super-resolution microscopy - uzh

SUPER-RESOLUTION MICROSCOPY

Dr. Nathalie Garin

Content

Motivation for superresolution

Superresolution, nanoscopy, …: definition

Structured Illumination Microscopy (SIM)

Localization microscopy

STimulated Emission Depletion (STED)

Combined technics

PSFs melt together...

... not a good representation of the structure

Rolf Borlinghaus / Leica

The diffraction limit

Why Superresolution Microscopy?

Diffraction Limit

Cell Bacterium Mitochondrium Influenza Virus Titin GFP

sin2nx

Content

Motivation for superresolution

Superresolution, nanoscopy, …: definition

Structured Illumination Microscopy (SIM)

Localization microscopy

STimulated Emission Depletion (STED)

Combined technics

Abbe limit: Pushing or breaking?

Confocal superresolution Nanoscopy Confocal

Content

Motivation for superresolution

Superresolution, nanoscopy, …: definition

Structured Illumination Microscopy (SIM)

Localization microscopy

STimulated Emission Depletion (STED)

Combined technics

Horn et al., (2013) JCB 202: 1023-1039

KASH5 SCP3

Slide courtesy: G. Wright, Singapor H. Horn, Journal of Cell Biology (2013)

f (x) a0 an cos(nx

Ln )

n1

Fourier series (1D)

Vincent Studer IINS - Bordeaux

ˆ f (r k ) f (

r )e2i

r k .

r d2 r

f (r ) ˆ f (

r k )e

2ir k .

r d2

r k

Fourier transform

FT

Inverse FT

Real Space

Fourier space

r r (x,y,z) (

r ,z)

r k (kx,ky,kz) (

r k ,kz)

r r (x,y,z) (

r ,z)

f (r )

r k (kx,ky,kz) (

r k ,kz)

ˆ f (r k )

kx

ky

Vincent Studer IINS - Bordeaux

Two superimposed patterns (in this case the illumination pattern and the structures in the sample) interfere with each other and produce a third, characteristic pattern: the Moiré fringes. The Moiré fringes have a lower spatial frequency than the original structures within the sample. Therefore, the fringes can be transmitted by a normal objective lens.

Creating a Moiré Fringe

TF

TF

x OTF=

x OTF=

Vincent Studer IINS - Bordeaux

Optical Sectioning

Vincent Studer IINS - Bordeaux

Optical Sectioning

Vincent Studer IINS - Bordeaux

Optical-Sectioning

Vincent Studer IINS - Bordeaux

keratin-14

3D-SIM

Applied Precision

Gustafsson et al. 2008. BioPhy J

SIM: take home message

Wide field technique

Works with any fluorophore

Sub-diffraction resolution (130 nm x 350 nm, Zeiss website)

Un‐modulated areas of the sample due to out of focus fluorescence do

not contribute to the signal but they add shot noise ! Problem with thick samples.

‐> TIRF – SIM is therefore of great interest for low level fluorescence

where shot noise is important

Content

Motivation for superresolution

Superresolution, nanoscopy, …: definition

Structured Illumination Microscopy (SIM)

Localization microscopy

STimulated Emission Depletion (STED)

Combined technics

Galectin-3 accumulates in Flotillin-positive endosomes

Slide courtesy: Ralf Jacob, Marbrug

WF single molecule superresolution

The solution with diffraction limited resolution

Diffraction limited resolution FWHM of the PSF

NNAx

2

Localization resolution

(N= number of photons)

Thompson et al., Biophys. J. 2002

Movie courtesy: Claudio Dellagiacoma, EPFL, Lausanne

S1

S0

T1

t ~ 100ms

t ~ 3ns

10k to 100k frames – calculate centre of mass – Sum

Fölling, J., Bossi, M., Bock, H., Medda, R., Wurm, C.

A., Hein, B., Jakobs, S., Eggeling, C. and Hell, S. W.,

Nat Methods. 5(2008), 943 - 945

Localization microscopy acquisition

Acronymes

Sam Hess

PALM PhotoActivation Light

Microscopy

uPAINT Universal Point Accumulation Imaging in the

Nanoscale Topography

N-STORM Stochastic Optical

Reconstruction Microscopy

Ground state depleted

GSDiM / (D)STORM The dark state of fluorescence – not bleaching

3D: different possibilities

Astigmatism Double helix

biplane microscopy US 7880149 B2

Localization techniques: take home message

Widefield technique

Calculated SR

Works with any fluorophores

Sub-diffraction resolution:

o 20 nm in xy

o 50nm in z

Often in TIRF

Content

Motivation for superresolution

Superresolution, nanoscopy, …: definition

Structured Illumination Microscopy (SIM)

Localization microscopy

STimulated Emission Depletion (STED)

Combined technics

Does it colocalize?

