Special Techniques 1. Mark Scott FILM Facility
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1 Special Techniques 1 Mark Scott FILM Facility
2 SPECIAL TECHNIQUES Multi-photon microscopy Second Harmonic Generation FRAP FRET FLIM In-vivo imaging
3 TWO-PHOTON MICROSCOPY Alternative to confocal and deconvolution microscopy Two photons of half the energy combine Emission equivalent to being excited with a single photon laser
4 TWO-PHOTON MICROSCOPY Leica SP5 Upright Leica SP5 Inverted Newport Spectra-Physics Mai Tai Laser nm
5 TWO-PHOTON MICROSCOPY Much higher energy required Pulsed laser ~1,000,000x greater Femtosecond pulses High power per pulse Low average power Wave-form power output (2.7W peak)
6 TWO-PHOTON MICROSCOPY No pinhole required (no out of focus light excited) NDD detectors higher sensitivity (less light path = less light lost) Less photo-bleaching/photo-toxicity (lack of excitation above and below the focal plane) Less scattering of light (Red vs Blue) Deeper penetration Protein uncaging specialist application Image resolution/thin specimens
7 TWO-PHOTON MICROSCOPY
8 TWO-PHOTON STAIN SELECTION Spectral profile differs (peak may change) Dye Single Photon Ex/Em Two Photon Ex GFP 488/507nm 860<960nm DsRed 543/580nm 900<1064nm DAPI 350/470nm 780>820nm FITC 490/525nm 780>820nm CY3 550/570nm 780nm CY5 649/670nm 780>820nm
9 THREE-PHOTON EXCITATION Only 10x more power needed from 2-photon, not the million-fold increase going from 1- photon to 2-photon excitation. Requires 3 photons ~1/3 of normal excitation 1020nm 340nm (510nm)
10 SECOND HARMONIC GENERATION (Frequency Doubling) Photon's interact to form single photons (double the wavelength) Only non-centrosymmetric structures
11 SECOND HARMONIC GENERATION Gives structural information without staining Useful for morphological information in whole tissue Gain structural information of surrounding when tracking cells in-vivo without needing staining (live specimens) Can be combined with other microscopy techniques (Anisotropic imaging, Autofluorescence, Lifetime imaging)
12 SECOND HARMONIC GENERATION Wavelength dependency for different structures Useful to distinguish between tissue composition without staining (with other techniques) Commonly imaged structures: Collagen I/III(840/930nm) Elastin (740nm) NAD(P)H (680nm)
13 760nm 840nm 930nm
14 760nm 840nm 930nm
15 SHG 840nm Collagen I antibody staining
16 Pancreatic Islets showing two-photon SHG generation of intracellular NAD(P)H
17 SECOND HARMONIC DYES SHRIMPS (Second Harmonic Radiation Imaging Probes) Development of structurally significant molecules Specific wavelengths of SHG generation used to excite Structural interference/amplification Excellent possibilities for extreme long term imaging
18 PHOTO-BLEACHING Photochemical destruction of fluorescent molecules Differs between fluorophores 2P excitation reduces this bleaching by limiting exposure
19 FLUORESCENCE RECOVERY AFTER PHOTO- BLEACHING FRAP Bleaching of specific areas Diffusion/transport experiments
20 FORSTER RESONANCE ENERGY TRANSFER FRET Transfer of energy between fluorophores Typically under 15nm Quantitative measure of co-localisation Only molecules within strict distances will FRET FRET efficiency determined by distance between molecules
21 FRET
22 FRET Specific fluorophore pairings Based on spectral overlap Emission of Donor must overlap Excitation of Acceptor FRET efficiency determines Forster distance Common Donor-Acceptor Pairs: FITC Rhodamine (4.9nm) CY3 CY5 (>5.0nm) PE CY5 (7.2nm) CY5 CY5.5 (>8.0nm)
23 FRET Photobleaching effects the FRET transfer Ratio-metric analysis Acceptor bleached, Donor emission increases Two-photon bleaching more precise Only bleaches in plane of focus so able to specify the 3D area of bleaching much more precisely
24 FRET Donor Pre-bleach Acceptor Pre-bleach Donor Post-bleach Acceptor Post-bleach
25 FLUORESCENCE LIFETIME IMAGING MICROSCOPY FLIM Pulsed mode-locked laser (2P FILM Confocal 3 and 4) Measure of excitation decay
26
27 FLIM FOR FRET Lifetime of fluorophores effected by energy transfer Lifetime also changes with conditions of sample
28 IN-VIVO IMAGING Confocal 3 Imaging whole tissue or animals Problematic due to light scatter, movement...
29 IN-VIVO IMAGING High quality lens 25x water immersion lens 0.95 N.A. High confocal zoom possible Allows low power rapid scanning and high power area imaging
30 IN-VIVO IMAGING 63x Objective No zoom 25x Objective 6x zoom
31 IN-VIVO IMAGING Ensure specimen does not move Longer scan time possible Reproducibility increased
32 IN-VIVO IMAGING 2P laser excitation deeper penetration, less scatter, less damage... Steady specimen holder increased scan time possible Multiple imaging techniques able to be combined
33 IN-VIVO IMAGING
34 Thank You...
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