Sponsored Content: GAIA: Deep Live Cell Imaging in Super Resolution
In theory, super resolution (SR) encompasses microscopy techniques surpassing the diffraction limit, defined by Abbe’s law as d = wavelength/2NA. Practically, SR achieves resolution better than 200-240 nm using high NA objectives (ca. 1.50), typically characterized by high magnification and short working distance (WD). Importantly, resolution depends solely on NA, not magnification.
NA/WD challenge is the central obstacle for deep SR imaging, the higher the NA, the better the resolution yet the shorter the WD of an objective. An NA 1.50 objective offers a lower diffraction limit and better resolved structures than NA 1.05 one, yet the former has a WD of 120 microns allowing imaging only close to the cover slip whereas the latter can image up to 800 microns deep into the sample.
For live cell imaging, most SR techniques (e.g., STORM, ExM, STED) face limitations due to sample preparation, long acquisition or high laser power. SR techniques providing resolution between 100 and 200 nm: SIM, fluctuation based (SOFI, SRRF) and pixel reassignment (PR) techniques are more cell friendly. PR techniques have proven to be the least phototoxic in SR and, therefore, the most suitable for live cell SR imaging (Jacquemet et al, J Cell Sci 2020).
Rescan confocal microscopes stand out among PR techniques for its advantages in both deep and live cell SR imaging. The latest microscope developed by Confocal.nl, Point REscan GAIA, excels in this. GAIA, optimized for SR imaging with objectives ranging from 30x-100x, employs objectives with longer WD, thereby enhancing deep SR imaging. Its more open pinhole (≥1.5 AU without resolution loss) and high quantum efficiency detector minimize phototoxicity rendering GAIA superior for live cell imaging.
GAIA was used to image a living zebrafish 400 microns in depth using 30xSil objective with 1.05NA. Since imaging at 30 micron depth is already considered deep in super resolution, we refer to imaging at 400 micron and more in depth as profound. Profound SR imaging with GAIA revealed dynamic events at a whole-animal scale, exposing organ intricacies from the spinal cord to the beating heart. Switching to a 60xSil objective with 1.30 NA provided more detailed structures of pectoral fin neurons.
GAIA Point REscan is the perfect solution for NA/WD challenge as it is equipped with a switchable pinhole enabling ideal confocality for a full range of objectives. Optimized for VIS and NIR imaging provides versatility with applications and model systems. Whether the sample is cell line, growing organoid, embryo or an entire zebrafish, GAIA ensures live cell friendly imaging in super resolution over long periods and deeper than 500 microns into the sample. GAIA can be mounted on any microscope body. Adding a camera and a laser gives rise to fully equipped confocal super resolution microscope optimized for profound live cell imaging.