Advanced Fluorescence & Confocal Microscopy 08/2007 Lecture by Dr. Dirk Lang Dept. of Human Biology UCT Medical School Room Phone:

Fluorescence Stokes 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 nm Fluorescein molecule Fluorescnece Intensity 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 Emission Filter Excitation Filter Confocal Microscope

Ethidium PE cis-Parinaric acid Texas Red PE-TR Conj. PI FITC 600 nm300 nm500 nm700 nm400 nm Common Laser Lines

How a Confocal Image is Formed Condenser Lens Pinhole 1 Pinhole 2 Objective Lens Specimen Detector Modified from: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989

Optical Sectioning

Z-Projection

Laser Confocal Scanning Microscopy ProsCons Allows for higher resolutionLimited 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 sectionsLoss of image intensity Better control for bleedthrough/autofluorescenceFairly expensive Faster than deconvolutionProne 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 S0S0 S’ 1 Energy S1S1 hv ex hv em 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

FRAP Intense laser Beam Bleaches Fluorescence Recovery of fluorescence 10 seconds 30 seconds Zero time Time %F

Energy Transfer: FRET Effective between Å only Emission and excitation spectrum must significantly overlap Donor transfers non-radiatively to the acceptor

Fluorescence Resonance Energy Transfer Intensity Wavelength Absorbance DONOR Absorbance Fluorescence ACCEPTOR Molecule 1Molecule 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 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 Antibodies on 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 Microscopy operates 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

Effect of image deconvolution: Immunolabelled axons in a wholemount nerve preparation

Deconvolution Microscopy ProsCons Allows for higher ResolutionComputationally intense if iterative Allow collection of stacks of image planesMinimal penetration of thick sections Hg Bulb allows for variety of filter combinations Sensitive to spherical aberrations Can get superior sensitivityCan amplify noise, produce artifacts Cheaper than confocalRequires 3D data set Pros and Cons of Deconvolution Microscopy RawDeconvolved

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)