Advancing Biosystems Imaging
To fully comprehend the complex mechanistic framework that forms the developing organism, the Nano Bio Imaging Laboratory develops cutting-edge imaging technologies, assays, and probes for carrying out qualitative and quantitative imaging with (i) high spatiotemporal resolution at the single-cell level and with (ii) sensitivities down to individual proteins.
Taking advantage of localized multiphoton photoactivation of paGFP, we previously established a fluorescence decay after photoactivation (FDAP) assay to analyze the kinetics of Oct4 in individual nuclei of a developing mammalian embryo. We reported that transcription factor kinetics, rather than expression levels, are a measure of developmental heterogeneity that predicts cell lineage patterning in the early mouse embryo.
To further elucidate the elaborate protein and cell dynamics that underlie development, we are expanding quantitative imaging to green-to-red photoconvertible proteins, as they combine the strength of global analysis of the non-converted population with partial analysis of the photoconverted population as exemplified in PhOTO Zebrafish. To accomplish spatially confined analysis in whole-animal imaging, we recently invented an imaging technology termed Confined Primed Conversion that allows efficient color switching after photons from two distinct wavelength sources are sequentially absorbed. Currently, we apply confined primed conversion (i) to unravel complex structural information of neurons of the developing zebrafish brain in real time and (ii) to dissect the cellular dynamics of complex tissue regeneration in adult zebrafish.
Given that in many experimental settings fluorescent probes fall short of their potential due to dye bleaching, dye signal saturation, and tissue autofluorescence, the Nano Bio Imaging Laboratory introduced Second Harmonic Generating (SHG) nanoprobes as a superior imaging probe. We are currently engineering SHG nanoprobes to be used as imaging probes for in vivo molecular dynamic studies, enabling straightforward detection and tracking of individual cells and even molecules.