Linking the genotypes and phenotypes of cancer cells in heterogenous populations via real-time optical tagging and image analysis.

Linking single-cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, could help unravel molecular mechanisms driving the growth of tumour-cell subpopulations. Here we show that a custom-built optical microscope with an ultrawide field of view, fast automated image analysis and a dye activatable by visible light enables the screening and selective photolabelling of cells of interest in large heterogeneous cell populations on the basis of specific functional cellular dynamics, such as fast migration, morphological variation, small-molecule uptake or cell division. Combining such functional single-cell selection with single-cell RNA sequencing allowed us to (1) functionally annotate the transcriptomic profiles of fast-migrating and spindle-shaped MCF10A cells, of fast-migrating MDA-MB-231 cells and of patient-derived head-and-neck squamous carcinoma cells, and (2) identify critical genes and pathways driving aggressive migration and mesenchymal-like morphology in these cells. Functional single-cell selection upstream of single-cell sequencing does not depend on molecular biomarkers, allows for the enrichment of sparse subpopulations of cells, and can facilitate the identification and understanding of the molecular mechanisms underlying functional phenotypes.

Lipids-Fluorophores Interactions Probed by Combined Nonlinear Polarized Microscopy.

Studying the structural dynamics of lipid membranes requires methods that can address both microscopic and macroscopic characteristics. Fluorescence imaging is part of the most used techniques to study membrane properties in various systems from artificial membranes to cells: It benefits from a high sensitivity to local properties such as polarity and molecular orientational order, with a high spatial resolution down to the single-molecule level. The influence of embedded fluorescent lipid probes on the lipid membrane molecules is however poorly known and relies most often on molecular dynamics simulations, due to the challenges faced by experimental approaches to address the molecular-scale dimension of this question. In this work we develop an optical microscopy imaging method to probe the effect of fluorophores embedded in the membrane as lipid probes, on their lipid environment, with a lateral resolution of a few hundreds of nanometers. We combine polarized-nonlinear microscopy contrasts that can independently address the lipid probe, by polarized two-photon fluorescence, and the membrane lipids, by polarized coherent Raman scattering. Using trimethylamino derivative 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS) as model probes, we show that both probes tend to induce an orientational disorder of their surrounding lipid CH-bonds in 1,2-dipalmitoylphosphatidylcholine (DPPC) lipids environments, while there is no noticeable effect in more disordered 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid membranes.

Shot-noise limited tunable dual-vibrational frequency stimulated Raman scattering microscopy.

We present a shot-noise limited SRS implementation providing a >200 mW per excitation wavelength that is optimized for addressing two molecular vibrations simultaneously. As the key to producing a 3 ps laser of different colors out of a single fs-laser (15 nm FWHM), we use ultra-steep angle-tunable optical filters to extract 2 narrow-band Stokes laser beams (1-2 nm & 1-2 ps), which are separated by 100 cm-1. The center part of the fs-laser is frequency doubled to pump an optical parametric oscillator (OPO). The temporal width of the OPO's output (1 ps) is matched to the Stokes beams and can be tuned from 650-980 nm to address simultaneously two Raman shifts separated by 100 cm-1 that are located between 500 cm-1 and 5000 cm-1. We demonstrate background-free SRS imaging of C-D labeled biological samples (bacteria and Drosophila). Furthermore, high quality virtual stimulated Raman histology imaging of a brain adenocarcinoma is shown for pixel dwell times of 16 µs.

Lipid Order Degradation in Autoimmune Demyelination Probed by Polarized Coherent Raman Microscopy.

