The Nikon Eclipse Ti N-STORM Superresolution system combines the latest advances in photonics, sample preparation and image processing to allow researchers to visualise nanometric biological structures using fluorescence microscopy. Historically, our ability to resolve optically structures has been limited by diffraction to a maximum of approximately ~200nm laterally (xy) and ~600nm axially (z). Superresolution techniques such as STORM use the selective activation of fluorescent markers to map their position with up to 10 times more accuracy. The N-STORM system is based on the methods devised in the laboratory of Xiaowei Zhuang (Harvard University) and can achieve a resolution of approximately 20nm laterally and 50nm axially and can also be used to perform two colour super-resolution imaging. With the aim of promoting the use nanoscale imaging in biomedicine, Bionand has established one of Spain´s first N-STORM superresolution platforms dedicated to practical applications.

N-STORM super-resolution

The key to the localisation-based super resolution techniques such as N-STORM is to be able to cyclically activate only a small subpopulation of fluorescent molecules at a time and thus avoid simultaneously activating adjacent fluorophores. The more events (photons) that can be detected for each individual fluorescent molecule the better is the effective resolution. The N-Storm approach uses the cyclical activation and inactivation of bright “reporter” fluorochromes such as Alexafluor 647 (Molecular Probes) combined with high quality optics and a very sensitive EM-CCD camera to capture thousands of activation events. These events can then be analysed to generate the super-resolution image. The Nikon N-Storm approach also has the advantage of permitting two colour imaging by combining different activator fluorochromes such as Alexafluor 405 (Molecular Probes) and Cy3 (GE). More information on how the N-Storm technique works can be found in the links below. The Bionand N-STORM superresolution imaging platform aims to assist end users by constantly working to optimise sample preparation and imaging protocols for different real world applications.

Specifications

• Inverted Nikon Ti Eclipse fully motorised microscope.
• Andor iXon3 897 EM-CCD camera
• 100x Plan Apo Oil immersion Objective 1.49 NA
• 4 Lasers in the visible range:
  • Violet 405nm
  • Argon-Ion 457nm, 477nm, 488nm, 514nm
  • Yellow Diode 561nm
  • Red 647nm (300mW)
• High precision motorized stage.
• TIRF optical system (used to improve contrast with N-STORM samples)
• Removable cylindrical lens for 3D superresolution.
• Nikon PFS (Perfect Focus System) for improved stability
• NIS-Elements with N-STORM acquisition
• Offline workstation with additional processing module]

Applications

• Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution
  • Löschberger et al. Journal of Cell Science 125, 570-575 (2012)
• 3D Multicolor Super-Resolution Imaging Offers Improved Accuracy in Neuron Tracing
  • Lakadamyali et al. PLoS One. 7(1): e30826. (2012) ^{Open access}
• Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes
  • Kopek et al. PNAS 109 (16): 6136–6141(2012) ^{Open access}
• Superresolution Imaging of Chemical Synapses in the Brain
  • Dani et al. Neuron, Volume 68, Issue 5, 843-856 (2010)
• Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes
  • Bates et al. Science Vol. 317 no. 5845 pp. 1749-1753 (2007)

Helpful information

• Open Nikon Nd2 files with Fiji or ImageJ and the Bioformats plugin. Also available to local users via in the GGI Software folder on the intranet.
• Superresolution introduction (Nikon)
• Superresolution Microscopy (Wikipedia)

Related resources

• Nikon Eclipse Ti TIRF