maxim s. pshenichnikov maxim s. pshenichnikov

Microsoft PowerPoint - Superresolution Optical Microscopy final.pptZernike Institute for Advanced Materials
University of Groningen Zernike Institute for Advanced Materials
Maxim S. Pshenichnikov Maxim S. Pshenichnikov Federal Agency for
Science and Innovations, Russia (grant 02.740.11.5155)
Zernike Institute for Advanced Materials
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Superresolution Optical Microscopy Vladivostok, 6 October 2010
University of Groningen Zernike Institute for Advanced Materials
Maxim S. Pshenichnikov Maxim S. Pshenichnikov
Outline
Molecules
Enhanced optical field
Diffraction limited spot
Scanning-probe microscopiesNear-filed microscopy
- Basic principles of fluorescent microscopy - Resolution of optical microscope and the Abbe limit - Redefinition of the problem - Single fluorophore detection with nm accuracy - High-resolution imaging with photobleaching - Conclusions
NOT to be considered
ALL microscopies are about getting the contrast!
100% intensityBackground
intensityBackground-intensitySpecimen Contrast ⋅=
Contrast enhancing: 1. Decrease background intensity
2. Increase specimen intensity
Stokes shift Brief history:
1852: Sir George G. Stokes 1914: Stanislav Von Provazek 1940’s: Albert Coons - the new field of
immunofluorescence 2000’s: fluorescence microscopy made
a revolution in cell biology
Common Fluorophores in Microscopy
Fluorophores for Microscopy
Laser
Confocal Microscope
plane
Laser
Spatial resolution: a half wavelength (~300 nm)
Can go single-molecule!
Why doing single molecule detection ?
-Why measuring many single molecules when one can get the result just from an ensemble experiment and save a lot of time ???
Bulk experiment
• Characterization of distributions and fluctuations of molecular properties, devoid of ensemble averaging
• Observation of transient intermediates, elucidate reaction mechanisms (no dephasing!)
No synchronization needed for measuring time dependent phenomena
Getting rid of ensemble averaging Single molecule experiments reveals heterogeneities in the probed population by measuring distributions
10/s
Fluorescence imaging of single molecules
(Y ild iz , S el vi n, A cc .C he
m .R es . 2 00
5)
How to do single molecule microscopy?
Resolution of the Microscope
Rayleigh criterion
20x
Can we beat the Abbe limit? – NO! Can we do better? - YES! if the initial problem is redefined
Redefinition of the Problem
We are NOT to resolve fine specimen details but to locate the position of a single chromophore
Webb&Co, Biophys. J. 82(5) 2775 (2002)
Accuracy of the mean values of x0, y0:
0
)()( 2
(x0, y0)
background
Potentially, single molecules can be localized with an arbitrarily high precision
A priory information Image of Cy3-dye immobilized onto
glass coverslip
N s
Point-spread function 200 nm
Yildiz&Selvin, Acc.Chem.Res. 38, 574 (2005)
1-nm accuracy
Myosin: a linear motorMyosin: a linear motor
Kinesin Molecular Motor
Hand-over-hand Inchworm
Phosphorescence and Photobleaching Coupling to triplet state Timescale for phosphorescence
↑↓Spin
↑↑Spin
High-Resolution Imaging with Photobleaching
Gornon et al., PNAS 101 (17), 6462 (2004)
Fit with One 2D GaussianFit with Two 2D Gaussians
( )1 0
Separation (nm)
Separation (nm)
Limitation: signal-to-noise ratio ! Qu et al., PNAS (101) 11298 (2004)
Three Alexa 488-labeled bis-PNA
7-base bis-PNA binds to specific recognition sequences in double-stranded DNA
Fluorophores that photobleached in the same CCD frame are shown with the same color
A priory information
Photoswitchable Molecules
“Dark” state: no fluorescence
Switching to the “bright” state by a
switching laser
Photoactivated Localization Microscopy (PAL-M)
Reporters (fluorescence / Raman)
PALM: Concept
Number of activated molecules <=1 in diffraction limited area Determination of center with nanometer resolution
Dedecker et al, Angewandte Chemie International Edition 2007, Nov 11
PALM: How It Is Working
Visualization of Rotary Visualization of Rotary NanoNano--MotionMotion 3-4 nm
Fluorophore
Isomerisation
ω
Conclusions
Concept of the fluorescence imaging with nm resolution - a priori information
- modern methods of excitation (lasers) and detection (CCD’s) - (a PC) computer power
Further developments - 3D nanolocalization (also in transversal direction)
- angular single-molecule resolution - obtaining system dynamics
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maxim s. pshenichnikov maxim s. pshenichnikov

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