Hi everyone,
I am working on a CyCIF project and need to image a large tissue area (approx. 2cm x 2cm). I am using an Olympus FV3000 point-scanning confocal.
In previous pilot runs on smaller areas, I used a 20x (NA 0.75) objective with the Galvano scanner. However, scaling this to 2cm x 2cm results in scan times exceeding 10 hours per round, which is not sustainable for a multi-round CyCIF workflow. The resolution I am imaging at is 1024x1024. I am doing Z-stacks with 5 steps in each image, but before processing we do a z-projection.
Is 10x magnification (NA 0.4) generally sufficient for MCMICRO/cell segmentation and immune phenotyping in tissue sections, or is the loss of NA too detrimental?
Also, do you have any suggestions on how to reduce the imaging time?