Two-photon Fluorescence Lifetime Imaging Research Articles

Chloride accumulation in mammalian olfactory sensory neurons.

The generation of an excitatory receptor current in mammalian olfactory sensory neurons (OSNs) involves the sequential activation of two distinct types of ion channels: cAMP-gated Ca(2+)-permeable cation channels and Ca(2+)-gated Cl(-) channels, which conduct a depolarizing Cl(-) efflux. This unusual transduction mechanism requires an outward-directed driving force for Cl(-), established by active accumulation of Cl(-) within the lumen of the sensory cilia. We used two-photon fluorescence lifetime imaging microscopy of the Cl(-)-sensitive dye 6-methoxy-quinolyl acetoethyl ester to measure the intracellular Cl(-) concentration in dendritic knobs of OSNs from mice and rats. We found a uniform intracellular Cl(-) concentration in the range of 40-50 mm, which is indicative of active Cl(-) accumulation. Functional assays and PCR experiments revealed that NKCC1-mediated Cl(-) uptake through the apical membrane counteracts Cl(-) depletion in the sensory cilia, and thus maintains the responsiveness of OSNs to odor stimulation. To permit Cl(-) accumulation, OSNs avoid the "chloride switch": they do not express KCC2, the main Cl(-) extrusion cotransporter operating in neurons of the adult CNS. Cl(-) accumulation provides OSNs with the driving force for the depolarizing Cl(-) current that is the basis of the low-noise receptor current in these neurons.

Two-Photon Fluorescence Lifetime Imaging of the Skin Stratum Corneum pH Gradient

Two-photon fluorescence lifetime imaging is used to identify microdomains (1–25 μm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime τ (pH 4.5, τ = 2.75 ns; pH 8.5, τ = 3.90 ns) is pH dependent over the pH range of the stratum corneum (pH 4.5 to pH 7.2). Hairless mice (SKH1-hrBR) are used as a model for human skin. Images (≤50 μm × 50 μm) are acquired every 1.7 μm from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (∼1 μm in diameter with variable length) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space of the corneocytes in mid-stratum corneum (25 μm diameter) approaches neutrality (average pH 7.0). The surface is acidic. The average pH of the stratum corneum increases with depth because of a decrease in the ratio of acidic to neutral regions within the stratum corneum. The data definitively show that the stratum corneum acid mantle results from the presence of aqueous acidic pockets within the lipid-rich extracellular matrix.

Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing.

Two-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC-BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to approximately 3.0 ns. Control experiments using fluorescein conjugated poly-L-lysine and poly-D-lysine demonstrated that the increase in fluorescence parameters observed with FITC-BSA were due to intracellular proteolysis since addition of the inert D-isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC-dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.