Notes

Slide Show

Outline

1	Imaging in 3D: Confocal vs. NLO Microscopy?
2	Without Optical Sectioning Images Are Blurred
3	Some Names PMT Photo Multiplier Tube T-PMT Transmitted light PMT META Multichannel detector for spectral imaging/dye separation NDD Non-Descanned Detector FLIM Fluorescence Lifetime Imaging FRET Fluorescence (Förster) Resonance Energy Transfer FCS Fluorescence Correlation Spectroscopy
4	Microscopy Techniques Available at the Imaging Center
5	Techniques Coming to the Imaging Center
6	Components of a Laser Scanning Microscope
7	Confocal: The Pinhole Suppresses Out-of-Focus Light
8	Confocal: The Pinhole Suppresses Out-of-Focus Light
9	Confocal: The Pinhole Suppresses Out-of-Focus Light
10	NLO does Not Need Pinhole
11	Get 3D Information
12	The Principle of 2 (or Multi-) Photon Excitation
13	1 Photon Excitation (Confocal Microscope)
14	1 Photon Excitation (Confocal Microscope)
15	Multiphoton Excitation (Nonlinear Excitation, NLO)
16	Multiphoton Excitation (Nonlinear Excitation, NLO)
17	Multiphoton Excitation (Nonlinear Excitation, NLO)
18	NLO: Biocytin traced Nerve Fibers Nerve fibers traced with biocytin in slice culture Staining: avidin-AMCA Excitation: 770 nm (Coherent Chameleon) Detection: KP 685 nm (short pass) Pinhole open
19	Non-Descanned Detection
20	NLO: No Out of Focus Bleaching
21	Reduced Bleaching allows Quantification
22	Biological Material is Transparent to NIR-Light
23	NLO: Imaging Thick Samples Drosophila-embryo Exc.780nm Hoechst Projection of 120 slices 122 μm thick 521 μm x 305 μm
24	NLO Allows Life-Cell Imaging
25	Effect of to High Laser Power
26	Effect of to High Laser Power
27	2 Photon Excitation of Fluorescence Proteins
28	NLO: One Laser Wavelength Excites Multiple Dyes FluoCells prepared slide #3 (Molecular Probes) Staining: Alexa Fluor 488 wheat germ agglutinin Alexa Fluor 568 phalloidin DAPI Excitation: 790 nm (Coherent Chameleon) Detection: META 562 - 516 nm META 498 - 530 nm BP 435 - 485 nm Pinhole open
29	NLO: One Laser Wavelength Excites Multiple Dyes
30	Excitation Fingerprint of GFP-labeled Mouse Embryo
31	Excitation Fingerprint of GFP-labeled Mouse Embryo
32	Excitation Fingerprint of Unlabeled Mouse Embryo
33	Point Spread Function in XY and Z
34	Point Spread Function : Size of E. coli Bacterium
35	Point Spread Function and Resolution
36	High N.A. Increases Resolution
37	Shorter Wavelength Increases Resolution
38	Confocal vs. 2 Photon: Similar Resolution
39	Imaging in 3D: Confocal vs. NLO Microscopy?
40	Thanks ! Imaging Facility Cameron Cooper Danny Stark Jennifer Kasemeier Jessica Teddy Joel Schwartz Paul Kulesa Paul Rupp Rebecca McLennan Bill Neaves Earl Glynn Peter Baumann Teri Johnson