In many situations when using fluorescence microscopy, theoretically obtainable sensitivity is never achieved due to autofluorescence from solvents, solutes, cells, tissues, the fixatives employed, and optical components of the microscope system. This is a common problem affecting essentially all studies employing fluorescence microscopy, whether in living cells, fixed tissues, or clinical samples. Autofluorescence effectively decreases the signal-to-noise ratio of detection. This autofluorescence has a defined lifetime (i.e. the average time that a molecule remains in an excited state prior to returning to the ground state), and is on the order of 1-100 nsec. DLM enables the separation of true fluorescence emitted from the fluorescence probe of interest from autofluorescence from solvents, solutes, cells, tissues, the fixatives employed, and optical components of the microscope system. The principle of DLM is to label the structure of interest with a long-lifetime probe (lifetimes on the order of 1 µsec - 1 msec), and observe the emitted fluorescence of the structure of interest after the autofluorescence has decayed (lifetimes on the order of 1-100 nsec). DFM requires that the molecular markers to be used be labeled with phosphorescent or fluorescence compounds with long lifetimes as probes, excitation of the sample with a short pulse of light, and detection of the emitted fluorescence from the long lived probes delayed to the micro- or millisecond range.

In many clinical situations, the ability to detect disease at the earliest stage possible results in a better prognosis for the patient. Unfortunately, detection of many diseases remains problematic due to the low level of pathogen present or the low number of cells infected. One potential solution to this problem is the development of technologies that can detect low levels of disease pathogens with high specificity and sensitivity and require little patient material to accomplish. For example, we have recently developed a FISH assay for HPV detection that can detect as little as one copy of HPV/cell in fresh cervicovaginal cell preparations but cannot be used on standard PAP smears (i.e. those already stained and read by a cytopathologist), due to the fact that the absorption stains used by cytopathologists are intensely (auto)fluorescent and interfere with the FISH signal. This (auto)fluorescence has a defined lifetime (i.e. the average time that a molecule remains in an excited state prior to returning to the ground state); and recently we have successfully conjugated an HPV cDNA probe to a compound which is fluorescent but whose fluorescence continues to be emitted after the fluorescence from the absorption stains has decayed. This allows the observation and quantitation of HPV in standard PAP smears without interference from the routine histological stains. This technique (DLM), enables the separation of true fluorescence emitted from HPV cDNA probes from autofluorescence of PAP absorption stains. The principle behind this technology is such that it will find use in many other types of fluorescence microscopy where signal to noise ratio is of critical concern. For example, DLM could find applications in protein detection, antigen-antibody visualization, cell biology, pathology, physiology as well as clinical diagnostics. Use of this technology in living cells would increase signal to noise ratio by doing away with autofluorescence allowing detection of lower number of biologically relevant molecules and less damage to the cell as very short pulses of excitation light (rather than continuous excitation) would be employed.