| dc.description.abstract | Practical applications of optical biomedical imaging/diagnostics have been rapidly growing, stimulated by the promise of supreme sensitivity. Fluorescence detection has reached the ultimate sensitivity level of a single molecule, but only in highly purified samples in extremely restricted volumes (below femtoliter). These conditions are not applicable to real, unprocessed physiological samples. Detecting biological objects and physiological processes with high spatial and temporal resolution in real-world conditions remains a significant challenge. Over 15 years ago, it became obvious that the problem was neither the signal strength nor the sensitivity of the detector but the background. Unrelated physiological components in a typical biomedical sample overwhelm the desired sample signal. In practice, any sample at physiologically acceptable conditions is dominated by high scattering, high intrinsic fluorescence of physiological components, and Raman scattering of water. Therefore, achieving a good signal-to-background ratio (SBR) is a significant challenge. There are two options to increase the SBR; (1) increase the signal of the probe or (2) reduce the background signal. To increase the signal of the probe, we can increase its concentration, but this perturbs the physiological system. Instead, we can improve the brightness of fluorescent probes, which have already reached theoretical limits. Alternatively, to improve SBR, one could reduce the background contribution. This can only be done by intensive sample purification that would harshly alter the physiological conditions. For some probes that present long fluorescence lifetimes (much longer than the lifetimes of physiological components), an experimental approach based on time-gated detection significantly increases SBR. However, a substantial part of the probe signal is also lost. This becomes a problem since long-lived probes often present very low brightness.
Herein, we present an approach that enhances the long-lived probe signal, suppresses the background, and greatly improves the SBR. We demonstrated the potential for imaging and localization of an amount of DNA equivalent to a single cell. | en_US |
