This repository contains HILO microscopy images of either differentiated immortalized mouse brown preadipocytes or differentiated 3T3-L1 cells, as indicated in each image file name. Cells were transiently transfected with either YFP-tagged free fatty acid receptor 4 (FFA4) or Halo-tagged β2-adrenergic receptor (B2AR), labelled with JF646. These fluorescently tagged receptors were used to train a machine learning–based pixel classifier in Ilastik to categorize receptor pixels as either plasma membrane or intracellular.

In some images, cells were also co-transfected with mini-G protein probes, which translocate from the cytoplasm to active receptors upon stimulation. MATLAB was used to quantify mini-G translocation to FFA4 or B2AR at both the plasma membrane and intracellular membranes.

Each file name includes, in order: the corresponding figure, cell line, transfected overexpressed proteins, laser excitation wavelength, stimulant used, and cell number.