Comparison of Widefield imaging with RCM on living Huvecs stained for Bacmam 2.0 Mitochondria-GFP. It can be clearly observed that RCM is a much more gentle imaging method since there is no observable bleaching. Here we used the Hamamatsu Flash 4 V3 camera for both imaging modalities. Illumination intensity was such that the readout of the emission intensity was the same.
Sample courtesy: Rio Juni, Amsterdam UMC, location VUmc
Mouse hippocampal neurons (DIV14) stained with Bacmam 2.0 mitochondria GFP (green), lentiviral cellfiller mCherry (red) and SiR-DNA (magenta). Look closely and you will see mitochondrial fission/fusion, Nuclear rotation and axonal trafficking. This is a z-stack in time, deconvolved with on-the-fly Microvolution software and subsequently max projected. The neurons were imaged for approximately 3 hours, acquiring close to 4000 frames. Colors were acquired simultaneously, using the Multisplit V2 from Cairn Research. Laserpower was measured to be 3 microwatt at sample plane for the 488 laser, 4 microwatt for the 561 and 3 microwatt for the 640.
Sample Courtesy: Vera Wiersma (VU, CNCR, Amsterdam)
HO1N1 cells stained with Bacmam 2.0 Mito-GFP (green), ER-tracker (blue) and SiR-DNA (red). Sequential colors were taken every 10 seconds for 1.5 hours, giving a total of 1000 images. Because the RCM is very sensitive laser power can be kept very low (few microwatts), enabling long-term imaging. The native lateral resolution of the RCM is 170 nm. After deconvolution by SVI Huygens the resolution is improved to 120 nm!
Movie taken by Jeroen Kole (Confocal.nl), sample courtesy Dandan Ma (ACTA, Amsterdam), equipment provided by Marko Popovic (Nikon Center of Excellence, Amsterdam University Medical centers, VUmc, Amsterdam.)
HO1N1 cells expressing Mitochondria-RFP through the Bacmam expression system. One image was taken every 10 seconds for 61 hours (giving a total of almost 22,000 images!). Laser power was measured to be 1 microwatt at the sample plane. Movie taken by Jeroen Kole (Confocal.nl), sample courtesy Dandan Ma (ACTA, Amsterdam), equipment provided by Marko Popovic (Nikon Center of Excellence, Amsterdam University Medical centers, VUmc, Amsterdam.)
The first video shows the RAW data from RCM, the entire 61 hour time lapse.
The second video shows the benefits of deconvolution. We used SVI Huygens CMLE deconvolution to improve the resolution to 120 nm and have an even better signal-to-noise ratio.
Tip: watch in HD!
Maximum-projection of a Z-stack in time. Arabidopsis hypocotyl cells expressing GFP-EB1b – the protein binds to +ends of microtubuli and indicates their growth (so called comet-like movement). EB1b also binds to nuclear membrane, as seen by the high intensity structures. Sample courtesy: Dr S L Shaw. Acquired during the AQLM course at the MBL in Woods Hole, MA, USA.