Fluorescence Nanoscopy
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1 Fluorescence Nanoscopy Keith A. Lidke University of New Mexico panda3.phys.unm.edu/~klidke/index.html
2 Optical Microscopy 30 µm
3 Fluorescent Probes Michalet et. al. Science. Vol 307 p. 538 QD cell membrane 5 nm protein zeiss-campus.magnet.fsu.edu/articles/probes/fpintroduction.html
4 Fluorophore Energy Levels
5 Epi-Fluorescence Setup Objective Lens Dichroic Mirror Sample Microscope Slide Single Mode Fiber Emission Filter Tube Lens 488 nm Laser CCD Camera
6 Imaging in a light microscope Point Spread Function (PSF) Point Source Objective Lens Tube Lens n=1.5 NA=1.4 lambda=.55 CCD Camera
7 Point Spread Function and Optical Transfer Function PSF OTF k max =2 NA/λ OTF is the Fourier Transform of the PSF
8
9 Wide Field Immunofluorescence
10 Wide Field Immunofluorescence 1 µm
11 Single-Molecule Localization-Based Super-Resolution f(palm), (d)storm, Pointillism, GSDIM, PAINT, Blink Microscopy,
12 Single Molecule Localization λ em =emission wavelength γ=collection efficiency A= emission rate Ober, R.J., et al, Biophysical Journal, (2): p
13 Single Molecule Super-Resolution Concept Single Molecule Localization Photo-Activation Photo-Switching or Blinking t
14 Actin in HeLa Cell
15 Mechanisms for on / off switching (f)palm = (fluorescence) Photo-Activation Localization Microscopy Betzig/ H.Hess, Science 2006 S. Hess, Biophysical Journal 2006 PA-FP (Kaede, meos,...) photobleaching
16 Mechanisms for on / off switching STORM = STochastical Optical Reconstruction Microscopy (Zhuang, Nat. Methods 2006) dimer construct (Cy3/Cy5,...)
17 Mechanisms for on / off switching GSDIM = Ground State Depletion followed by single molecule IMaging (Hell, PRL 2007)
18 Mechanisms for on / off switching dstorm = "direct" STORM (Heilemann, Angew. Chem. 2008) any fluorophore O 2 O 2 reduction fluorescence oxidation
19 Image Processing Pipeline
20 3D Localization Techniques Dual Focal Plane -1000nm -500nm 0nm Juette, Nat. Meth.,2008 Ram, Biophys. J., 2008 Astigmatism 500nm Double-helix PSF Huang, Science, 2008 Pavani, PNAS, 2009
21 Dealing with reality: Aberrations Image of a point source at various defocus Pupil Function Data Model Data Model 10 um
22 Dual focal plane setup Emission light from microscope
23 Dual Focal Plane 3D SR Data
24 3D Super-resolution using Dual Focal Planes
25 Can we image multiple targets with a single fluorophore?
26 Crosstalk NaBH4 and Photo-bleaching Original SR reconstruction Post-photodestruction No significant cross-talk
27 Brightfield registration and alignment Sequential imaging of β-tubulin and α-tubulin, overlay using BF alignment α-tub Photodestruction β-tub
28 Localization of EGFR within clathrin-coated vesicles
29 Four color super-resolution imaging with a single fluorophore, and negligible crosstalk Valley CC, Liu S, Lidke DS, Lidke KA (2015) Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore. PLoS ONE
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