Myelin around axons is currently widely studied by structural analyses and large-scale imaging techniques, with the goal to decipher its critical role in neuronal protection. Although there is strong evidence that in myelin, lipid composition, and lipid membrane morphology are affected during the progression of neurodegenerative diseases, there is no quantitative method yet to report its ultrastructure in tissues at both molecular and macroscopic levels, in conditions potentially compatible with in vivo observations. In this work, we study and quantify the molecular order of lipids in myelin at subdiffraction scales, using label-free polarization-resolved coherent anti-Stokes Raman, which exploits coherent anti-Stokes Raman sensitivity to coupling between light polarization and oriented molecular vibrational bonds. Importantly, the method does not use any a priori parameters in the sample such as lipid type, orientational organization, and composition. We show that lipid molecular order of myelin in the mouse spinal cord is significantly reduced throughout the progression of experimental autoimmune encephalomyelitis, a model for multiple sclerosis, even in myelin regions that appear morphologically unaffected. This technique permits us to unravel molecular-scale perturbations of lipid layers at an early stage of the demyelination progression, whereas the membrane architecture at the mesoscopic scale (here ∼100 nm) seems much less affected. Such information cannot be brought by pure morphological observation and, to our knowledge, brings a new perspective to molecular-scale understanding of neurodegenerative diseases.

Quantitative analysis of light scattering in polarization-resolved nonlinear microscopy.

Polarization resolved nonlinear microscopy (PRNM) is a powerful technique to gain microscopic structural information in biological media. However, deep imaging in a variety of biological specimens is hindered by light scattering phenomena, which not only degrades the image quality but also affects the polarization state purity. In order to quantify this phenomenon and give a framework for polarization resolved microscopy in thick scattering tissues, we develop a characterization methodology based on four wave mixing (FWM) process. More specifically, we take advantage of two unique features of FWM, meaning its ability to produce an intrinsic in-depth local coherent source and its capacity to quantify the presence of light depolarization in isotropic regions inside a sample. By exploring diverse experimental layouts in phantoms with different scattering properties, we study systematically the influence of scattering on the nonlinear excitation and emission processes. The results show that depolarization mechanisms for the nonlinearly generated photons are highly dependent on the scattering center size, the geometry used (epi/forward) and, most importantly, on the thickness of the sample. We show that the use of an un-analyzed detection makes the polarization-dependence read-out highly robust to scattering effects, even in regimes where imaging might be degraded. The effects are illustrated in polarization resolved imaging of myelin lipid organization in mouse spinal cords.

Molecular orientational order probed by coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy: a spectral comparative study.

We investigate how to extract information on the orientational order of molecular bonds in biological samples from polarized coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy. Experimentally, the mean orientation of the molecular angular distribution, as well as its second and fourth orders of symmetry, are estimated by monitoring intensity signals under a varying incident polarization. We provide a generic method of analysis of polarized signals in both CARS and SRS contrasts, and apply it to imaging of lipid bonds' orientational order in multilamellar vesicles. A comparison of the two contrasts in the lipid region around 3000 cm(-1) shows that while SRS allows retrieving pure molecular order information, CARS is generally tainted by a bias from the nonresonant contribution.

Ultimate use of two-photon fluorescence microscopy to map orientational behavior of fluorophores.

The orientational distribution of fluorophores is an important reporter of the structure and function of their molecular environment. Although this distribution affects the fluorescence signal under polarized-light excitation, its retrieval is limited to a small number of parameters. Because of this limitation, the need for a geometrical model (cone, Gaussian, etc.) to effect such retrieval is often invoked. In this work, using a symmetry decomposition of the distribution function of the fluorescent molecules, we show that polarized two-photon fluorescence based on tunable linear dichroism allows for the retrieval of this distribution with reasonable fidelity and without invoking either an a priori knowledge of the system to be investigated or a geometrical model. We establish the optimal level of detail to which any distribution can be retrieved using this technique. As applied to artificial lipid vesicles and cell membranes, the ability of this method to identify and quantify specific structural properties that complement the more traditional molecular-order information is demonstrated. In particular, we analyze situations that give access to the sharpness of the angular constraint, and to the evidence of an isotropic population of fluorophores within the focal volume encompassing the membrane. Moreover, this technique has the potential to address complex situations such as the distribution of a tethered membrane protein label in an ordered environment.