7. class: superresolution 1 Flashcards by Thomas Hasiweder

Q

Why super?

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dimensions of a membrane

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Émile Verdet

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Resolving power of optical microscopy

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Rayleigh criteria

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Diffraction limited image

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Superresolution fluorescence microscopy

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Localization microscopy

2D

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Processes at the single molecule level are stochastic!

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Stochastic process resulting in 2 states + accurate position determination

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Localization microscopy

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Localization microscopy

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PALM concept

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Different ways of doing Localization microscopy

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NALMS fluorescence microscopy Nanometer localized multiple single molecule

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NALMS fluorescence microscopy

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Photo-activation localization microscopy (PALM)

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Photo-activation localization microscopy (PALM)

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HeLa cell expressing β-actin-KikGR

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Stroboscopic PALM (S-PALM)

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Stroboscopic PALM (S-PALM)

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Stroboscopic PALM (S-PALM)

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Stroboscopic PALM (S-PALM)

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Multi-color PALM

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STORM

Multi-color STORM (stochastic optical reconstruction)

3D Localization microscopy

3D Localization microscopy

Labelling density issue

Localization microscopy: advantages and disadvantages

Membrane rafts/micro-domains

Probes for membrane rafts

Probes for membrane rafts

Photo-switchable FPs: photo-chromic proteins that can be switched many times

Probes for membrane rafts

Cholesterol-enriched membrane raft stained with θ-D4-Dronpa (HeLa cell, fixed)

Comparison between cholesterol- and sphingomyelinenriched membrane rafts

Dual color super-resolution imaging

Dual color confocal images of HeLa cell stained with Alexa647-EGF & θ-D4-Dronpa

Dual PALM image of Alexa647-EGF & θ-D4-Dronpa, (HeLa cell)

Dual PALM image of Alexa647-EGF

The HIV-virus life cycle

Labeling HIV-1 with photoactivatable proteins

Structural features of HIV-1 particles

ENV forms clusters on HIV-1 particles

Diffusion of polymer chains

An experiment suggested by P.G. de Gennes

Poly(isocyanopeptides)

Acid Catalyzed Polymerization of Isocyanopeptides

Polyisocyanopeptides

Single chain dynamics

Single chain dynamics

Single chain dynamics

Data analysis

Fitting parameters

Single chain dynamics

Single chain dynamics

Zeolites: ZSM-5

ZSM-5: in situ loading with dyes

High resolution microscopy?

High resolution microscopy

Reactions at the nanoscale

Convolution

Stimulated emission depletion microscopy (STED)

STED setup

Saturated depletion

Intensity distribution at focus

Phase retardation by a film

Electric field and intensity with and without phase shaper

Simulation of 3D intensity distribution

Phase shapers for doughnut mode beam

Fabrication of phase shapers

Experimental setup for phase shift analysis

Interferogram of phase shapers

Phase shift by phase shapers

Phase shaper for 633 nm

Intensity distribution with different beam size (f = 100 mm)

Experimental setup for analysis of intensity distribution by single molecule fluorescence

Fluorescence image of single TDI molecules

STED-Microscopy of pTDI

It’s all about photophysics

3D STED (1)

3D STED (2) isoSTED or 4pi STED

3D STED (2) isoSTED or 4pi STED

List of dye used for STED

STED vs. Localization microscopy

RESOLFT

More about STED

POSH microscopy

Photo-Switchable Fluorescent Protein Dronpa

High resolution microscopy with Dronpa

High resolution microscopy with Dronpa

Experimental results

Theoretical resolution improvement

More about STED RESOLFT

STED-FCS

Principles of MINFLUX

Setup, measurement strategy, and various application fields of the 2D MINFLUX implementation

Localization precision of MINFLUX measured on a single ATTO 647N molecule

MINFLUX nanoscopy of labeled DNA origamis.

Single-molecule MINFLUX tracking in living E. coli bacteria

Fourier transforms

Sample structure vs Fourier

patterns

FFT iFFT

The Fourier transform of the PSF

Structured illumination: Frequency domain

Structured illumination: Frequency domain vs spatial domain

the beat pattern

angle

phase

Structured illumination: Frequency domain

3D structured illumination

Targeted fluorescence depletion

SOFI

Array

sofi mathematics

sofi 1-4

Advantages of SOFI: & disadvantages

Different order of SOFI

SOFI in bio-application 1

Improvement in SNR

3D imaging

image acquisition

Expansion microscopy

Expansion microscopy 2

(3) Expansion microscopy: Pre- VS Post-expansion

(3) Expansion microscopy: tubulin anti-body (Alexa488)

Expansion microscopy • Purpose:

Expansion microscopy • HeLa histone 2b-eGFP: Nucleus

Expansion microscopy - Chromosomes

Numerical aperture

Why aren’t we all using these techniques?

ACCESSIBILITY