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Fluorescence imaging is an essential tool in biological research, which enables the visualization of proteins in live and fixed cells. The resolution of images using conventional techniques, such as confocal microscopy, is limited by the microscope and by the diffraction properties of light to around 200 nm. The last two decades have seen the development of super-resolution microscopy (SRM) techniques, such as photoactivated localization microscopy (PALM), and direct stochastic optical reconstruction microscopy (dSTORM), which “cheat” the diffraction limit of light resulting in improvements in resolution of up to ten-fold. Both PALM and dSTORM are single molecule imaging techniques in which individual fluorescent molecules are sequentially and repeatedly activated and deactivated using light of specific wavelengths. The localized positions of thousands of individual fluorophores are then digitally reconstructed into a “super-resolved” image.
Fluorescence imaging requires the precise labeling of biomolecules with bright fluorophores. Luke Lavis’ lab at the Janelia Research Campus, Howard Hughes Medical Institute (HHMI), recently developed a range of new rhodamine-based fluorescent dyes, known as Janelia Fluor®dyes, which were first described by Grimm et al. in 2015.
These dyes are suitable for use with conventional imaging techniques, such as confocal microscopy, as well as in SRM techniques, including dSTORM. In addition photoactivatable derivatives have been developed that are applicable in PALM (see Grimm et al. 2016).
Janelia Fluor® dyes are very bright and photostable and importantly, they are cell-permeable, enabling live-cell intracellular imaging. The small molecule fluorophores are supplied as NHS esters allowing for conjugation to an antibody of choice, and are also compatible with self-labeling tag systems. Tocris acquired a license to the Janelia Fluor® dyes and their photoactivatable derivatives from HHMI, Janelia Research Campus, and made them commercially available in 2017.
The image shows a single isolated Wistar rat cardiomyocyte stained against α-actinin, displaying its periodic structure localized at the ends of sarcomeres (Z-discs). Wide-field fluorescence of Janelia Fluor® 549 (left panel), prior to illuminating with a 561 nm laser to induce photoswitching and produce a dSTORM super-resolution image (right panel). Scale bar: 1 µm. Image kindly provided by Prof. Christian Soeller, University of Exeter, UK, acquired by Alex Clowsley and Anna Meletiou.