Super Resolution Microscopy Structured Illumination

Transcription

1 Super Resolution Microscopy Structured Illumination Bo Huang Department of Pharmaceutical Chemistry, UCSF CSHL Quantitative Microscopy, 10/31/ years to extend the resolution Confocal microscopy (1957) Near field scanning optical microscopy (1972/1984) Multiphoton microscopy (1990) 4 Pi microscopy / I 5 M ( ) Structured illumination microscopy (2000) Negative refractive index (2006) Near field scanning optical microscopy Excitation light β 2 adrenergic receptor clusters on the plasma membrane Optical fiber ~ 50 nm Aperture Sample Ianoul et al.,

2 4 Pi / I 5 M d 2 NA NA = n sin Major advantage: Similar z resolution as x y resolution Patterned illumination Detector Detector = x Excitation Detection x Structured Illumination Microscopy (SIM) 9 images Reconstruction WF SIM 2 = Gustafsson, J Microscopy

3 Being (slightly) more rigorous about SIM Fourier transform and spatial frequencies =? Fourier transform and spatial frequencies + Summed image 3

4 Fourier transform and spatial frequencies + + Summed image Fourier transform and spatial frequencies Discrete spatial frequencies Summed image G(x) = Σ F(k) sin(k x) Fourier transform and spatial frequencies y k y x k x Original Image (real space) Fourier transform (frequency space) 4

5 Fourier optics and microscope resolution Sample Objective Back focal plane f f α k = f sinα Fourier optics and microscope resolution Sample Objective Back focal plane f f x I(x) α A(k) k Phase delay from the mid point Δφ = x sinα / 2πλ = x k/2πλf assuming refractive index = 1 Light intensity at the sample plane Fourier Transform! I(x) = ΣA(k) sin(δφ) = Σ A(k)sin (xk/2πλf) Fourier optics and microscope resolution Sample Objective Back focal plane f f α k max Spatial frequency = k / 2πλf Size of the back focal plane k max = f sinα max = f NA Resolution = λ / 2NA 5

6 Extending the measurable freq. range Excitation(x) Sample(x) = Observed Signal(x) x = Freq = 30 Freq = 25 Freq = 55 & 5 sina sinb = (cos (A B) cos(a + B)) / 2 Extending the measurable freq. range Excitation(x) Sample(x) = Observed Signal(x) x = Freq = 30 Freq = 25 Freq = sina sinb = (cos (A + B) cos(a B)) / 2 Extending the measurable freq. range k y k ex k max k y k max k k ex k max kex k x k max k x k + k ex k max 6

7 Extending the measurable freq. range k y k ex k max k y k max k k ex k max kex k x k max k x Gustaffson et al., J. Microscopy, 2000 k + k ex k max Generating the illumination pattern Sample plane Objective Back focal plane Grating Spatial light modulator 3D SIM: better resolution + optical sectioning Schermellech et al., Science 2008, Gustafsson et al., Biophys J

8 Multicolor SIM Same as conventional fluorescence microscopy! Live imaging with SIM Kner, Chhun et al., Nat Methods, 2009 Shao et al., Nat Methods, 2011 The diffraction limit still exists 1 d 2 2NA Confocal 4Pi / I 5 M SIM 8

9 Breaking the diffraction barrier Breaking the diffraction barrier Confocal 4Pi / I 5 M SIM Stimulated Emission Depletion (STED) Detector Excitation Fluorescence h Stimulated Emission 2h FL 0 FL0 FL 1 I STED / I s Send to a dark state 0 I s 9

10 STED microscopy Detector Excitation Fluorescence Light modulator Depletion Stimulated Emission Excitation Excitation STED pattern = Effective PSF? Hell 1994, Hell 2000 Saturated depletion D 1 1 I / I NA s 2 STED pattern Saturated Depletion I STED = I2 100 S I S I S I S zero point STED images of microtubules Wildanger et al.,

11 3D STED Harke et al., Nano Lett, 2008 Muticolor STED Excitation Excitation 2 STED STED 2 2 color isosted resolving the inner and outer membrane of mitochondria 1 µm Schmidt et al., Nat Methods 2008 Live STED Westaphl et al., Science, 2008 Nagerl et al., PNAS,

12 The use of two opposing objectives I 5 S isosted Shal et al., Biophys J Pi scheme Near isotropic 3D resolution ipalm Schmidt et al., Nano Lett 2009 Shtengel et al., PNAS 2009 Super resolution optical microscopy Hell, Science, 2007; Hell, Nat Methods, 2008 Rust, Bates & Zhuang, Nat Methods, 2006 Betzig et al., Science, 2006 Hess, Girirajan and Mason, Biophys. J., 2006 Gustafsson, PNAS., 2005 STED SSIM STORM/(F)PALM The patterned illumination approach Multiple cycles Ground state Triplet state Isomerization etc. Excitation Depletion pattern = 12

13 Saturated SIM FL Fluorescence saturation WF Deconvolution I ex Saturation level SIM SSIM Saturated illumination pattern Sharp zero lines 50 nm resolution Suffers from fast photobleaching under saturated excitation condition Gustaffson, PNAS 2005 The single molecule switching approach (STORM/PALM etc.) Multiple photons Photoswitching Blinking Diffusion Binding etc. + Stochastic Switching = Super resolution microscopy spec sheets 13

14 3D spatial resolution x y (nm) z (nm) Opposing objectives (nm) Two photon Conventional Pi: µm deep SIM I 5 S: 120 xyz STED ~30 ~100 isosted: 30 xyz 100 µm deep STORM/PALM ipalm: 20 xy, 10 z 10 µm Multicolor imaging Conventional SIM STED STORM/PALM Multicolor capability 4 colors in the visible range 2 colors so far 3 activation x 3 emission Time resolution 2D Spatial resolution Time resolution SIM Wide field 120 nm 9 frames (0.09 sec) STED Scanning 60 nm 1 x 2 µm: 0.03 sec 10 x 20 µm: 3 sec STORM/PALM Wide field 60 nm 3000 frames (6 sec) 3D Spatial resolution Time resolution SIM Wide field 120 nm 15 frames x 10 (1.5 sec) STED Scanning 60 nm 1 x 2 x 0.6 µm: 0.6 sec 10 x 20 x 0.6 µm: 60 sec STORM/PALM Wide field 60 nm 3000 frames (6 sec) no scan! 14

15 Practical issues SIM STED STORM/PALM Fluorophore limitation x x Instrument complexity xx xxx x Data analysis xxx xx Cost (rapidly changing) xx xxx x With the creation of new tools 15