To unravel dynamic processes underpinning key functions in cell biology, it is essential to develop imaging technologies able to track the movement of individual bio-nano-objects under physiologically relevant conditions, hence at high speed (ms) and in 3D. We demonstrate interferometric gated off-axis reflectometry (iGOR) which detects the back-scattered light of the structure of interest using an external off-axis reference, enabling label-free high-speed tracking of nanoparticles and suspended membranes in 3D volumes. Employing coherence time-gating by femtosecond pulses, the axial extension of the detected volume is controlled. We show tracking of single nanoparticles down to 10nm size freely diffusing in volume, which allows us to determine their geometrical and hydrodynamic radius as well as non-sphericity. We also show the spatiotemporal dynamics of suspended lipid bilayers, and the influence of lipid phase transitions on these dynamics with sub-nm thickness precision.
Quantitative differential interference contrast (qDIC) microscopy is applied to the study of the main phase transition of dipentadecanoylphosphatidylcholine (DC15PC) supported lipid bilayers. We measure thickness changes of about 1nm occurring in the bilayer with sub-nanometre resolution and show how the presence of fluorescently labelled lipids, even at small concentrations, can broaden the phase transition.
We present a label-free vibrational microscopy technique recently developed by us, which offers backgroundfree chemically-specific image contrast, shot-noise limited detection, and phase sensitivity enabling topographic imaging of interfaces. The technique features interferometric heterodyne detection of coherent anti-Stokes Raman scattering (CARS) in epi-geometry, as well as multi-modal acquisition of stimulated Raman scattering and forward-emitted CARS intensity in the same instrument. As an important biologically-relevant application, epi-detected heterodyne CARS imaging of individual lipid bilayers is demonstrated. We show that we can resolve a single lipid bilayer, distinct from a double bilayer, and measure the phase of its susceptibility, which provides information about the topography of the bilayer with nanometer resolution. As an additional application example, we show imaging of silicon oil droplets surrounded by an aqueous environment at the glass-water interface, where three different signal generation pathways are distinguished. Our epi-detected heterodyne CARS microscope setup thus paves the way to exciting new experiments pushing the sensitivity and resolution limits of vibrational microscopy to the nanoscale.
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