Super Resolution Microscopy STORM/PALM Bo Huang Department of Pharmaceutical Chemistry, UCSF CSHL Quantitative Microscopy, 1/31/211 Looking into microscopic world of life 1 µm 1 µm 1 nm 1 nm 1 nm 1 Å Naked eye: ~ 5 1 μm 1 µm 1 µm 1595, Zaccharias and Hans Janssen First microscope, 9x magnification Antonie van Leeuwenhoek (1632 1723), 2x Ernst Abbe (184 195) The physical diffraction limit 1 nm 1 nm Compound microscope >1x 1 nm 1 Å 16 17 18 19 2 1
The diffraction barrier 1 µm 1 µm Diffraction limit: ~ 25 nm lateral ~ 6 nm axial 1 nm 1 nm 1 nm 1 μm 1 Å Naked eye: ~ 5 1 μm 1 µm 1 µm 1 nm 1 nm 1595, Zaccharias and Hans Janssen First microscope, 9x magnification Antony Van Leeuwenhoek (1632 1723), 2x Compound microscope >1x D d Diffraction S Ernst Abbe (184 195) The physical diffraction limit Super resolution Deconvolution Optical 4 Pi microscopy Microscopy... 1 nm 16 17 18 19 2 1 Å Super resolution optical microscopy Hell, Science, 27; Hell, Nat Methods, 28 Rust, Bates & Zhuang, Nat Methods, 26 Betzig et al., Science, 26 Hess, Girirajan and Mason, Biophys. J., 26 Gustafsson, PNAS., 25 STED SSIM STORM/(F)PALM 2
The single molecule switching approach Rust, Bates & Zhuang, Nat Methods, 26 Betzig et al., Science, 26 Hess, Girirajan and Mason, Biophys. J., 26 STORM/(F)PALM STORM: Super resolution by localization Fluorescence image Underlying structure Single molecule image FWHM 32 nm Single molecule localization precision 1 photon D d / N 1 photons 1 photons 1 photons 3
STORM: Super resolution by localization Fluorescence image Raw images Deactivation Activation STORM Image Localization Photoswitchable molecules 2x real time Stochastic Optical Reconstruction Microscopy = STORM Also named as PALM (Betzig et al., Science, 26) and FPALM (Hess et al., Biophys. J. 26) Rust, Bates & Zhuang, Nat. Methods, 26 Bates, Huang, Dempsey & Zhuang, Science, 27 Reconstructed from 4, frames, 3,35,37 localization points 5 μm B SC 1 cell, Microtubules stained with anti β tubulin Cy3 / Alexa 647 secondary antibody Bates, Huang, Dempsey and Zhuang, Science, 27 5 nm 5 μm Bates, Huang, Dempsey and Zhuang, Science, 27 4
FWHM 32 nm 15 FWHM = 24 nm stdev = 1 nm Number of points 1 5-8 -4 x (nm) 4 8-4 -8 8 4 y (nm) 5 μm Bates, Huang, Dempsey and Zhuang, Science, 27 In a 2D world Satellite image of??? Google maps 5
3D Imaging: Localization in the Third Dimension (x, y, y) z) In focus z (nm) 4 Scan z = 6 to 6 nm 2 2γ 2 EMCCD 4 Huang, et al., Science, 28 3D Imaging of the Microtubule Network 6 z (nm) 3 Scale bar: 2 nm 5 μm Huang, Wang, Bates and Zhuang, Science, 28 3D Imaging of the Microtubule Network 6 z (nm) Small, isolated clusters FWHM 22 nm 28 nm 55 nm 3 5 μm Huang, Wang, Bates and Zhuang, Science, 28 6
Other 3D localization method Astigmatic imaging (x, y, y) z) z (nm) 4 Bi plane imaging 2 2 4 Huang et al., Science 28 SLM Double helical PSF Juette et al., Nat Methods 28 EMCCD EMCCD z (nm) 9 5 6 14 Pavani et al., PNAS 29 Better 3D by two objective interference Shtengel et al., PNAS 29 Photoswitchable fluorophores 7
Photoswitchable probes readily available 4 5 6 7 nm Cyanine dye + thiol system Alexa647 Cy5 Cy5.5 Cy7 Bates et al., 25, Bates et al., 27, Huang et al., 28 Rhodamine dye + redox system Alexa488 Atto565 Alexa532 Atto59 Atto52 Alexa568 Atto655 Atto7 Heilemann et al., 29 Photoactivatible fluorescent proteins PS CFP2 PA GFP Dronpa EYFP Dendra2 Dreiklang meosfp2 PAmCherry PAtagRFP Reviews: Lukyanov et al., Nat. Rev. Cell Biol., 25 Lippincott Schwartz et al., Trends Cell Biol., 29 Photoswitching of red cyanine dyes Fluorescent 65 nm N + N + thiol Cy5 / Alexa 647 photoactivation Deactivation 36 nm 65 nm Dark Bates eta l., PRL 25, Bates et al., Science 27, Dempsey et al., JACS 29 Multi color Imaging 8
