Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach

Abstract

We use the lipophilic fluorescence probe Laurdan to study cell membranes. The generalized polarization (GP) of Laurdan-labeled cells contains useful information about membrane fluidity and polarity. A high GP is usually associated with low fluidity, low polarity, or high cholesterol content of the membranes, and a low GP is the opposite. We have combined the GP method and two-photon fluorescence microscopy to provide an alternative approach to study cell membranes. Using two-photon excitation in a conventional microscope offers great advantages for studying biological samples. These advantages include efficient background rejection, low photodamage, and improved depth discrimination. We performed GP measurements on mouse fibroblast cells and observed that both intensity and GP images are not spatially uniform. We tested for possible GP artifacts arising from cellular autofluorescence and lifetime quenching, using a procedure for background fluorescence subtraction and by direct lifetime measurements in the microscope. GP measured in a single cell displays a broad distribution, and the GP of 40 different cells grown on the same cover glass is also statistically distributed. The correlations between intensity and GP images were analyzed, and no monotonic dependence between the two was found. By digitally separating high and low GP values, we found that high GP values often associate with the regions of the plasma membrane and low GP values link with the nuclear membranes. Our results also show local GP variations within the plasma and nuclear membranes.

Two-photon microscopy of aorta fibers shows proteolysis induced by LDL hydroperoxides

“…LAURDAN emission was analyzed by calculating the GP value [28,29] at each pixel, thus producing a pseudo-color GP image. Figure 2A is a pseudo-color representative image of the LAUR-DAN GP, in the aorta cross-section ring, where red (high GP) endothelial cells are facing the lumen and yellowgreen (low GP) smooth muscle cells are visible in the space between the matrix fibers.…”

“…From the images at the two emissions, the generalized polarization (GP) [28] image was calculated as previously described [29]. The lifetime images were obtained using the microscope configuration described in [19] and using the harmonic response technique.…”

Relationship between Erythrocyte Membrane Phase Properties and Susceptibility to Secretory Phospholipase A2

“…The two-photon excitation images were collected on an Axiovert 35 inverted microscope (Zeiss, Thornwood, NY) at the Laboratory for Fluorescence Dynamics (Urbana, Il) as described previously (12,13,21). The excitation source was a titanium-sapphire laser (Coherent, Palo Alto, CA) tuned to 780 nm and pumped by a FIGURE 1: Effects of temperature on fluorescence emission of laurdan in erythrocytes.…”

“…To obtain laurdan GP values, dual images were collected simultaneously using two detectors, a dichroic beam-splitter, and emission interference filters (Ealing 440 and 490) (21). In preparation for the images, washed cells (about 2 × 10 6 cells) and 2 mL MBSS were combined in a dish and incubated 5 min at the desired temperature.…”

Modulation of Nicotinic Acetylcholine Receptor Conformational State by Free Fatty Acids and Steroids

“…The physical origin of Laurdan spectral properties resides in its capacity to sense the polarity and the molecular dynamics in its environment because of the effect of dipolar relaxation processes (43,44). The principal dipoles sensed by Laurdan in the membrane are water molecules (51). Thus, GP values depend on the extent of water penetration allowed by the local membrane packing, and in this sense Laurdan GP is extensively used to sense changes in phospholipid order (52).…”