Biophotonics lecture23. November 2011

Last week:

- Fluorescence microscopy in general, labeling, etc…- How to do optical sectioning and fill the missing cone, the confocal microscope

Today:

- Structured illumination: an alternative approach to optical sectioning- Super resolution techniques: beyond the Abbe limit

- High-resolution structured illumination microscopy

Optical sectioning alternatives: structured illumination microscopy (SIM)

- Sample is illuminated with a structured illumination pattern, i.e. a line grid.- This can be produced by placing a grid in a conjugate image plane in the

illumination pathway.- Three images are taken for three different (lateral) positions of the illumination

grid.- These images can be computed into a final, optically sectioned image.

For single-shotsectioning

In-focussample slice

Out-of-focussample slice

Full-field illuminationStructured illumination

Acquired wide-field imageAcquired structured illumination image

x

y

x

z

x

y

x

y

Acquired structured illumination images

I1(r) I2(r) I3(r)

Ifinal(r) = max{I1(r) , I2(r) , I3(r)} - min{I1(r) , I2(r) , I3(r)}

Result: sectioned image Wide-field image

Advantages:- Cheaper: no need for laser, scanner, PMTs, AOTFs, etc.- Potentially faster: for a large field of view 3 wide-field images can be acquired

faster than a point-wise scan.- Less photo-bleaching: less light is lost as compared to using a pinhole

Wide-field Sectioning SIM

Frequency support of sectioning SIM

- Filled missing cone- Higher axial support- Higher lateral support (1 direction), more on this later

But this isNOT an OTF !!

𝐼 (𝑟 )=𝑆(𝑟 )⨂h𝑐𝑜𝑛𝑓𝑜𝑐𝑎𝑙 (𝑟 )

𝐼 (𝑟 )=𝑆(𝑟 )⨂h𝑤𝑖𝑑𝑒𝑓𝑖𝑒𝑙𝑑 (𝑟 )

𝐼 (𝑟 )=𝑆(𝑟 )⨂h𝑆𝐼𝑀 (𝑟 )

Wide-field

Confocal

Sectioning SIM

The image formation in section SIM cannot be written as a convolution ofthe sample with an intensity PSF.

Advantages:- Cheaper: no need for laser, scanner, PMTs, AOTFs, etc.- Potentially faster: for a large field of view 3 wide-field images can be acquired

faster than a point-wise scan.- Less photo-bleaching: less light is lost as compared to using a pinhole

Disadvantage:- Not a linear imaging modality. Not as useful for quantitative analysis.

Imaging beyond the Abbe-limit: the true strength of SIM

“Sample“ for simulation Fourier transform of “Sample“

Sample will be “repainted” with a blurry brush rather than a point-like brush.

Real space Fourier spaceLimited resolution in conventional, wide-field imaging

Moiré effect

high frequencydetail

high frequencygrid

low frequencymoiré patterns

Moiré effect

Structured Illumination Microscopy

Illumination with periodic light pattern down-modulates high-frequency sample information and makes it accessible for detection.

Sample

Illumination

Laser

CCD

x

zTubelens

Filter

Dichromaticreflector

Tubelens

Objective

Sample

Diffraction grating,SLM, etc…

Sample Illumination

Structured Illumination Micropscopy

Sample with structured illumination

Multiplication of sample and illumination

Structured Illumination Micropscopy

Multiplication of sample and illumination

Real space Fourier space Convolution of sample and illumination

Sample Illumination

Structured Illumination Micropscopy

Sample

Structured Illumination Micropscopy

Structured Illumination Micropscopy

Sample Sample & llumination

Sample Sample & llumination

Imaging leads to loss of high frequencies (OTF)

Structured Illumination Micropscopy

Separating the components…

Sample

Structured Illumination Micropscopy

Separating the components…Shifting the components…

Sample

Structured Illumination Micropscopy

Separating the components…Shifting the components…

Recombining the components…

Sample

Structured Illumination Micropscopy

Separating the components…Shifting the components…Recombining the components… using the correct weights.

Sample Reconstructed sample

Structured Illumination Micropscopy

sample wide-field SIM (x only)

Missing cone – no optical sectioning

Full-field illumination

1 focus in back focal plane

Missing cone – no optical sectioning

2-beam structured illumination

2 foci in back focal plane

Missing cone filled – optical sectioning

2-beam structured illumination

3 foci in back focal planebetter

z-resolution

1 mm

Fourier space

(percentile stretch)

Liisa Hirvonen, Kai Wicker, Ondrej Mandula, Rainer Heintzmann

WF: 252 nm

SIM: 105 nm

99 beads averaged

wide-field

SIM

2 µm

excite 488nm, detect > 510 nm24 lp/mm = 88% of frequency limitPlan-Apochromat 100x/1.4 oil iris Samples Prof. Bastmeyer, Universität Karlsruhe (TH)

Axon Actin (Growth Cone)

excite 488nm, detect > 510 nm24 lp/mm = 88% of frequency limitPlan-Apochromat 100x/1.4 oil iris

2 µm

Samples Prof. Bastmeyer, Universität Karlsruhe (TH)

Axon Actin (Growth Cone)

Doublets in Myofibrils

Isolated myofibrils fromrat skeletal muscleTitin T12 – Oregon green

L. Hirvonen, E. Ehler, K. Wicker, O. Mandula, R. Heintzmann, unpublished results

1 µm 124 nm

1 mm

Molecules in space and time

living COS1 cell

L. Hirvonen, K. Wicker., O. Mandula and R. Heintzmann, Structured illumination microscopy of a living cell, Europ. Biophys. J. 38, 807-812, 2009

Marie Walde, James Monypenny (cooperation G. Jones), King‘s College London

f-ActinVinculin

Are podosomes arranged as "sticks and joints"?

5 mm

Podosomes

0

magnitude

spatial frequency

Support region of OTF

0

magnitude

spatial frequency

-K0 K0-2K0

-K0 K0

Linear Excitation (low intensity)

Non-Linear Excitation (high intensity)

-3K0

Support region of OTF

Conventionalmicroscopy

Saturatedstructured illumination

1 µm

Linearstructured illumination

1 µm1 µm

Mats Gustafsson, UCSF50 nm microscpheresnonlinearity: fluorescence saturation, 53J/m2

3 extra harmonics

M.G.L. Gustafsson (2005), PNAS, 37, 13081-13086

NonlinearStructured Illumination Micropscopy

But:Artefacts possible!

0.5 µm

0.5 µm But:Artefacts possible!

Sophisticated algorithms are needed!