B SC 1 cell, anti β tubulin Commercial Alexa 647 secondary antibody 5 nm 5 μm Multicolor STORM/PALM 561 nm 642 nm meosfp2 Alexa647 575 nm 675 nm 635DRLPXR meos2 tubulin Alexa 647 anti β tubulin Drosophila S2 cells 2 µm 9
Alexa 647 anti β tubulin meos2 tubulin Multicolor STORM/PALM: Emission n 1 n 2 5% SRA545 + 5% SRA617? 1% SRA577? n 1 = n 2 Single molecule detection! 3 color imaging with one excitation wavelength and two detection channels Bossi et al., Nano Lett, 28 Multicolor STORM/PALM: activation Fluorescent 65 nm Cy5 Cy3 photoactivation Deactivation 36 nm 65 nm 532 nm Dark Cy5 Cy3 1
Controlling the activation of Cy5 Fluorescent 65 nm Cy5 photoactivation Deactivation Cy3 Cy2 Alexa 45 Fluorescence 532 457 45 nm Activation pulses Dark 1 time (s) 2 3 Cy3 / Alexa 647: Clathrin Cy2 / Alexa 647: Microtubule Crosstalk subtracted Laser sequence Cy3 A647 Cy2 A647 532 457 1 μm Bates, Huang, Dempsey and Zhuang, Science, 27 Multicolor imaging approaches By emission wavelengths Simple fluorophores Low crosstalk Continuous imaging Multi channel detection optics Needs nanometer scale image alignment By activation wavelengths Dye pairs Crosstalk from nonspecific activation Laser sequences Single channel detection Images naturally aligned 11
The limit of Super Resolution Unbound theoretical resolution D = d Diffraction S STORM/PALM S N 6, photons 5 nm 1, photos during Cy5 life time < 1 nm Effective resolution: Probe matters Antibodies: ~ 1 nm Fluorescent Proteins: ~ 3 nm Small fluorophores: ~ 1 nm Measured FWHM by antibody: Actual microtubule diameter: Measured FWHM by FP: 58 nm 25 nm 43 nm 1 nm 1 nm 1 nm ~ 6 photons < 1 photons ~ 6 photons 12
Fluorescent protein vs. Antibody Fluorescent protein fusion Live sample labeling High specificity High labeling efficiency Genetically encoded Lower S/N Multicolor imaging so far challenging Antibody immunofluorescence Fixed sample Potential nonspecific labeling Lower labeling efficiency Labeling endogenous proteins High signal = high localization precision More versatile for multicolor imaging Effective resolution: Density matters Frames for image reconstruction: 2 5 1, 5, 4, Nyquist criteria Point to to point distance < ½ Feature size This labeling density limit of resolution applies to all fluorescence microscopy methods Effective resolution: Contrast matters Fluorescent 65 nm e.g. 1% 1% means Sparsely labeled sample photoactivation Deactivation Densely labeled sample 65 nm Dark e.g. 99% 13
Effective resolution: Contrast matters Fluorescent 65 nm e.g. 1% 1% means Homogeneous sample Microtubule 65 nm photoactivation Dark Deactivation e.g. 99% Common blinking dyes: >3% Cy5 + mercaptoethylamine:.1.2% meosfp:.1% Average point to point distance: 4 nm 14 nm Live Cell Imaging Live cell STORM meos2 labeled microtubule in live S2 cells Time step 4 Camera image frames Time step 3 Time step 2 Time step 1 1 µm 6 frames/sec 12 frames/step (2 sec time resolution) 5x real time 14
Live cell imaging of plasma membrane DiD stained plasma membrane Diameter: 62 nm 1 frames, 1 sec total time 1 μm 12 frames / sec, 3 frames (25 sec) 1x real time 3 mm mercaptoethylamine 1 μm Spatial temporal resolution trade off FWHM 32 nm Assuming: 1 molecule occupies 5 5 nm On average.1 point /.25 µm 2 frame 7 nm resolu on 2 frames 1 fps = 2 sec time resolution What s next? More friendly to biologists? Deeper (thinker) 3D imaging? Molecular scale resolution? Live cell single molecule dynamics? Combining with other single molecule methods? 15