Confocal

STED

29

Centrioles in U2OS cells visualized by indirect immunofluorescence. Co-localization of Centrin3 – Alexa Fluor 594 (green) and Cep152 – Alexa Fluor 647 (red) in confocal (left) and deconvolved STED image (right). Sample courtesy of Ella Fung, CRUK/MRC Oxford Institute for Radiation Oncology, UK. The anti-Cep152 antibody was kindly provided by E. Nigg, Biozentrum, University of Basel, Switzerland (Sonne KF et. al. J Cell Science 2013).

500 nm

The diffraction limit

Point

Detector

Lens Scan

Ernst Abbe, 1873 Consequence:

Diffraction prevents to separate objects closer than 200nm

(= ½ wavelength of visible light)

sin2nx

Breaking the diffraction limit

Point

Detector

Lens Scan

Stefan W. Hell, Inventor of STED-microscopy

Reducing the area of effective excitation, in combination with a scanning microscope breaking the diffraction limit becomes possible

STED Microscopy – PSF shaping

•

On state Off state

STED: a switch off process

Pyridine 2

The driving forces: laser and dye development

~ 200 nm

~ 150 nm

~ 75 nm

Gated STED

Lifetime distribution within STED CW focal spot

STED Confocal

Gated STED: The Principle

For CW STED resolution has a life time dependence

Short lived states cause somehow blurry appearance and reduce the contrast of STED CW images

Gated STED only records fluorescence from long living states

Improved resolution

Clearer images/better contrast

Standard STED CW with pulsed Excitation

Time (ns)

Fluorescence

Excitation

Detection

Confocal

DNA Origami 70nm distance

Standard STED CW with pulsed Excitation

Time (ns)

Fluorescence

Excitation

STED

Detection

STED CW

STED laser not used at full power

DNA Origami 70nm distance

Gated STED

Time (ns)

Excitation

STED

Detection

STED laser not used at full power

DNA Origami 70nm distance

Gated STED

Time (ns)

Excitation

STED

Detection

STED laser not used at full power

DNA Origami 70nm distance

Deuterosomes: platform for centriole amplification

Work done with C. Boutin, Institut de Biologie du Développement de Marseille Centriole marker: yellow/Alexa488 Deuterosome marker: Cyan/Alexa568, magenta/Alexa514

1mm

Cleared kidney sample: 45-55 µm inside

Confocal 3D STED

Sample courtesy of David Unnersjö Jess, KTH, Stockholm More on clearing on Leica Science Lab

STED: take home message

Confocal technique

Optical SR

Works with any fluorophores depleted by 592, 660 or 775nm

Sub-diffraction resolution:

o <50 nm in xy

o < 130 nm in z

Ideal for thick and living samples

Content

Motivation for superresolution

Superresolution, nanoscopy, …: definition

Structured Illumination Microscopy (SIM)

Localization microscopy

STimulated Emission Depletion (STED)

Combined technics

Lattice light sheet

Technic combining:

o Light sheet fluorescence microscopy

o Bessel beam microscopy

o Structured Illumination Microscopy (SIM)

https://www.janelia.org/lab/betzig-lab

Lattice light sheet

https://www.janelia.org/lab/betzig-lab

Confocal focus cross section

500 nm

Combining the Numerical Aperture of 2 opposing objectives

Volume reduction by factor 3 – 5 compaired to confocal

Interference side lobes are removed mathematically

4Pi focus cross section

110 nm

x

z z

4Pi microscopy (Stefan Hell)

4Pi measurements in living cells

4Pi microscopy

Conventional fluorescence microscopy

Summary

Modified from JBC Review 2010 Lothar Schermelleh, Rainer Heintzmann and Heinrich Leonhardt

Gated STED

<50

560 70

/GSDIM

70

3D STED

<130

<130

Sample courtesy: Anne Aubusson-Fleury, CNRS, Gif sur Yvette, France

From microscopy to nanoscopy

Some references

Stefan W. Hell & Jan Wichmann (1994). "Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy". Optics Letters M. L. Bossi, J. Fölling, M. Dyba, V. Westphal, S. W. Hell (2006). "Breaking the diffraction resolution barrier in far-field microscopy by molecular optical bistability" New J. Phys. Lothar Schermelleh et al. (2010). A guide to super-resolution fluorescence microscopy J Cell Biol. Review Gustafsson, M. G. L. (2000). Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. Journal of Microscopy. Bi-Chang Chen et al. (2014). Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution. Science