Super-Resolution Fluorescence Microscopy Clif Thivierge 08/12/10 Prof. Kevin Burgess Texas A&M University

The Bane of Imaging: Diffraction Limit practical limit obtained when imaging very small objects by magnification diffraction causes blurring of objects when imaging smaller than ~200-500 nm (diffraction limit) “broadening” of a point caused by diffraction is known as the “point spread function” () x-y= (0.61 /( sin())  = refractive index medium  = half-cone angle of focused light

Examples of Diffraction Limit

Ways to Circumvent Limit Near Field Microscopy (NSOM) Far Field Microscopy Confocal, 4pi and I5M, SIM Super-Resolution Spatially Patterned Excitation STED RESOLFT SSIM Localization Methods STORM PALM FPALM

Near Field Imaging (NSOM) -place microscope distance less than 1 wavelength from sample -20-50 nm resolution problem: cannot image into sample because of wavelength restriction

Far Field: Confocal Microscopy Non-linear 2-photon excitation and pinhole detection decrease SPF beyond classical limits 21/2 improvement in resolution problem: 2-photon excitation uses high wavelengths which increase SPF: x-y= (0.61 /( sin())

Structured-Illumination Micropscopy (SIM) 100 nm resolution possible

Conclusions methods use common dyes (good) confocal is easiest, most widely used best resolution obtainable only 100 nm (SIM) single molecule is problematic

Super-Resolution Microscopy Goal: obtain sub-100 nm resolution pioneered by Stefan Hell in mid-1990s Max Plank Institute (Germany) two methods: Spatially Patterned Excitation STED, RESOLFT, SSIM (ii) Localization Methods STORM, PALM, FPALM

Stimulated Emission Depletion Microscopy (STED) Spontaneous VS Stimulated Emission when an excited molecule encounters a photon matching it’s emission energy, another “clone” photon is created and ground state results

STED sample is excited with laser and blur is obtained due to diffraction (exc) another “doughnut” shaped laser excites at emission wavelength (STED) of dye and switches outside dyes to “dark state” observing in between exc and STED, very resoved image is produced observe exc STED

STED Microscopy resolutions 20 - 30 nm common, best 6 nm

STED Dyes -dyes need to be very photostable excitation laser: 107 W/cm2 STED laser: 109 W/cm2 -dye needs large stimulated emission cross-section -most common: Atto 532 and Atto 647N Rhodamine derivatives?

Dyes Used for STED

Reversible Saturable Optically Linear Fluorescence Transitions (RESOLFTs) same concept as STED but dyes are made to “dark state” by other mechanisms: -switch to triplet state -switch to ground state -use reversibly photoswitchable dyes advantages: -less powerful lasers need to be used (100 W/cm2) -this leads to many more dyes and even fluorescent proteins being used

Single Molecule Imaging the exact location of single dyes can be determined by doing multiple excitation/emission cycles

Things Become More Complicated with Multiple Dyes Considerable overlap. Hard to identify individual fluors in real live.

Super-Resolution Single Molecule Localization Methods Stochastic Optical Reconstruction Microscopy (STORM) Photoactivated Localization Microscopy (PALM) Fluorescence Photoactivated Localization Microscopy (FPALM) all work by same principle: image only some dyes at one time

Consider Previous Example Considerable overlap. Hard to identify individual fluors in real live.

Most Dyes in “Off-state” localization of “on-state” dyes possible

Switch the State of the Dyes localization of other dyes possible

Combine the Images position of each dye is known

Dyes for Localization Microscopy have on and off state easily able to switch from on/off state on/off can be non-fluorescent or have a change in either excitation or emission wavelengths best if reversible but not necessary

Common Dyes for Localization Mic.

Examples of Photoswitchable Dyes

Conclusions on Localization Microscopy using this technique 3D localization of labels can be achieved with 20 nm resolution

Multicolor Imaging methods discussed so far are valuable in the elucidation of structures to study interactions, multicolor imaging can be used multicolor imaging has been done with both STED and Localization Microscopy

Multicolor STED 2 methods: (i) use set of dyes with non-overlapping excitation, emission, and STED wavelengths (hard to find) (ii) find dyes with same STED excitation but non-overlapping absorbance

Multicolor STED: Synaptic Proteins Synaptophysin (red) Syntaxin 1 (green)

Conclusions still very young technology and best dyes have yet to be discovered impact is big since imaging is such a popular tool a lot of opportunity for innovation/development

The End