Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity

Abstract

Ultrasensitive detection of DNA is achieved via two-photon excitation of a fluorescent base analogue.

Getting DNA and RNA out of the dark with 2CNqA: a bright adenine analogue and interbase FRET donor

“…A remaining challenge with the utilization of fluorescent base analogues is their lower brightness ( ϵ Φ F ; molar absorptivity multiplied by the fluorescence quantum yield) compared with conventional external fluorophores such as those mentioned above. However, recent studies show that the brightness gap between internal and external fluorophores is decreasing ( 26–28 ).…”

Single-Molecule Detection of a Fluorescent Nucleobase Analogue via Multiphoton Excitation

“…This demonstrated the feasibility of single-molecule detection, albeit improvements in brightness were still required. 23 Here we demonstrate that an extended 6-aza-uridine ribonucleoside, DMA th aU (Chart 1), adopts a highly fluorescent form, in which the brightness following three-photon (3P) excitation is over an order of magnitude greater than that of pA under 2P excitation, allowing, for the first time, single-molecule detection via multiphoton excitation. Chart 1.…”

“…The 2P cross-section (σ 2 ) of DMA th aU in dioxane, measured relative to rhodamine 6G, is 90.0 ± 5.0 GM with a corresponding 2P brightness of 18 GM. This cross-section is higher than that of any other FBA studied previously, including pA (21 GM in ethanol 23 ) and the brightness is three times greater than that of pA. Given the existence of DMA th aU in a highly fluorescent state (albeit with a low population) we were encouraged to pursue single-molecule detection in buffer.…”

“…Furthermore, the magnitude of the calculated 3P cross sections are in agreement with estimated 3P cross sections 29 and those measured for fluorophores of similar size. 30 Next, we examined the limits of detection of DMA th aU in buffer, using fluorescence correlation spectroscopy (FCS) and photon-counting with a multichannel scaler (MCS), similar to our recent work with pA. 23 Excitation powers of 10 mW, where the 3P excitation begins to saturate, gave the best SBR; the pulses were compressed to 8 fs at the focal plane. In FCS measurements there was a striking discrepancy between the known sample concentration and the number of molecules observed in the laser focus.…”

Single-molecule fluorescence detection of a tricyclic nucleoside analogue

“…8,9,[15][16][17] This lack of brightness has rendered them largely unsuitable for singlemolecule fluorescence studies. [18][19][20] Furthermore, with the current rapid development of spatially-resolved transcriptomics and genomics, there is clearly a future need for fluorescent analogues that can act as effective single-molecule probes. 21,22 Conventional fluorophores typically exhibit superior optical properties to fluorescent nucleobase analogues, primarily because of their larger extinction coefficients-sometimes in excess of 10 5 -but they need not be larger molecules.…”