On-the-fly decoding luminescence lifetimes in the microsecond region for lanthanide-encoded suspension arrays.

Significant multiplexing capacity of optical time-domain coding has been recently demonstrated by tuning luminescence lifetimes of the upconversion nanoparticles called 'τ-Dots'. It provides a large dynamic range of lifetimes from microseconds to milliseconds, which allows creating large libraries of nanotags/microcarriers. However, a robust approach is required to rapidly and accurately measure the luminescence lifetimes from the relatively slow-decaying signals. Here we show a fast algorithm suitable for the microsecond region with precision closely approaching the theoretical limit and compatible with the rapid scanning cytometry technique. We exploit this approach to further extend optical time-domain multiplexing to the downconversion luminescence, using luminescence microspheres wherein lifetimes are tuned through luminescence resonance energy transfer. We demonstrate real-time discrimination of these microspheres in the rapid scanning cytometry, and apply them to the multiplexed probing of pathogen DNA strands. Our results indicate that tunable luminescence lifetimes have considerable potential in high-throughput analytical sciences.

Time-gated orthogonal scanning automated microscopy (OSAM) for high-speed cell detection and analysis.

We report a new development of orthogonal scanning automated microscopy (OSAM) incorporating time-gated detection to locate rare-event organisms regardless of autofluorescent background. The necessity of using long-lifetime (hundreds of microseconds) luminescent biolabels for time-gated detection implies long integration (dwell) time, resulting in slow scan speed. However, here we achieve high scan speed using a new 2-step orthogonal scanning strategy to realise on-the-fly time-gated detection and precise location of 1-µm lanthanide-doped microspheres with signal-to-background ratio of 8.9. This enables analysis of a 15 mm × 15 mm slide area in only 3.3 minutes. We demonstrate that detection of only a few hundred photoelectrons within 100 µs is sufficient to distinguish a target event in a prototype system using ultraviolet LED excitation. Cytometric analysis of lanthanide labelled Giardia cysts achieved a signal-to-background ratio of two orders of magnitude. Results suggest that time-gated OSAM represents a new opportunity for high-throughput background-free biosensing applications.

Managing SRS competition in a miniature visible Nd:YVO4/BaWO4 Raman laser.

We demonstrate the operation of a compact and efficient continuous wave (CW) self-Raman laser utilizing a Nd:YVO4 gain crystal and BaWO4 Raman crystal, generating yellow emission at 590 nm. We investigate the competition that occurs between Stokes lines in the Nd:YVO4 and BaWO4 crystals, and within the BaWO4 crystal itself. Through careful consideration of crystal length and orientation, we are able to suppress competition between Stokes lines, and generate pure yellow emission at 590 nm with output power of 194 mW for just 3.8 W pump power.

Efficient, miniature, cw yellow source based on an intracavity frequency-doubled Nd:YVO4 self-Raman laser.

We report cw yellow emission from a miniature self-Raman laser using highly doped Nd:YVO4 crystals combined with intracavity frequency doubling. Pump-limited 587.8 nm output of 220 mW was obtained from an 18 mm long resonator, pumped by a 3.8 W diode laser.

Automated detection of rare-event pathogens through time-gated luminescence scanning microscopy.

Many microorganisms have a very low threshold (<10 cells) to trigger infectious diseases, and, in these cases, it is important to determine the absolute cell count in a low-cost and speedy fashion. Fluorescent microscopy is a routine method; however, one fundamental problem has been associated with the existence in the sample of large numbers of nontarget particles, which are naturally autofluorescent, thereby obscuring the visibility of target organisms. This severely affects both direct visual inspection and the automated microscopy based on computer pattern recognition. We report a novel strategy of time-gated luminescent scanning for accurate counting of rare-event cells, which exploits the large difference in luminescence lifetimes between the lanthanide biolabels, >100 µs, and the autofluorescence backgrounds, <0.1 µs, to render background autofluorescence invisible to the detector. Rather than having to resort to sophisticated imaging analysis, the background-free feature allows a single-element photomultiplier to locate rare-event cells, so that requirements for data storage and analysis are minimized to the level of image confirmation only at the final step. We have evaluated this concept in a prototype instrument using a 2D scanning stage and applied it to rare-event Giardia detection labeled by a europium complex. For a slide area of 225 mm(2) , the time-gated scanning method easily reduced the original 40,000 adjacent elements (0.075 mm × 0.075 mm) down to a few "elements of interest" containing the Giardia cysts. We achieved an averaged signal-to-background ratio of 41.2 (minimum ratio of 12.1). Such high contrasts ensured the accurate mapping of all the potential Giardia cysts free of false positives or negatives. This was confirmed by the automatic retrieving and time-gated luminescence bioimaging of these Giardia cysts. Such automated microscopy based on time-gated scanning can provide novel solutions for quantitative diagnostics in advanced biological, environmental, and medical sciences.

Miniature wavelength-selectable Raman laser: new insights for optimizing performance.

We report a miniature, wavelength-selectable crystalline Raman laser operating either in the yellow (588 nm) or lime (559 nm) selected simply by changing the temperature of an intracavity LBO crystal. Continuous-wave (CW) output powers are 320 mW and 660 mW respectively, corresponding to record diode-visible optical conversion efficiencies of 8.4% and 17% for such miniature devices. The complex laser behavior arising from interplay between nonlinear processes is studied experimentally and theoretically. We show that the interplay can lead to complete suppression of the first-Stokes field and that the phase matching conditions for maximum visible powers differ markedly for different length LBO crystals. By using threshold measurements, we calculate the round-trip resonator losses and show that crystal bulk losses dominate over other losses. As a consequence, Raman lasers utilizing shorter LBO crystals for intracavity frequency mixing can produce higher visible output power. These are new considerations for the optimum design of CW intracavity Raman lasers with visible output.