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TIRF/PALM/dSTORM High Resolution Microscope

LS
07/05/2017
Partager
Single-Molecule Super-Resolution Imaging

STORM reconstructs a super-resolution fluorescent image by combining precise localization information for individual fluorophores in complex fluorescent microscope specimens.
This powerful technology enables the visualization of molecular interactions at the nanoscopic level, opening up new worlds of scientific understanding.
n-storm-nikon

Equipment Configuration

Illumination

  • ROPER laser bench
    • 405nm, 50mW
    • 491nm, 100mW
    • 561nm, 1000mW
    • 633nm, 300mW

Detectors

Objectives

100x

CFI Plan Apo TIRF

NA 1.49

WD 0.12

DIC

Oil

Chroma Bloc Filter

Associated devices

Techniques

  • Fluorescence
  • Live Imaging 2D and 3D
  • TIRF
  • Photoactivable fluorophore

Applications

  • Cell Biology

Principle

Single Molecule Localization Microscopy

STORM,Stochastic Optical Reconstruction Microscopy, is one of a family of super-resolution Single Molecule Localization Microscopies (SMLM) for the visualization of biological systems with an optical resolution measured in the tens of nanometers (nm) in the x, y, and z directions.

STORM and other SMLMs are conceptually similar techniques: the photochemical properties of the fluorophore are exploited to induce a weakly emissive or non-emissive “dark” state. From the dark state, very small populations of (ideally) fluorophore are returned to an emissive state, excited, and detected. However, in order to be identified, emission profile must exhibit minimal overlap in each image. The centroid position of each identified molecule is statistically fitted, most often to a Gaussian function, and with a level of precision scaling with the number of detected photons. By imaging and fitting single emitters to a sub-diffraction limited area over many thousands of single images, eventually the user will have enough data to create a composite reconstruction of all identified emitters. The majority of SMLMs operate using this common generalized concept, requiring virtually identical hardware and data analysis steps, but differing in the exact fluorophore chemistry exploited to realize such “on-off” switching behavior. More...

The technique of direct stochastic optical reconstruction microscopy (dSTORM) differs by employing conventional carbocyanine dyes for cellular staining and does not rely on the proximity of two fluorophores attached to the same targeting antibody in a specific ratio. The original research report on dSTORM used Cy5 and Alexa Fluor 647 to demonstrate the novel single-molecule superresolution technique, which requires substantial laser power to ensure effective transition of the photoswitchable fluorophores into a dark state. More...

Similar in concept to STORM, PALM photoactivated localization microscopy, utilizes optical highlighter fluorescent proteins to stochastically switch on a sub-population of molecules for sequential single-molecule readout. The fundamental principle behind PALM and related methodology is that the activated state of a photoswitchable molecule must lead to the consecutive emission of sufficient photons to enable precise localization before it enters a dark state or becomes deactivated by photobleaching.