Microscope

Dichroic Filter

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter

EPI-Illumination

Problems and Challenges

• Blur: Out of focus light decreases resolution

• Bleaching: Excited fluorophores react to become nonfluorescent

• Phototoxicity: Light can harm cells

• Background/Autofluorescence: Cells have fluorophores too. May look like the ones you want to examine!

• Bleedthrough: Broad peaks cross over into one another. One fluorophore comes off in 2 channels

Fluorescent Microscope

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter

Objective

Laser

Emission Pinhole

Excitation Pinhole

PMT

EmissionFilter

Excitation Filter

Confocal Microscope

Ethidium

PE

cis-Parinaric acid

Texas Red

PE-TR Conj.

PI

FITC

600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488 Common Laser Lines

How a Confocal Image is Formed

CondenserLens

Pinhole 1 Pinhole 2

ObjectiveLens

Specimen

Detector

Modified from: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989

Optical Sectioning

Z-Projection

Laser Confocal Scanning Microscopy

Pros Cons

Allows for higher resolution Limited EX peaks on lasers

Allow collection of stacks of image planes and 3D reconstruction

Phototoxicity

(up to a 40degree C temp jump at focal point)

Laser penetrates somewhat thick sections Loss of image intensity

Better control for bleedthrough/autofluorescence Fairly expensive

Faster than deconvolution Prone to Photobleaching

Precise Laser Positioning (FRAP)

Pros and Cons of Confocal Microscopy

No Pin Hole 50um Pin Hole

Two-Photon Confocal Microscopy

Two-photon excitation

S0

S’

1

En e

r gy

S1

hvex hvem

Two photons of half the necessary excitation energy (double the wavelength) arrive simultanously

Excitation, as with a single photon of high energy (short excitation wavelength)

Applications

Release of “Caged” Compounds

UV Beam

Release of “Cage”

Culture dish

FRAPIntense laser BeamBleaches Fluorescence

Recovery of fluorescence

10 seconds 30 secondsZero time

Time

%F

Energy Transfer: FRET

• Effective between 10-100 Å only

• Emission and excitation spectrum must significantly overlap

• Donor transfers non-radiatively to the acceptor

Fluorescence Resonance Energy

Transfer

Inte

nsi

ty

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence Fluorescence

ACCEPTOR

Molecule 1 Molecule 2

Applications of FRET

Fluorescent Proteins• GFP - Green Fluorescent Protein

– GFP is from the chemiluminescent jellyfish Aequorea victoria

– excitation maxima at 395 and 470 nm (quantum efficiency is 0.8) Peak emission at 509 nm

– contains a p-hydroxybenzylidene-imidazolone chromophore generated by oxidation of the Ser-Tyr-Gly at positions 65-67 of the primary sequence

– Major application is as a reporter gene for assay of promoter activity

– requires no added substrates– now modified forms available: yellow, red, cyan and blue

fluorescent proteins– Often used in FRET

Multiphoton Microscopy in Neuroscience:

Axon pathfinding in embryogenesis and regeneration

Signal transduction in growing axons

Analysis of brain cytoarchitecture

Intravital imaging of neuronal signalling

Analysis of axon pathfinding in live zebrafish brain

Micropipette(antibodies or morpholinos)

Hindbrain

Injection of Function-blocking Antibodieson the Retinotectal Projection:

Signal Transduction in Neuronal Growth Cones:

Does Nogo-A Receptor (red) signal through raft domains (green)?

FRET

Analysis of Brain Cyto-Architecture:

Deep z-stack acquisition and stereology in live or fixed brain tissues allows detailed analysis of developmental or pathological changes:

Cell numbers

Neurone morphology and connectivity

Subcellular changes (e.g. synaptic spines)

Multiphoton microscopy of brain in vivo:Alzheimer plaques and vascularisation

(Christie et al., 2001)

Miniaturization of multiphoton microscope for brain imaging of live animals

(Helmchen et al., 2001)

Analysis of Neuronal Signalling:

Rapid scanning techniques in the study of brains or live brain slices allow intravital analysis of physiological processes:

Synaptic structure and physiology

Calcium dynamics

Receptor mapping and trafficking

Non-Confocal Methods of Removing

„Blur“

Deconvolution Microscopyoperates on the principle of a point spread function (PSF). As one moves away from focal plane in which an object lies the light from it will spread in a predictable manner

Image Deconvolution

Image Deconvolution can produce optical sections of near-confocal quality on a conventional fluorescence microscopy system

Deconvolution Microscopy

Pros Cons

Allows for higher Resolution Computationally intense if iterative

Allow collection of stacks of image planes Minimal penetration of thick sections

Hg Bulb allows for variety of filter combinations

Sensitive to spherical aberrations

Can get superior sensitivity Can amplify noise, produce artifacts

Cheaper than confocal Requires 3D data set

Pros and Cons of Deconvolution Microscopy

Raw Deconvolved

Structured Illumination – “Apotome”

Structured Illumination – “Apotome”

High-Resolution Fluorescence Microscopy

Resolution Limits of Fluorescence Microscopy

(Jaiswal & Simon, 2007)

Total Internal Reflection Fluorescence (TIRF)

Pushing the Resolution Limit…

(Hell, 2007)

Pushing the Resolution Limit…

(Donnert et al., 2006)