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 microscopy

Solution: 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=1000nmx

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=1000nmx

z

x

z

2-photon, l=1000nmx

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

Podosomenbildung400nm

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