Pawel Wodnicki

Time‐resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system

The design and implementation of a time‐resolved fluorescence lifetime imaging microscope (TRFLIM) for the biomedical sciences are described. The measurement of fluorescence lifetimes offers many benefits, among which is that they are independent of local signal intensity and concentration of the fluorophore and they provide visualization of the molecular environment in a single living cell. Unlike single photon counting, which employs a photomultiplier as the detector, TRFLIM uses a nanosecond‐gated multichannel plate image intensifier providing a two‐dimensional map of the spatial distribution of fluorescent lifetime in the sample under observation. Picosecond laser pulses from a tunable dye laser are delivered to the fluorophore inside living cells on the stage of a fluorescent microscope. Images of the fluorescence emission at various times during the decay of the fluorescence are collected using a high‐speed gated image intensifier and the lifetimes are calculated on a pixel‐by‐pixel basis. Lifetimes measured by TRFLIM are compared with those measured by conventional methods.

Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy

Time-resolved fluorescence lifetime microscopy (TRFLM) allows the combination of the sensitivity of fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells, as well as providing more accurate, sensitive, and specific diagnosis of certain clinical diseases and chemical analyses. Here we discuss two applications of TRFLM: (1) the use of nonratiometric probes such as Calcium Crimson, for measuring Ca2+; and (2) quantification of protein interaction in living cells using green and blue fluorescent protein (GFP and BFP, respectively) expressing constructs in combination with fluorescence resonance energy transfer microscopy (FRET). With respect to measuring Ca2+ in biological samples, we demonstrate thatintensity-based measurements of Ca2+ with single-wavelength Ca2+ probes such as Calcium Crimson may falsely report the actual Ca2+ concentration. This is due to effects of hydrophobicity of the local environment on the emission of Calcium Crimson as well as interaction of Calcium Crimson with proteins, both of which are overcome by the use of TRFLM. The recent availability of BFP (P4-3) and GFP (S65T) (which can serve as donor and acceptor, respectively) DNA sequences which can be attached to the carboxy-or amino-terminal DNA sequence of specific proteins allows the dual expression and interaction of proteins conjugated to BFP and GFP to be monitored in individual cells using FRET. Both of these applications of TRFLM are expected to enhance substantially the information available regarding both the normal and the abnormal physiology of cells and tissues.

Fluorescence lifetime imaging in cell biology

The measurement of fluorescence lifetimes offers the advantages of being independent of local intensity and concentration of the fluorophore, and can provide information regarding the molecular environment in a single living cell. Historically, measurements of fluorescence lifetimes have employed photomultipliers as detectors, providing high sensitivity but sacrificing spatial information. Fluorescence Lifetime Imaging Microscopy (FLIM) provides a 2- or 3D spatial map of the distribution of fluorescent lifetime(s) in the sample under observation. Picosecond laser pulses from a tunable dye laser are delivered to fluorophore containing living cells on the stage of a fluorescent microscope, and images of the fluorescence emission at various times during the decay of the fluorescence lifetime are collected using a high speed nanosecond-gated multichannel plate image intensifier. FLIM promises to substantially enhance the information obtainable from living cells and tissues, and will allow observations of the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques.

Highly sensitive detection of human papillomavirus type 16 DNA using time-resolved fluorescence microscopy and long lifetime probes

We have been interested in the role of Human Papillomavirus (HPV) in cervical cancer and its diagnosis; to that end we have been developing microscopic imaging and fluorescent in situ hybridization (FISH) techniques to genotype and quantitate the amount of HPV present at a single cell level in cervical PAP smears. However, we have found that low levels of HPV DNA are difficult to detect accurately because theoretically obtainable sensitivity is never achieved due to nonspecific autofluorescence, fixative induced fluorescence of cells and tissues, and autofluorescence of the optical components in the microscopic system. In addition, the absorption stains used for PAP smears are intensely autofluorescent. Autofluorescence is a rapidly decaying process with lifetimes in the range of 1-100 nsec, whereas phosphorescence and delayed fluorescence have lifetimes in the range of 1 microsecond(s) ec-10 msec. The ability to discriminate between specific fluorescence and autofluorescence in the time-domain has improved the sensitivity of diagnostic test such that they perform comparably to, or even more sensitive than radioisotopic assays. We have developed a novel time-resolved fluorescence microscope to improve the sensitivity of detection of specific molecules of interest in slide based specimens. This time-resolved fluorescence microscope is based on our recently developed fluorescence lifetime imaging microscopy (FILM) in conjunction with the use of long lifetime fluorescent labels. By using fluorescence in situ hybridization and the long lifetime probe (europium), we have demonstrated the utility of this technique for detection of HPV DNA in cervicovaginal cells. Our results indicate that the use of time-resolved fluorescence microscopy and long lifetime probes increases the sensitivity of detection by removing autofluorescence and will thus lead to improved early diagnosis of cervical cancer. Since the highly sensitive detection of DNA in clinical samples using fluorescence in situ hybridization image is useful for the diagnosis of many other type of diseases, the system we have developed should find numerous applications for the diagnosis of disease states.