Analysis of Aquaporin-Mediated Diffusional Water Permeability by Coherent Anti-Stokes Raman Scattering Microscopy

Abstract

Water can pass through biological membranes via two pathways: simple diffusion through the lipid bilayer, or water-selective facilitated diffusion through aquaporins (AQPs). Although AQPs play an important role in osmotic water permeability (P(f)), the role of AQPs in diffusional water permeability remains unclear because of the difficulty of measuring diffusional water permeability (P(d)). Here, we report an accurate and instantaneous method for measuring the P(d) of a single HeLa S3 cell using coherent anti-Stokes Raman scattering (CARS) microscopy with a quick perfusion device for H(2)O/D(2)O exchange. Ultra-high-speed line-scan CARS images were obtained every 0.488 ms. The average decay time constant of CARS intensities (τ(CARS)) for the external solution H(2)O/D(2)O exchange was 16.1 ms, whereas the intracellular H(2)O/D(2)O exchange was 100.7 ± 19.6 ms. To evaluate the roles of AQP in diffusional water permeability, AQP4 fused with enhanced green fluorescent protein (AQP4-EGFP) was transiently expressed in HeLa S3 cells. The average τ(CARS) for the intracellular H(2)O/D(2)O exchange in the AQP4-EGFP-HeLa S3 cells was 43.1 ± 15.8 ms. We also assessed the cell volume and the cell surface area to calculate P(d). The average P(d) values for the AQP4-EGFP-HeLa S3 cells and the control EGFP-HeLa S3 cells were 2.7 ± 1.0 × 10(-3) and 8.3 ± 2.6 × 10(-4) cm/s, respectively. AQP4-mediated water diffusion was independent of the temperature but was dependent on the expression level of the protein at the plasma membrane. These results suggest the possibility of using CARS imaging to investigate the hydrodynamics of single mammalian cells as well as the regulation of AQPs.

Direct visualization and quantitative analysis of water diffusion in complex biological tissues using CARS microscopy

“…However, we confirmed that WT-MDCK cysts did not express endogenous AQP4 under normal circumstances. In WT-MDCK cysts, the water permeability of the luminal membrane (1.2 ± 0.2 × 10 −3  cm/s) was similar to that measured previously in single Hela cells (8.3 ± 2.6 × 10 −4  cm/s)13. The mAQP4-MDCK cells formed cysts with enhanced expression of mAQP4 protein in the basolateral membrane (Fig.…”

“…Since we established our CARS microscopy system and confirmed its reliability using single mammalian cells13, we have developed a novel strategy to characterize the dynamics of water diffusion in complex biological tissues. In the current study, we applied the CARS technique to epithelial 3D cysts formed by Madin-Darby canine kidney (MDCK) cells14 and renal cortical collecting duct (CCD)-origin M-1 cells15 and succeeded in directly observing H 2 O/D 2 O exchange across the epithelial cysts.…”

Direct visualization of the arterial wall water permeability barrier using CARS microscopy

“…However, fs pulses provide a better match with the broad Raman resonance of water. Water and D 2 O CARS imaging has previously been performed in simple systems, using either ps (34,40,41) or fs (42) pulses, although no direct comparison has been presented. We tested both excitation schemes for water imaging and compared their performances.…”

Molecular Imaging of Aquaglycero-Aquaporins: Its Potential for Cancer Characterization

“…2,16) On the other hand, radioactivity did not change statistically with time in muscle where only AQP1 and AQP4 are expressed. 17) As water exchange through AQP was reported to be quite fast, 26) glycerol exchange between extra-and intra-cellular space by the cells expressing high level of AQP3/AQP9 can be expected to be much faster compared with the time resolution of in vivo measurement in this study, which could explain the nearly equilibrated tissue […”