Sample preparation

Remember- you are imaging a physical interaction between light and the chosen label for your target. Even the most sophisticated microscope will only give you good/reliable enough data as your sample quality!

After phrasing your question, and before diving into tedious imaging sessions and analysis optimizations, take the time to think about your sample preparation:

Choice of labelling: 
   - Affinity, Size – Consider the effect on the interaction with your target.
   - Fluorescent Proteins/Fluorescent Dyes
   - Spectral properties: aim for good signal to noise ratio, avoid crosstalk for co-localization.
   - For long-term imaging: consider phototoxicity and photobleaching 
   - Are you interested in Correlative light-electron microscopy? Make sure your sample label fits both.

Improving your sample’s optical properties:
   - Clearing 
   - Expansion  Microscopy- a bench strategy for Super-resolution 

Including controls for quantitative measurements
   - Find the center- using fluorescent beads for co-localization and deconvolution 
   - Always use a single label for multi-label imaging
   - Try to also include a biological control to verify your observation
   - Check antibody specifity with no primary antibody control!
   - Statistics Statistics Statistics!!