High-throughput time-correlated single photon counting

Abstract

We demonstrate time-correlated single photon counting (TCSPC) in microfluidic droplets under high-throughput conditions. We discuss the fundamental limitations in the photon acquisition rate imposed by the single photon detection technique and show that it does not preclude accurate fluorescence lifetime (FLT) measurements at a droplet throughput exceeding 1 kHz with remarkable sensitivity. This work paves the way for the implementation of innovative biomolecular interaction assays relying on the FLT detection of nanosecond-lived fluorophores for high-throughput biotechnological applications, including high-throughput screening or cell sorting potentially allowed by droplet microfluidics or other fast sample handling facilities.

Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

“…DWR, however, several applications of TCSPC were recently demonstrated for accurate FLT detection under high throughput conditions. (36)(37)(38) In particular, using a microfluidic flow cytometer designed for efficient FLT detection by TCSPC at a throughput up to 3000 particles per minute, Nedbal et al (37) reported a comprehensive series of experiments demonstrating the superiority of FLT over FLINT for biomolecular interaction sensing in individual cells, based on the detection of FRET (Förster Resonant Energy Transfer) between ns-lived chromophores including fluorescent proteins. We instead have implemented TCSPC in microfluidic droplets and investigated the fundamental limitations imposed by the single photon detection technique on the photon acquisition rate and on the achievable signal-to-noise ratio in high throughput conditions.…”

Single-photon avalanche diode imagers in biophotonics: review and outlook

“…In TCSPC mode, the detection system's limitations introduce pile-up effects at detection rates >0.1 photon/laser cycle, causing underestimation of the lifetime. Pile-up correction using standard SPAD detectors has been discussed in detail by Léonard et al [60], who show that low count rates are not necessarily needed to avoid pile-up, at the price of an increase in the lifetime estimation variation. In integrated SPAD detectors, multiple subsystems contribute to the pile-up.…”

“…Linear arrays can also be used as single point detectors, for example by means of optical 1D to 2D transformations, to reduce the effect of single SPAD dead time and increase the throughput in FLIM measurements [59]; an example of the corresponding results is shown in Figure 3(f, g). This and other approaches could open the way to high-throughput biotechnological applications, such as high-throughput screening or cell sorting [60,68], based on nanosecond-lived fluorophores.…”

Nouvelles unités d’enseignement autour de l’étude et de la conception de laboratoires sur puces

“…Les modèles compacts de réactions biologiques sont décrits selon le formalisme développé par Gendrault et al [24,27]. Chaque réaction a été modélisée avec un module comportant un réseau électrique de composants élémentaires: la résistance (dégradation des espèces chimiques), le condensateur (stockage d'une espèce donnée dans un volume donné) et la source de courant commandée en tension (taux d'espèces en fonction de synthèse de la concentration des autres espèces).…”

“…Dans la suite du travail de l'étudiant, ces algorithmes sont en cours d'implémentation sur FPGA pour pouvoir traiter la grande quantité de données en temps réel. Comme l'a montré une étude expérimentale et théorique [27], environ 1000 photons de fluorescences peuvent être détectés par goutte de 150 pL avec des concentrations en molécule fluorescente de 300 nM, compatible avec l'exigence de ne pas endommager la cible biologique sur laquelle agit la molécule. L'écart type relatif de la mesure (CV) est alors suffisamment bon (< 4%) pour réaliser des criblages biologiques.…”