Two-photon fluorescence lifetime for label-free microfluidic droplet sorting

Abstract

Microfluidic droplet sorting systems facilitate automated selective micromanipulation of compartmentalized micro- and nano-entities in a fluidic stream. Current state-of-the-art droplet sorting systems mainly rely on fluorescence detection in the visible range with the drawback that pre-labeling steps are required. This limits the application range significantly, and there is a high demand for alternative, label-free methods. Therefore, we introduce time-resolved two-photon excitation (TPE) fluorescence detection with excitation at 532 nm as a detection technique in droplet microfluidics. This enables label-free in-droplet detection of small aromatic compounds that only absorb in a deep-UV spectral region. Applying time-correlated single-photon counting, compounds with similar emission spectra can be distinguished due to their fluorescence lifetimes. This information is then used to trigger downstream dielectrophoretic droplet sorting. In this proof-of-concept study, we developed a polydimethylsiloxane-fused silica (FS) hybrid chip that simultaneously provides a very high optical transparency in the deep-UV range and suitable surface properties for droplet microfluidics. The herein developed system incorporating a 532-nm picosecond laser, time-correlated single-photon counting (TCSPC), and a chip-integrated dielectrophoretic pulsed actuator was exemplarily applied to sort droplets containing serotonin or propranolol. Furthermore, yeast cells were screened using the presented platform to show its applicability to study cells based on their protein autofluorescence via TPE fluorescence lifetime at 532 nm. Graphical abstract

Fluorescence lifetime activated droplet sorting (FLADS) for label-free sorting of Synechocystis sp. PCC6803

“…were not yet able to reach the single-cell level and were limited to low sorting frequencies of 1-2 Hz. 31,32 This is now targeted in the current study to distinguish and sort living and dead cyanobacterial cells. We choose Synechocystis sp.…”

“…This is especially true for the field of high-throughput sorting, which is limited by the ability to determine fluorescence lifetimes at high rates. In our previous works 31,32 we developed an approach for label-free fluorescence lifetime activated droplet sorting and showed its potential to distinguish model substances. In this study, we also demonstrated the suitability of the approach for cell sorting of yeast cells using two-photon excitation, but we were not yet able to reach the single-cell level and were limited to low sorting frequencies of 1–2 Hz.…”

“…In this study, we also demonstrated the suitability of the approach for cell sorting of yeast cells using two-photon excitation, but we were not yet able to reach the single-cell level and were limited to low sorting frequencies of 1–2 Hz. 31,32…”

High precision, high throughput generation of droplets containing single cells

“…), and produces lower throughputs (max 50 Hz sorting rate). [29][30][31][32][33] Until now, it is considered difficult, if not impossible, to overcome the Poisson limit of droplet microfluidics with an easy and versatile solution.…”

High precision, high throughput generation of droplets containing single cells

“…The maximal sorting throughput (based on detection methods such as image processing, Raman spectrum, UV-vis spectrum etc.) is around 300 cell-loaded droplets per minute [29][30][31][32][33]. Currently, there is not a general solution for high throughput, label-free, and deterministic single cell encapsulation into droplets.…”