Dye-Specific Wavelength Offsets to Resolve Spectrally Overlapping and Co-Localized Two-Photon Induced Fluorescence.

The fundamentally important fluorescent imaging has one major limitation, resolution of over three dyes. This limitation is in part due to the overlap of the broad emission profile of each of the emitters used in fluorescence detection. The overlapping emission contaminates each emitter's detection channel, referred to as cross-talk. To reduce fluorescence cross-talk for two photon applications, we present an innovative Two-Photon-Dye-Specific Excitation-Emission Offset (TP-DSO) method. TP-DSO selectively detects each dye by synchronously scanning the excitation and emission wavelengths at defined wavelength offsets. This technique advances multicolor analysis significantly by resolving dyes with highly overlapping spectral profiles. We identified three benefits: reduced excitation spectral bandwidth, reduced emission cross-talk between colocalized emitters with closely overlapping fluorescence, and validated use of thin-film variable optical emission filters for tuning the bandwidth and center wavelength. TP-DSO will advance multicolor analysis for many applications.

Detection of miRNA using a double-strand displacement biosensor with a self-complementary fluorescent reporter.

Design of rapid, selective, and sensitive DNA and ribonucleic acid (RNA) biosensors capable of minimizing false positives from nuclease degradation is crucial for translational research and clinical diagnostics. We present proof-of-principle studies of an innovative micro-ribonucleic acid (miRNA) reporter-probe biosensor that displaces a self-complementary reporter, while target miRNA binds to the probe. The freed reporter folds into a hairpin structure to induce a decrease in the fluorescent signal. The self-complementarity of the reporter facilitates the reduction of false positives from nuclease degradation. Nanomolar limits of detection and quantitation were capable with this proof-of-principle design. Detection of miRNA occurs within 10 min and does not require any additional hybridization, labeling, or rinsing steps. The potential for medical applications of the reporter-probe biosensor is demonstrated by selective detection of a cancer regulating microRNA, Lethal-7 (Let-7a). Mechanisms for transporting the biosensor across the cell membrane will be the focus of future work.