Extra kinetic dimensions for label discrimination.

Due to its sensitivity and versatility, fluorescence is widely used to detect specifically labeled biomolecules. However, fluorescence is currently limited by label discrimination, which suffers from the broad full width of the absorption/emission bands and the narrow lifetime distribution of the bright fluorophores. We overcome this limitation by introducing extra kinetic dimensions through illuminations of reversibly photoswitchable fluorophores (RSFs) at different light intensities. In this expanded space, each RSF is characterized by a chromatic aberration-free kinetic fingerprint of photochemical reactivity, which can be recovered with limited hardware, excellent photon budget, and minimal data processing. This fingerprint was used to identify and discriminate up to 20 among 22 spectrally similar reversibly photoswitchable fluorescent proteins (RSFPs) in less than 1s. This strategy opens promising perspectives for expanding the multiplexing capabilities of fluorescence imaging.

Out-of-Phase Imaging after Optical Modulation (OPIOM) for Multiplexed Fluorescence Imaging Under Adverse Optical Conditions.

Fluorescence imaging has become a powerful tool for observations in biology. Yet it has also encountered limitations to overcome optical interferences of ambient light, autofluorescence, and spectrally interfering fluorophores. In this account, we first examine the current approaches which address these limitations. Then we more specifically report on Out-of-Phase Imaging after Optical Modulation (OPIOM), which has proved attractive for highly selective multiplexed fluorescence imaging even under adverse optical conditions. After exposing the OPIOM principle, we detail the protocols for successful OPIOM implementation.

Size-based characterization by the coupling of capillary electrophoresis to Taylor dispersion analysis.

In this work, a new methodology is presented for performing capillary electrophoresis (CE) coupled to Taylor dispersion analysis (TDA). The CE step allows the separation of the different compounds of the injected mixture, while the diffusion coefficient related to each sample zone can be derived from the subsequent TDA step. TDA is an absolute and straightforward nonseparative method allowing the determination of the diffusion coefficient (or hydrodynamic radius) from the peak dispersion obtained in an open tube under Poiseuille laminar flow conditions. With a mass concentration sensitive detector, the hydrodynamic radius derived from TDA is a weight average value calculated upon all the molecules present in the sample zone. Since CE can be hardly coupled to light scattering detection for technical reasons (low volumes, short detection path length), TDA represents an interesting alternative for the size characterization, without calibration, of sample mixtures using CE-based separation techniques. The coupling of CE to TDA can be implemented on a commercial CE apparatus.