###### Abstract  
Intracellular mechanical work facilitates multiple cell functions, such as material transport, cell motility, etc., and is indicative of the cell's physiological condition. Still, the characterization of intracellular mechanical work and resultant dynamics remain hard to determine in intact label-free cells. For that, we imaged live T cells via bright-field microscopy and studied fluctuations in the homogeneity of their intracellular medium. Specifically, we characterized medium homogeneity and dynamics by using the information entropy of its related intensity gray levels (termed Gray Level Information Entropy (GLIE)) and spectral analysis of GLIE fluctuations, respectively. First, we provide simple examples of particle motion, to demonstrate the utility of our approach. Using this approach, we could further study and distinguish mitochondrial dysfunction and ATP depletion state in live Jurkat cells. The relation of our results to intracellular dynamics was confirmed by comparison to image correlation spectroscopy (ICS) results in the same cells. Importantly, GLIE fluctuations combined with spectral analysis enabled differentiation of malignant Jurkat cells from benign lymphocytes with 86% accuracy for single cells and 95% for populations of 10 cells each. Our approach can serve for label-free live-cell study and diagnostics of important pathophysiological conditions, such as mitochondrial dysfunction and malignancy. malignancy; medium homogeneity; information entropy; microscopy; image processing; spectral analysis +
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of metabolism (e.g., normal vs. malignant cell metabolism) [4; 9; 10]. They also relate to intracellular rheology parameters, such as elasticity and viscosity [3]. Previously, [PERSON] et al. [4] described how mechanical work and motion are reduced after cellular ATP depletion. These parameters were higher in malignant cells, relative to benign cells. Their work utilized tracking of intracellular injected nano-particles, vesicles and proteins complexes in multiple benign and malignant cells. Indeed, intracellular particle tracking is a powerful experimental tool for evaluation of cell dynamics [9; 10; 11; 12]. The analysis of that intracellular dynamic's characteristics could reveal cellular pathophysiology conditions, such as malignancy, activation, or mitochondrial dysfunction and ATP depletion. Still, this method has some prominent disadvantages that prevent the application of this method for clinical use. Disadvantages include the need to apply thresholds to the original images in order to rely on a specific range of pixel intensities (while ignoring the other pixels data). Then, this process requires collecting a large number of trajectories that usually have a broad distribution of motion characteristics. The analysis in this approach is very time consuming and difficult to automate. This makes that approach more suitable for laboratory research than for clinical use. Other experimental methods to analyze intracellular dynamics, such as Optical Coherence Tomography (OCT) speckle decorrelation [13] or Diffusion NMR spectroscopy [14], require much more advanced experimental setup. In addition to particle tracking, time-dependent fluctuations in general characteristics of the medium, such as homogeneity or information entropy, may depend on intracellular motion. By that, they could also applied for the study of intracellular dynamics [15]. Through this approach, all pixels of a cell image can be considered for analysis. In contrast, when using particles tracking, only a selection of specific pixels that characterize the contour of particles are followed. That kind of process, which relies on a small number of selected pixels, requires high resolution and low errors of localization. Monitoring changes in pixels' homogeneity or in their Gray Level Information Entropy (GLIE) is less optically demanding because there is no need to follow the motion of the delicate contour line of particles.