Living cells are crowded with macromolecules and organelles. Yet, it is not fully understood how macromolecular crowding affects the myriad of biochemical reactions, transport and the structural stability of biomolecules that are essential to cellular function and survival. These molecular processes, with or without electrostatic interactions, in living cells are therefore expected to be distinct from those carried out in test tube in dilute solutions where excluded volumes are absent. Thus there is an urgent need to understand the macromolecular crowding effects on cellular and molecular biophysics towards quantitative cell biology. In this report, we investigated how biomimetic crowding affects both the rotational and translation diffusion of a small probe (rhodamine green, RhG). For biomimetic crowding agents, we used Ficoll-70 (synthetic polymer), bovine serum albumin and ovalbumin (proteins) at various concentrations in a buffer at room temperature. As a control, we carried out similar measurements on glycerolenriched buffer as an environment with homogeneous viscosity as a function of glycerol concentration. The corresponding bulk viscosity was measured independently to test the validity of the Stokes-Einstein model of a diffusing species undergoing a random walk. For rotational diffusion (ps–ns time scale), we used time-resolved anisotropy measurements to examine potential binding of RhG as a function of the crowding agents (surface structure and size). For translational diffusion (μs–s time scale), we used fluorescence correlation spectroscopy for single-molecule fluctuation analysis. Our results allow us to examine the diffusion model of a molecular probe in crowded environments as a function of concentration, length scale, homogeneous versus heterogeneous viscosity, size and surface structures. These biomimetic crowding studies, using non-invasive fluorescence spectroscopy methods, represent an important step towards understanding cellular biophysics and quantitative cell biology.

Living cells are known to be crowded with organelles, biomembranes, and macromolecules such as proteins, DNA, RNA, and actin filaments. In such crowded environments it is reasonable to believe that cellular viscosity is heterogeneous, which is likely to influence biomolecular diffusion, protein-protein interactions, protein-substrate interaction, and protein folding. In this contribution, we investigate the difference between bulk viscosity and microviscosity in crowded environments and their effects on both rotational (ps-ns) and translational (ms-s) diffusion of rhodamine green (as a probe) using time-resolved fluorescence anisotropy (TRFA) and fluorescence correlation spectroscopy (FCS), respectively. For biomimetic crowding, Ficoll-70, BSA and ovalbumin were used as crowding agents and compared with glycerol-rich solutions as a homogeneous environment. Assuming a Stockes-Einstein model, the microviscosity was calculated using TRFA and FCS, assuming no binding, and the results are compared with the bulk viscosity, which was measured using a conventional viscometer. Our results indicate that the micro- and bulk viscosities in a homogeneous environment like glycerol-rich solutions are similar over the 1-20 cP range. In Ficoll-70, BSA and ovalbumin-crowded environments, the microviscosity differs from the corresponding bulk viscosity, depending on the nature of crowding agents (i.e., proteins versus polymers) and the concentration of crowding agents. These results are discussed in terms of both non-specific binding and heterogeneous viscosity in crowded solutions, which in return provide an apparent deviation from the Stokes-Einstein model (i.e., Brownian diffusion). Our findings provide a foundation for FCS and TRFA-based studies of diffusion and binding of biomolecules in the crowded milieu of living cells.

Mitochondria play vital roles in energy metabolism, apoptosis, oxidative stress, aging, and neurodegenerative disease [1]. In this contribution, we probe different aspects of cellular response to chemical-induced oxidative stress in living C3H10T1/2 cells using hydrogen peroxide, rotenone, and excess glucose. Using two-photon fluorescence lifetime imaging microscopy (2P-FLIM), we exploit the autofluorescence dynamics of natural coenzymes such as nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and flavoproteins as intrinsic biomarkers for oxidative stress. The effects of polarization selectivity in 2P-FLIM measurements are being investigated towards the development of a quantitative, genuine non-invasive 2P-FLIM of patho-physiological changes in living cells. The efficiency of 2P-FLIM cellular autofluorescence for monitoring changes in the metabolic and redox states of the cells is compared with conventional assays such as MitoSOX Red, JC-1, and Rhodamine-123 that are routinely used for oxidative stress studies. Our results help in the collective effort to establish cellular autofluorescence as a natural biomarker for biological and biomedical studies. 1. Heikal, A.A. Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies. Biomarkers in Medicine, 4(2): 241-63 (2010).

Native coenzymes such as the reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide play pivotal roles in energy metabolism and a myriad of biochemical reactions in living cells/tissues. These coenzymes are naturally fluorescent and, therefore, have the potential to serve as intrinsic biomarkers for mitochondrial activities, programmed cell death (apoptosis), oxidative stress, aging, and neurodegenerative disease. In this contribution, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) and time-resolved anisotropy imaging of intracellular NADH for quantitative, non-invasive biochemistry on living cells in response to hydrogenperoxide- induced oxidative stress. In contrast with steady-state one-photon, UV-excited autofluorescence, two-photon FLIM is sensitive to both molecular conformation and stimuli-induced changes in the local environment in living cells with minimum photodamage and inherently enhanced spatial resolution. On the other hand, time-resolved, two-photon anisotropy imaging of cellular autofluorescence allows for quantitative assessment of binding state and environmental restrictions on the tumbling mobility of intrinsic NADH. Our measurements reveal that free and enzyme-bound NADH exist at equilibrium, with a dominant autofluorescence contribution of the bound fraction in living cells. Parallel studies on NADH-enzyme binding in controlled environments serve as a point of reference in analyzing autofluorescence in living cells. These autofluorescence-based approaches complement the conventional analytical biochemistry methods that require the destruction of cells/tissues, while serving as an important step towards establishing intracellular NADH as a natural biomarker for monitoring changes in energy metabolism and redox state of living cells in response to environmental hazards.