Solvation Dynamics of Biological Water in a Single Live Cell under a Confocal Microscope

Abstract

Time-resolved confocal microscopy has been applied to study the cytoplasm and nucleus region of a single live Chinese hamster ovary (CHO) cell. To select the cytoplasm and the nucleus region, two different fluorescent probes are used. A hydrophobic fluorescent dye, DCM, localizes preferentially in the cytoplasm region of a CHO cell. A DNA binding dye, DAPI, is found to be inside the nucleus of the cell. The locations of the probes are clearly seen in the image. Emission maxima of the dyes (DCM in cytoplasm and DAPI in the nucleus) are compared to those of the same dyes in different solvents. From this, it is concluded that the polarity (dielectric constant, ε) of the microenvironment of DCM in the cytoplasm is ~15. The nucleus is found to be much more polar with ε ≈ 60 (as reported by DAPI). The diffusion coefficient (and hence viscosity) in the cytoplasm and the nucleus was determined using fluorescence correlation spectroscopy (FCS). The diffusion coefficient (D(t)) of the dye (DCM) in the cytoplasm is 2 μm(2) s(-1) and is ~150 times slower than that in bulk water (buffer). D(t) of DAPI in the nucleus (15 μm(2) s(-1)) is 30 times slower than that in bulk water (buffer). The extremely slow diffusion inside the cell has been ascribed to higher viscosity and also to the binding of the probes (DCM and DAPI) to large biological macromolecules. The solvation dynamics of water in the cytoplasm (monitored by DCM) exhibits an average relaxation time [τ(sol)] of 1250 ± 50 ps, which is about 1000 times slower than in bulk water (1 ps). The solvation dynamics inside the nucleus (studied using DAPI) is about 2-fold faster, [τ(sol)] ≈ 775 ps. The higher polarity, faster diffusion, and faster solvation dynamics in the nucleus indicates that it is less crowded and less restricted than the cytoplasm.

Optical mapping of biological water in single live cells by stimulated Raman excited fluorescence microscopy

“…Inspired by this, we thus attributed higher in nucleus to a larger ratio of free water over bound water in nucleus than in cytoplasm. This qualitative trend indeed finds agreement with earlier spectroscopy results of faster solvation dynamics and more similarity to bulk-water dynamics inside nucleus than in cytoplasm 41–44 . Moreover, this attribution is also in the same trend with the concept of macromolecular crowding in which the cytoplasm is found to be somewhat more crowded than the nucleus 45,46 .…”

Using intracellular plasmonics to characterize nanomorphology in human cells

“…In addition to the different interactions of NPs with subcellular compartments, the differences in dielectric constants could be another reason for the plasmonic shifts between particles situated in the periphery and particles in the cytoplasm or the nucleus. Some studies show that the cytoplasm of cells has dielectric constants ten times higher than that of the cell membrane 41,44 , and in another study 45 , the dielectric constant of the nucleus was found to be four times higher than that of the cytoplasm. Nanostars tend to aggregate, and their LSPR peaks were broader than those of nanospheres.…”

Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs)

“…89 Dielectric constants of 15 and 60 were recently determined for the cytoplasm and cell nucleus of mammalian CHO cells, respectively. 90 …”