HIGHLIGHT

Fluorescence has become an essential observable in Biology and Medicine. The discrimination of a fluorescent label usually relies on optimizing its brightness and its spectral properties. Despite its widespread use, this approach still suffers from important limitations. First, extraction of a fluorescent signal is challenging in light-scattering and autofluorescent samples. Second, spectral deconvolution of overlapping absorption and emission bands can only discriminate a few labels, which strongly limits the discriminative power of emerging genetic engineering strategies, and falls short from the several tens needed for advanced bioimaging and highly multiplexed diagnostic assays. Our consortium of chemists, physicists, and biologists introduces the HIGHLIGHT concept (PHase-sensItive imaGing of reversibly pHotoswitchable Labels after modulatIon of activatinG ligHT) to achieve chromatic aberration-free highly multiplexed fluorescence imaging with only single and dual wavelength channels in emission and excitation. HIGHLIGHT aims at expanding the discriminative dimensions of fluorophore sets much beyond spectral and concentration information such as classically implemented in multicolor labeling approaches. In HIGHLIGHT, label discrimination will not necessitate anymore singular spectroscopic signatures, sophisticated reading-out instruments, or delicate data processing for signal unmixing. In contrast, it shifts towards designing reactive schemes and observables to selectively promote and retrieve the response of a targeted label. HIGHLIGHT exploits reversibly photoswitchable fluorescent proteins (RSFPs) as labels. Increasingly exploited in super-resolution microscopy and dynamic contrast, they are not only fluorescent but as well engaged in rich photocycles. The HIGHLIGHT protocols exploit their specific fluorescence responses to light modulation under well-designed conditions, which provides several dimensions of dynamic contrast to overcome the limitations encountered with spectral discrimination; These responses will serve as readouts either alone or combined using statistical machine learning strategies, which will enable us to perform real time multiplexed imaging of more than ten spectrally similar fluorescent labels and discriminate more than one hundred hues created by mixing these labels in variable amounts and cell territories. As a proof of principle, we propose to challenge HIGHLIGHT in two types of contexts where the paucity of spectrally distinct fluorescent markers has until now been a major hindrance: the analysis of the lineage of retinal cell subtypes and that of their connectivity. In this project, we will namely (i) design and implement a suite of transgenic tools enabling to express varied combinations of 6-12 RSFPs within a population of cells; (ii) design HIGHLIGHT protocols for wide-field and scanning microscopies as well as relevant barcoding strategies to discriminate different cells; (iii) evaluate the photoswitching properties of several tens of RSFPs with one- and two-photon excitation under various environments; (iv) validate HIGHLIGHT for its implementation in a commercial confocal microscope and in state-of-the art Single Plane Illumination scanning Microscopes to push forward acquisition depth and speed; and eventually (v) perform multiplexed clonal analysis in the vertebrate retina, and single-neuron tracing and analysis of axonal convergence. Eventually, the tools and protocols introduced in this project will have near-universal applicability in Biology for multiplexed fluorescence-based observations within biological samples.