Microscopy-based imaging screens can be applied in a high-throughput format to characterize a broad spectrum of cellular processes. They generate large-data sets and on of the key challenges is the analysis, including identification of cells (with many different shapes) under many different perturbation conditions.
Different fluorescent markers are used to identify cells, quantify changes in cell shape and viability, cell cycle, homeostasis as well as biophysical properties and kinetics of particular proteins. A short summary of fluorescent markers is listed below.
DAPI or Hoechst are used for counting cells per well (proliferation/viability) and for cell cycle analysis using plate cytometry or fluorescence microscopy. The cell cycle is analysed by measuring the total emitted fluorescence intensity per nucleus and subsequent gating from the distribution of cells in one well or all wells of a 384-well plate.
Tubulin and actin are visualized using fluorescence-labled antibodies. Cytoskelletal stains are used for image segementation (separation and identification of single cells in an image or well) and observation of cell morphology (phenotypic profiling).
Cytomplasmic stain is achieved by loading the cell with a cell-permeant dye (e.g. AM esters) or by transient or stable expression of recombinant GFP variants. This method is choosen for normalization purposes in combination with luminescence reporters or for kinetic measurements of cellular processes.
Sub-cellular compartments such as endosomes, lipid droplets, etc. are stained using loadeble dyes, antibodies or transfected GFP variants
GFP-fusion proteins and variants
GFP variants fused to target genes enable quantification of kinetics and localisation of highly conserved signaling pathways relevant in a vaiety of diseases.
Most of the mentioned reporters can be combined in one experiment and can serve in multiplexing experiment.