Imaging Guide

Microscope Configuration

To successfully image PRISM/SPRINTseq libraries, your microscope needs the following specs:

• Objective: 20x or 40x (Air or Oil depending on resolution needs). High NA (>0.75) recommended.
• Channels: DAPI, FITC (488), Cy3 (550), Cy5 (647), Cy7 (750).
• Camera: sCMOS recommended for large FOVs.

Image Acquisition

1. Focus: Auto-focus on the DAPI channel.
2. Exposure: Adjust exposure so signals are ~20-50% of dynamic range. Avoid saturation.
3. Z-Stacks: Typically 3-5 slices with 1 µm step size is sufficient for thin tissues (10 µm).

Data Storage

• Save as 16-bit TIFF.
• Naming convention: Experiment_Sample_Region_Channel.tif