Coherent anti-Stokes Raman scattering microscopy of D. discoideum. (a) CARS image of a single dictyostelium cell in aqueous medium. Pump laser was tuned to 636 nm, and Stokes beam was set to 800 nm to address the O—H-stretch vibration of water. The image is recorded in 5 s and consists of 16 averages per pixel. (Bar = 2 μm.) (b) Autofluorescence image measured simultaneously with a.

Comparison between experimental traces and simulation. (a) Experimentally determined water concentration profile in a single D. discoideum cell. Medium exchange starts at t = 0, indicated by dashed line. Vertical black lines mark the positions of the plasma membrane. Figure was corrected for variations in signal because of scattering at membranes and intracellular structures. The image is composed from the average of 24 individual measurements by correlative image analysis; the x axis is scaled proportional to the average cell diameter. (b) Simulation based on the model of a spatially dependent diffusion coefficient. The region of restricted water diffusion adjacent to the plasma membrane is set to 20% of the cell diameter. A diffusion constant of D_w = 5 μm²/s was used in the simulation for the zone of restricted water diffusion; outside this region, the diffusion was assumed to be water-like (D_w > 500 μm²/s). The membrane permeability was found to be P_d = 2.2 μm/s.

TPE fluorescence of D. discoideum that have been labeled with ANTS. (a) TPE fluorescence intensity image. Fluorescent probe is accumulated in cytosolic vesicles. (Bar = 2 μm.) (b) Fluorescence lifetime image of the same cell. Average lifetime is determined to be 4.5 ns, corresponding to an aqueous environment. (c) Simultaneous measurement of total intracellular CARS signal (solid line) and lifetime change of the ANTS fluorophore (dots) during a flushing event. Dashed line indicates the response time of the perfusion chamber. Inset shows the change in TPE fluorescence lifetime of a 10 mM ANTS solution for different volume fractions of D₂O relative to H₂O.

Vibrational spectra in the region of the O—H stretch vibration of water. Squares correspond to CARS spectrum of distilled water, whereas triangles refer to the CARS response from D₂O. The solid line represents the spontaneous Raman band profile.

Spatiotemporal CARS recording of a single cell that is initially immersed in aqueous medium and subsequently flushed with isotonic D₂O medium. The dashed line indicates the moment at which the perfusion chamber is flushed. Shaded horizontal lines correspond to the position of the plasma membrane. Arrows, 3 μm (horizontal, x), 1.5 s (vertical, t).

Analysis of plasma membrane permeability as determined from water efflux measurements. Distribution of membrane permeability constants of AX3 D. discoideum cells (gray bars) and of aquaporin-transformed act15∷rd28 cells (black bars).