Advanced Fluorescence & Confocal Microscopy 08/2007 Lecture by Dr. Dirk Lang Dept. of Human Biology...
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Transcript of Advanced Fluorescence & Confocal Microscopy 08/2007 Lecture by Dr. Dirk Lang Dept. of Human Biology...
Advanced Fluorescence &
Confocal Microscopy
08/2007
Lecture by Dr. Dirk Lang
Dept. of Human BiologyUCT Medical School
Room 6.10.1Phone: 406-6419E-Mail: [email protected]
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)