Eliminating the contribution of interfering compounds is a key step in chemical analysis. In complex media, one possible approach is to perform a preliminary separation. However purification is often demanding, long, and costly; it may also considerably alter the properties of interacting components of the mixture (e.g. in a living cell). Hence there is a strong interest for developing separation-free non-invasive analytical protocols. Using photoswitchable probes as labelling and titration contrast agents, we have demonstrated that the association of a modulated light excitation with a kinetic filtering of the overall observable is much more attractive than constant excitation to read-out the contribution from a target probe under adverse conditions (Figure 1).
Figure 1. Principle of temporally modulated light excitation and kinetic filtering for selective imaging of photoswitchable probes exchanging between two states of different brightness. Constant illumination reveals all spectrally similar probes of a mixture (top). In contrast, matching the parameters of the modulated illumination (mean intensity, radial frequency of modulation) with the dynamics of a targeted photoswitchable probe permits to selectively retrieve its contribution from the amplitude of the modulated components of the overall signal in the presence of spectrally interfering species, photoswitchable or not (bottom).
An extensive theoretical framework enabled us recently to optimize the out-of-phase concentration first-order response of a photoswitchable probe to modulated illumination by appropriately matching the average illumination and the radial frequency of the light modulation to the probe dynamics. Thus, we can selectively and quantitatively extract from an overall signal the contribution from a target photoswitchable probe within a mixture of species, photoswitchable or not. This simple titration strategy has been more specifically developed in the context of fluorescence imaging by introducing OPIOM (Out-of-Phase Imaging after Optical Modulation), which offers promising perspectives (Figure 2).
Figure 2. OPIOM application in mammalian HEK293 cells and in 24 hpf zebrafish embryos. a, b: Selective imaging of nuclear Dronpa-3 against membrane-localized EGFP; c, d: Selective imaging of Lifeact-Dronpa-3 against autofluorescence. Fixed (a) or live (b) cells, and zebrafish embryo (c, d) were imaged by epifluorescence (a–c) or single plane illumination microscope (SPIM, d; the dashed rectangles indicate the zone illuminated by the thinnest part of the light sheet) upon illuminating with sinusoidal (a–c) or square wave (d) light modulation of large amplitude tuned on the resonance of Dronpa-3. Images labeled Pre-OPIOM and OPIOM correspond to respectively the unfiltered and OPIOM-filtered images. Scale bars represent 50 μm.
J. Quérard, T. Le Saux, A. Gautier, D. Alcor, V. Croquette, A. Lemarchand, C. Gosse, L. Jullien, Kinetics of reactive modules adds discriminative dimensions for selective cell imaging, ChemPhysChem, 2016, 17, 1396 – 1413.
J. Querard, T.-Z. Markus, M.-A. Plamont, C. Gauron, P. Wang, A. Espagne, M. Volovitch, S. Vriz, V. Croquette, A. Gautier, T. Le Saux, L. Jullien, Photoswitching kinetics and phase sensitive detection add discriminative dimensions for selective fluorescence imaging, Angew. Chem. Int. Ed., 2015, 54, 2633 – 2637.
J. Querard, A. Gautier, T. Le Saux, L. Jullien, Expanding discriminative dimensions for analysis and imaging, Chem. Sci., 2015, 6, 2968 – 2978.