In this study we have modified the BioPhotonics workstation (BWS), which allows for using long working distance objective for optical trapping, to include traditional epi-fluorescence microscopy, using the trapping objectives. We have also added temperature regulation of sample stage, allowing for fast temperature variations while trapping. Using this modified BWS setup, we investigated the internal pH (pHi) response and membrane integrity of an optically trapped Saccharomyces cerevisiae cell at 5 mW subject to increasing temperatures. The pHi of the cell is obtained from the emission of 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, at 435 and 485 nm wavelengths, while the permeability is indicated by the fluorescence of propidium iodide. We present images mapping the pHi and permeability of the cell at different temperatures and with enough spatial resolution to localize these attributes within the cell. The combined capability of optical trapping, fluorescence microscopy and temperature regulation offers a versatile tool for biological research.