Characteristics of spatiotemporal fluctuation of gray values at a single-pixel level within bright field, label free image of a cell are explored in a variety of physiological and mitochondrial dysfunction states. From these fluctuations, the gray level information entropy (GLIE)  is calculated and its derivative measures such as standard deviation, autocorrelation and periodic aspect are analyzed. This is realized by a user-friendly combination of common bright field microscopy and a unique imaging dish, wherein cells are individually held untethered, each within a picoliter volume optical chamber in an array, which allows repeatable spatiotemporal observation before, during and after bio-manipulation in situ, at a single-cell resolution, while in a population.

First, system performance was validated on cell-free solutions. This was followed by examining the performance of the gray values fluctuation measures to distinguish between individual dead and live cells from various cell lines. Results, which were obtained on four types of cells, indicated the ability of the proposed measures to accurately evaluate cell's viability status.

GLIE fluctuation measures were than exploited to demonstrate the gradual dying of serum-deprived cells. Furthermore, these measures and mainly Discrete Fourier Transform (DFT) analysis based measures were utilized for the evaluation of cell's response to physiological stimulators: Phorbol Myristic Acetate (PMA), triiodothyronine (T3) thyroid hormone and mild hypothermia, and three different mitochondrial inhibitors as well: rotenone, carbonyl cyanide m-chlorophenyl hydrazine (CCCP) and oligomycin.

The GLIE fluctuation-based measures demonstrated the ability to (a) significantly distinguish cellular response to all six mediators, and (b) identify subgroups of cells according to their response to mild hypothermia.

On the whole, employment of high contrast microscopy approaches, i.e. Phase Contrast (PC) and Differential Interference Contrast (DIC), for tracing cellular events via the spatiotemporal fluctuation measures, did not show noticeable advantages over simple Bright Field (BF) microscopy.