Two-photon absorption cross-sections and time-resolved fluorescence imaging using porphyrin photosensitisers.

Three porphyrin systems have been characterised for use in two-photon fluorescence imaging of biological samples. We have determined the two-photon absorption cross sections (sigma(2)) of the di-cation, free-base and metallated forms of hematoporphyrin derivative (HpD), hematoporphyrin IX (Hp9) and a boronated protoporphyrin (BOPP) using the open-aperture Z-scan and the two-photon induced fluorescence (TPIF) techniques at an excitation wavelength of 800 nm. The insertion of either protons or a metal ion into the macrocycle is shown not to significantly influence the sigma(2) of the porphyrins.

Phase-locked 10 MHz reference signal for frequency domain time-resolved fluorescence measurements.

A complete electronic system that is suitable for use in megahertz frequency domain time-resolved fluorescence instruments based on mode-locked lasers is described. The circuit produces a 10 MHz signal, phase locked to the mode-locked laser pulse frequency, which is required by many commercial frequency synthesizers as the external reference signal. This device is particularly useful in conjunction with ultrafast gated intensified charge coupled device cameras capable of being frequency modulated for time-resolved fluorescence imaging.

In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia.

Multiphoton fluorescence lifetime imaging microscopy (FLIM) is a noninvasive, cellular resolution, 3-D functional imaging technique. We investigate the potential for in vivo precancer diagnosis with metabolic imaging via multiphoton FLIM of the endogenous metabolic cofactor nicotinamide adenine dinucleotide (NADH). The dimethylbenz[alpha]anthracene (DMBA)-treated hamster cheek pouch model of oral carcinogenesis and MCF10A cell monolayers are imaged using multiphoton FLIM at 780-nm excitation.

Time-resolved fluorescence microscopy of gunshot residue: an application to forensic science.

Time-resolved fluorescence microscopy has rapidly emerged as the technique of choice for many researchers aiming to gain specific insights into the dynamics of intricate biological systems. Although the unique advantages the technique provides over other methods have proven to be particularly useful in the biosciences, to date they have been largely unexploited by other research disciplines. In this paper, we demonstrate the capacity of time-resolved fluorescence microscopy as a practical analytical tool in the forensic sciences via the imaging of gunshot residues that are expelled when a firearm is discharged.

Simultaneous two-photon spectral and lifetime fluorescence microscopy.

When a fluorescence photon is emitted from a molecule within a living cell it carries a signature that can potentially identify the molecule and provide information on the microenvironment in which it resides, thereby providing insights into the physiology of the cell. To unambiguously identify fluorescent probes and monitor their physiological environment within living specimens by their fluorescent signatures, one must exploit as much of this information as possible. We describe the development and implementation of a combined two-photon spectral and lifetime microscope.