Biophotonics lecture 7. December 2011. Exam date ? Monday, 30 January 2012 or Wednesday, 1 February...
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Transcript of Biophotonics lecture 7. December 2011. Exam date ? Monday, 30 January 2012 or Wednesday, 1 February...
Biophotonics lecture7. December 2011
Exam date ?
Monday, 30 January 2012or
Wednesday, 1 February 2012
Last week:
- Stimulated emission depletion (STED) microscopy
Today:
- Imaging deep in tissue: 2-photon microscopy- Enlarging the NA: 4Pi microscopy- Super-resolution: Pointillism, STORM, PALM
Imaging deep in tissue:
2-photon microscopy
Refractive(and scattering)
tissue
objective lens
Refractive(and scattering)
tissue
Imaging single cells Imaging deep in tissue
well defined focal spot
well defined focal spot
well defined focal spot
not well defined focal spot
Imaging deep in tissue:
2-photon microscopy
The Problem:Scattering, aberrations, absorption
Rayleigh scattering: ~ l-4
Blue: Bad!
Red / Infrared: OK!
Imaging deep in tissue:
2-photon microscopySolution: imaging using longer wavelength
Refractive(and scattering )
tissue
not well defined focal spot
objective lens
well defined, but LARGER focal spot
Focal spot, l=500nm Focal spot, l=1000nm
x
z
x
z
ATF OTF ATF OTF
Jablonski diagram
Absorption…
… and spontaneous emission
Fluorescence
Jablonski diagram
NO absorption…
Fluorescence
Jablonski diagram
2-photon absorption…
… and spontaneous emission
2-photon fluorescence
2-photon fluorescence
- 2-photon absorption requires two photons to be present simultaneously
- The probability for this grows quadratically with intensity
- It will only occur where the local intensity is high
Focal spot, l=500nm Focal spot, l=1000nm
x
z
x
z
2-photon, l=1000nm
x
z
missing cone filledoptical sectioning
Zipfel, Williams, Webb, Nature Biotechnology 21, 1369 - 1377 (2003)
2-photon fluorescence
emission photons will still be multiply scattered and cannot be focussed on a pinhole
Non-descanned detection needed to maximize capture area
Wid
e Ar
ea D
etec
tor
at c
lose
des
tanc
e
DichromaticReflector
Two Photon Microscopy
• Much less absorption• Much less scattering• Fewer aberrations• Less out-of-focus bleaching• Inherent optical sectioning
Enlarging the NA:
4Pi Microscopy
Aperture increase: 4 Pi Microscope (Type C)Sample between
Coverslips
Illumination Emission
DetectorPinhole
HighSidelobes
FluorescenceIntensity
z
z
LaserDichromaticBeamsplitter
Stefan W. HellMax Planck Institute of Biophysical Chemistry
Göttingen, Germany
2 Photon Effect
ATF OTF
widefield
4Pi
widefield, l=500nm 4Pi, l=500nm
widefield, l=1000nm 4Pi, l=1000nm2-photon, l=1000nm 4Pi, l=1000nm, 2-photon
4Pi PSFs
Leica 4Pi
http://www.leica-microsystems.com
4Pi images
Deviding Escherichia Coli
From: Bahlmann, K., S. Jakob, and S. W. Hell (2001). Ultramicr. 87: 155-164.
Confocal (2-Photon ) 4Pi (2-Photon)
Thanks to: Elisabeth Ehler, Reiner Rygiel, Martin Fiala, Tanjef Szellas
4Pi images
Super-resolution:
Pointillism, STORM, PALM
Seurat: Tiger Douthwaite: Lewis Hamilton
Localization, not resolution
If particles can be separated, their relative positions can be measured accurately
If positions are know you can paint a picture!
PSF
Localization, not resolution
position
Localization, not resolution
position ?????
How to separate particles?
Spectral precision distance microscopy
Problems: Chromatic Aberrations, few dyes
Using fluorescence lifetime for separation (FLIM)
Problems: Lifetime depends on microenvironment
Use the blinking characteristics
M. Heilemann, D.P. Herten, R.Heintzmann, C. Cremer, C. Müller, P. Tinnefeld, K.D. Weston, J. Wolfrum and M. Sauer. Anal. Chem., 74, 3511-3517, 2002.
P. Edelmann, A. Esa, H. Bornfleth, R..Heintzmann, M. Hausmann, and C. Cremer. Proc. of SPIE , 3568:89-95, 1999
K.A. Lidke, B. Rieger, T.M. Jovin, R. Heintzmann Optics Express 13, 7052-7062, 2005.
How to separate particles?
Better:
Avoid overlap entirely by temporally separating the particles
E. Betzig, "Proposed method for molecular optical imaging", Opt. Lett. 20, 237 (1995)
Earth
Earth at night
Jena at night
Task: Localization of the university buildings
How: Each Professor has to turn on the light for one minute
Localizing is much moreprecise than resolution
Reso
ution
Jena at night
Separation over time
Without labelling:everything is bright
Labelling the university buildingswidefield: bad resolution
Pointillistic: accurate map
Separation over time
Pointillism, PALM, STORM
other techniques:STORM, FPALM
http://jcs.biologists.org/cgi/reprint/123/3/309.pdf
PALMPhoto-activation and localisation microscopy
MitochondriaCOS-7 ZellenCryo-Schnitte Cytochrom C Oxidase import Sequenz - dEosFP
E. Betzig et al., Science, DOI: 10.1126/science.1127344, Aug. 2006
WF PALMEM
Pointillism, PALM, STORM
Hochauflösende Struktur der Podosomen (Vinculin)
New, sophisticated algorithms,which can handle overlappingfluorophores
Pointillism, PALM, STORM
Podosomenbildung
400nm
Susan Cox, Edward Rosten, Marie Walde, James Moneypenny, Gareth Jones
Pointillism, PALM, STORM
Pointillism, PALM, STORM
Confocal microscopy
Widefield fluorescence
Structured illumination microscopy
dSTORM / B3
STED
1 m
Comparing some methods
Stochastic Optical Reconstruction Microscopy
Microtubules – (Cy3-Alexa647)
Science 319, 810 (2008); Bo Huang, et al. Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy
Localisation precision in pointilism:(for Gaussian PSFs)
- N photons collected from 1 fluorophore
- Positions of these photons are rn=rfluorophore ±with being the standard deviation defined by the PSF
- The fluorophore position is determined as the mean of all photon positionsrfluorophore=rn / N
- This mean position has an error ofrfluorophore with rfluorophore = /
- With N photons, the localisation precision is better than the resolution
Problem: sparseness of labelling