- RCM vs wide-field & confocal
- RCM vs PMT-based confocal system
- RCM Large FOV vs RCM High resolution
Comparison of RCM with wide-field and confocal imaging
In order to demonstrate the image improvement by RCM, we have made images of the same cell from deer skin fibroblast sample using wide-field, conventional confocal microscope and RCM. The image taken by RCM we have further improved using deconvolution.
Indian Muntjac – Deer Skin Fibroblast cells. Staining: Blue: DAPI, Green: Phalloidin-Alexa488, Red: Mitotracker CMXRos
Top: image from full field of view; bottom: zoom in. Images by Jeroen Kole (Confocal.nl).
|Wide-field||Zeiss Axiovert 200M||63x NA 1.4; oil||PCO sensicam (100.3nm/pixel)|
|Confocal||Nikon A1||60x NA 1.4; oil||GaAsP PMT||1 AU|
|RCM||RCM +Nikon TiE Eclipse||100x NA 1.45; oil||Hamamatsu Orca Flash 4.0||1 AU|
Deconvolution was carried out using SVI Huygens software, experimental PSF and CMLE algorithm.
Comparison of performance of RCM and a PMT based confocal system
The resolution and sensitivity of the raw RCM image is better, resulting in a better deconvolution result.
The light intensity at the sample plane was measured at: 4.5 microwatt (1.0 AU), 12.5 microwatt (0.3 AU) and 3.0 microwatt (RCM).
Nuclear spread from fixed mouse spermatocytes, immunostained for SYCP3 a component of the synaptonemal complex (Alexa 488-labelling). Upper panel: images of a PMT based confocal with 1.0 AU pinhole, 0.3 AU pinhole and RCM image. Lower panel: same images deconvolved using Huygens Essential RCM module (SVI), using an experimental point spread function. Scalebar: 1 micrometer. Sample courtesy of A. Agostinho – Advanced Light Microscopy Facility, Science for Life Laboratory. Imaging and deconvolution performed by Jeroen Kole (Confocal.nl).
RCM Large FOV vs RCM High resolution
RCM can be used in different imaging modes: Large Field of View allows maximizing the view, but at expense of resolution. In this mode, conventional confocal resolution is achieved. High Resolution, is the RCM mode providing improvement in resolution, but at the expense of the size of view.