What is the imaging depth using abbelight's system?

The imaging depth can go up to 5 um.

Which image file formats are generated by abbelight's software?

The image files and coordinate tables are generated in TIF and TXT.

How long does it take to deliver abbelight's buffer?

In Europe, delivery the next working day and in other countries, delivery takes 48h.

What is the size of the field of view?

abbelight's illumination system provides a 200x200 um² field of view.

What does abbelight's system include?

abbelight's system includes:
  • SAFe excitation (TIRF, HiLo, epi)
  • SAFe detection (DAISY technology, electronic control)
  • NEO SAFe software (live acquisition, treatment and analysis)

Which lasers are integrated in abbelight's system?

abbelight's SAFe module integrates 4 lasers (405 nm, 488 nm, 555 nm, 639 nm).

What is the resolution of abbelight's system?

An isotropic 15x15x15 nm3 localization precision can be achieved.

What is the difference between SMLM, PALM, STORM and PAINT?

Single molecule localization microscopy (SMLM) consists in inducing the fluorescence emission of only a subset of molecules at a given time in order to localize each of them individually. By repeating this process, you can accumulate enough localizations to reconstruct a final super-resolved image. STORM, PALM and PAINT are all based on SMLM principle. The only difference is the way to induce the stochastic emission of the fluorophores:
  • In STORM (Stochastic Optical Reconstruction Microscopy), fluorescent organic dyes (cyanines, rhodamines, …etc.) together with a specific imaging buffer (abbelight's buffer) are used to allow blinking of the fluorescent molecules;
  • In PALM (Photo-activated Localization Microscopy), photoactivatable, photoconvertible or photoswitchable proteins are used (ex: PA-GFP, PA-mCherry, mEOS, mMAPLE, …etc.);
  • In PAINT (Point Accumulation for Imaging in Nanoscale Topography), reversibly-binding fluorescent probes are used (ex: Nile Red).

Is the software limited to STORM imaging?

abbelight's NEO software is capable of handling localization in any type of single molecule data (PALM, PAINT, STORM, …).

Does abbelight's NEO software include analysis tools?

abbelight's NEO software software includes custom analysis tools to provide quantitative data. We implement clustering, co-localization and tracking algorithms.

Is there a way to know when a STORM image is optimal?

A decision-making tool is implemented in the software to determine when the STORM image is optimal and the acquisition can be stopped.

Is the module adaptable to classical microscopes? If so, does it compromise on existing imaging modalities?

abbelight's SAFe module is adaptable to any inverted microscope (epifluorescence, confocal, …etc.) without compromising on existing imaging modalities.

Is abbelight's system limited to TIRF excitation?

abbelight's system works with any type of wide-field excitation, including epi, HiLo, TIRF.

What the advantage of using abbelight's system?

abbelight's module is a dual-view system that exploits supercritical angle fluorescence emission (Bourg et al. Nature Photonics 2015) and strong astigmatic PSF engineering to extract axial information (Cabriel et al. BiorXiv 2018). The combination of these two techniques is reference-free and does not require calibration (Cabriel et al. Optics Letters 2017). The localization precision is 15x15x15 nm3, the field of view is 200 x 200 um² and the imaging depth can go up to 5 um.

How long can abbelight's buffer be conserved?

Once the sample is sealed, abbelight's buffer can be conserved for one week. Otherwise, it can be stored, as received, for 2 months at 4°C.

Which dyes do you recommend for dual–color 3D STORM imaging?

For the majority of immunostaining applications, we recommend the combination of AlexaFluor 647 and AlexaFluor 555 conjugated secondary antibodies. Additional combinations are possible.

Which coverslips are required for STORM imaging?

Coverslips n°1.5H (Marienfeld, precision cover glasses thickness No. 1.5H (tol. ± 5 μm), minimum size: 20*20 mm or 20 mm diameter, or equivalent)

Can abbelight's buffer be used for live imaging?

We recommend the use of our buffer on fixed samples, since the anoxic conditions needed for the photoswitching of organic fluorophores are mostly incompatible with live imaging of eukaryotic cells; however, we have successfully used it to image bacteria (Boudjemaa et al. AAC 2018).

Which fixation methods do you recommend for STORM imaging?

The best preservation of ultrastructure is in most cases obtained with chemical fixation with aldehyde (PFA, glutaraldehyde). However, in some cases, we recommend organic solvents (methanol). The choice is highly contingent on the structure of interest and, in the case of immunostaining, on the antibodies used. Thus the optimal fixation method needs to be optimized case by case.