FAQ

The imaging depth can go up to 5 um.

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

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

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

abbelight's system includes:

• SAFe excitation (TIRF, HiLo, epi)
• SAFe detection (DAISY technology, electronic control)
• NEO SAFe software (live acquisition, treatment and analysis)

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

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).

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

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

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

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

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

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.

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.

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

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)

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).

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.