3D Super Resolution Imaging (STORM/PALM)

15 Oct 2012

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For more details please contact Dr. Lin Wang.

 

​​Technique and applications	​Specifications
​Technique:  Single-molecule localisation super-resolution imaging. Down to 20 nm resolution in xy and 50 nm in z over 1.5 µm z range. z-resolution using phase ramp or biplane methods. TIRF, HILO and epi-illumination.​ Applications:  Biological and medical research, material science, chemistry	​ Lasers: Multicolour imaging with 488, 561, 642, 750 nm excitation and 405 nm activation Detectors: EMCCD and sCMOS cameras Objectives: Elyra- 63x oil and water immersion,100x oil and silicone immersion

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.

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Instrument	Zeiss Elyra PS1
Laser wavelengths (nm)	405 (for activation only), 488, 561, 642
Excitation power density	Moderate, depends on field of view
Compatible objective lenses	x100 oil immersion, x100 silicone oil immersion, x63 oil immersion, x63 water immersion
Compatible dyes	Restricted, depends on blinking characteristics and photon flux
Compatible sample mounts	#1.5 cover slip on microscope slide or glass bottom Petri dish
Configuration	Inverted microscope
Axial resolution method	Phase ramp
Multi-wavelength imaging	Up to three in 2D or 3D, always sequential
Detector	EMCCD
Temperature control	21-37°C
Lateral resolution	~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)
Axial range	1-1.5 microns
Temporal resolution	4 minutes (max)

Microscope can be used with epi or TIRF illumination.