In situlabel-free static cytometry by monitoring spatiotemporal fluctuations of image gray values

Abstract

Spatiotemporal fluctuation of homogeneity and randomness of gray values within an image was explored and utilized as a label-free means for cell examination. This was done by utilizing a user-friendly combination of simple bright field microscope and Cytocapture dish, wherein cells are individually held, each within a picoliter optical chamber, forming an array of cells to be repeatedly measured over time and biomanipulated in situ at single-cell resolution. First, the measured gray level information entropy (GLIE) was used and, based on the fact that living cells are not in a state of thermodynamic equilibrium but rather in a metastable state, two fluctuation-sensitive measures were proposed and examined: ASDE—the spatial average of temporal standard deviation (SD) of GLIE, and AA—the average time autocorrelation of GLIE. System performance was validated on cell-free solutions. This was followed by examining the performance of the measures AGLIE, ASDE, and AA to distinguish among individual live-still, dead and live cells from various cell lines, as well as between cells which were and were not induced to differentiate. Results, which were obtained on four types of cells, indicate advantages of the proposed measures which are believed to be significant additions to the microscope-based probe-free toolbox.

Fluctuations in the Homogeneity of Cell Medium Distinguish Benign from Malignant Lymphocytes in a Cellular Model of Acute T Cells Leukemia

“…Instead, GLIE fluctuations monitor the general chance of all image pixels to experience changes in gray levels due to particles motion. Evaluation of cell dynamics through GLIE fluctuation analysis enabled differentiation of live from dead cells [16] and exploration of a range of cellular physiological conditions that included malignancy [8,15].…”

Fluctuation of Information Entropy Measures in Cell Image

“…For instance, apoptotic and other recently dead cells have low entropy and GLIE fluctuations [18,20,27], while malignant cells have high entropy and GLIE fluctuation in comparison to matched normal healthy cells [2,29,30].…”

“…High Frequency Average Power spectrum amplitude (HFAP): GLIE fluctuation measure that was determined by calculating the average DFT power amplitudes of the frequency range 0.5-1 Hz, (dictated by previous results [20,21]) of 200 time serial GLIE25 values (HFAP25), yielding 100 values of HFAP25. Then, the average of these 100 HFAP25 values was defined as the HFAP value of a ROI.…”

“…Motivated by the fact that living cells are not in a state of thermodynamic equilibrium, but rather in a metastable state [4,19], our studies [20,21] focus on the examination of the fluctuations of the measure Gray Level Information Entropy (GLIE) and its informative value.…”

“…GLIE fluctuation parameters, based on standard deviation (SD) of temporal GLIE value distributions or autocorrelation of GLIE temporal changes, could be utilized to successfully distinguish dead cells from live ones in multiple cell lines with sensitivity and specificity values between 92% and 100% [20]. GLIE fluctuation parameters, based on discrete Fourier transform (DFT) analysis of the temporal GLIE changes enable evaluation of multiple cellular physiologic stimulations and mitochondrial dysfunction conditions [21].…”