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?
An important consideration: what XY resolution do you really need? Do you scan at nyquist resolution? Perhaps you can zoom out, making larger pixels and speed up image acquisition? The Nikon Resolution Calculator I find a useful tool to assess XYZ resolution for point scanners.
Fast confocal imaging of large areas may be better done on a spinning disk confocal or slit scanner confocal (AION). Do you have access to any at your institute or nearby?
At low magnification the pinhole/ slit is often too large, reducing the ‘confocal effect’, but the imaging time is much shorter as trade-off. Especially on a dual camera system and if imaging deeper with water immersion objective your imaging time should drop to < 1 hour.
Alternatively, point scanners with resonant capabilities may be useful too; especially if they do not require a minimal zoom so the confocal covers a large field of view at 2048x2048 pixel density.