Peter T. C. So

Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin

Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.

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 α 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.

Innovations in two-photon deep tissue microscopy

The goal of this article is to provide a review of deep tissue studies based on two-photon microscopy. We will further explore three new developments that have significantly enhanced the power of two-photon imaging: (1) simultaneous two-photon fluorescence and confocal reflected-light imaging, (2) two-photon video-rate imaging, and (3) fluorescent spectroscopic measurements in deep tissue.

Basic Principles of Multiphoton Excitation Microscopy

Multiphoton microscopy is one of the fastest growing areas in biomedical imaging. The potential of multiphoton excitation was first theorized by Maria GoppertMayer in 1931. Generating three-dimensionally resolved microscopic images based on nonlinear optical excitation was postulated in the 1970s (Gannaway and Sheppard, 1978; Wilson and Sheppard, 1984). The definitive experiment was done by Denk, Webb, and co-workers (1990), who accomplished two-photon, three-dimensional (3D) imaging of biological specimens. Furthermore, they demonstrated 3D localized uncaging and photobleaching using two-photon excitation to trigger a photochemical reaction in a subfemtoliter volume.