3D Super Resolution Imaging (STORM/PALM)
15 Oct 2012
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Details about super resolution imaging

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Zeiss Elyra PS1: STORM/PALM in the same microscope as structured illumination imaging, with an EMCCD detector. 3D imaging possible using the double-helical point-spread function method. Activation/excitation wavelengths are 405, 488, 561 and 640 nm.

 
Contacts: Dr. Lin Wang
 
Single molecule localisation microscopy is a family of super-resolution microscopy techniques, including stochastic optical reconstruction optical microscopy (STORM) and photoactivated localisation microscopy (PALM). A series of images (typically 5-15,000) are acquired, each of which contains a number of diffraction-limited spots. Each spot is assumed to be symmetrical and Gaussian in profile, allowing its centre to be localised down to ~10 nm. The resulting co-ordinates are aggregated and can be rendered as a super-resolved image. The challenge is to switch the fluorescent molecules on and off such that each appears in as few frames as possible, usually using a combination of a 405 nm activation laser and an appropriate buffer solution.


InstrumentZeiss Elyra PS1Bruker Vutara 350
Laser wavelengths (nm)405 (for activation only), 488, 561, 642405 (for activation only), 488, 561, 642, 750
Excitation power densityModerate, depends on field of viewVery high
Compatible objective lensesx100 oil immersion, x100 silicone oil immersion, x63 oil immersion, x63 water immersionx60 silicone immersion, x60 oil immersion
Compatible dyesRestricted, depends on blinking characteristics and photon fluxRestricted, depends on blinking characteristics and photon flux
Compatible sample mounts#1.5 cover slip on microscope slide or glass bottom Petri dish#1.5 cover slip on microscope slide or glass bottom Petri dish
ConfigurationInverted microscopeInverted microscope
Axial resolution methodPhase rampBiplane
Multi-wavelength imagingUp to three in 2D or 3D, always sequentialUp to two channels in 3D, up to four in 2D, always simultaneous
DetectorEMCCDsCMOS
Temperature control21-37°C21-37°C
Lateral resolution~20 nm (highly dependent on signal to noise/background ratios and labelling density)~20 nm (highly dependent on signal to noise/background ratios and labelling density)
Axial resolution~50 nm (highly dependent on signal to noise/background ratios and labelling density)~50 nm (highly dependent on signal to noise/background ratios and labelling density)
Axial range1-1.5 microns1-1.5 microns
Temporal resolution4 minutes (max)0.5 Hz (max)


Vutara SR-350: 2 colour biplane imaging with sCMOS detectors for fast 3D super-resolution microscopy. Activation/excitation wavelengths are 405, 488, 561, 640 and 750 nm.

 


Both microscopes can be used with epi or TIRF illumination. 

Contact: Wang, Lin (STFC,RAL,CLF)