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.
|Instrument||Zeiss Elyra PS1||Bruker Vutara 350|
|Laser wavelengths (nm)||405 (for activation only), 488, 561, 642||405 (for activation only), 488, 561, 642, 750|
|Excitation power density||Moderate, depends on field of view||Very high|
|Compatible objective lenses||x100 oil immersion, x100 silicone oil immersion, x63 oil immersion, x63 water immersion||x60 silicone immersion, x60 oil immersion|
|Compatible dyes||Restricted, depends on blinking characteristics and photon flux||Restricted, 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|
|Configuration||Inverted microscope||Inverted microscope|
|Axial resolution method||Phase ramp||Biplane|
|Multi-wavelength imaging||Up to three in 2D or 3D, always sequential||Up to two channels in 3D, up to four in 2D, always simultaneous|
|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 range||1-1.5 microns||1-1.5 microns|
|Temporal resolution||4 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.