Publications

Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1stdiffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0th and 1st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

JTD Keywords: cells, multicolor imaging, nanoscopy, particle tracking, point accumulation for imaging in nanoscale topography (paint), precision, single-molecule fo?rster resonance energy transfer (smfret), stochastic optical reconstruction microscopy (storm), Diffraction-limit, Multicolor imaging, Point accumulation for imaging in nanoscale topography (paint), Single-molecule förster resonance energy transfer (smfret), Single-molecule spectroscopy, Stochastic optical reconstruction microscopy (storm)

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

JTD Keywords: Developmental Biology, Zebrafish, Yolk, Atomic Force Microscopy, Cortical Tension, Microrheology, Nanoparticle tracking

Summary During definitive erythropoiesis, erythroid precursors undergo differentiation through multiple nucleated states to an enucleated reticulocyte, which loses its residual RNA/organelles to become a mature erythrocyte. Over the course of these transformations, continuous changes in membrane proteins occur, including shifts in protein abundance, rates of expression, isoform prominence, states of phosphorylation, and stability. In an effort to understand when assembly of membrane proteins into an architecture characteristic of the mature erythrocyte occurs, we quantitated the lateral diffusion of the most abundant membrane protein, band 3 (AE1), during each stage of erythropoiesis using single particle tracking. Analysis of the lateral trajectories of individual band 3 molecules revealed a gradual reduction in mobility of the anion transporter as erythroblasts differentiated. Evidence for this progressive immobilization included a gradual decline in diffusion coefficients as determined at a video acquisition rate of 120 frames/s and a decrease in the percentage of compartment sizes >100 nm. Because complete acquisition of the properties of band 3 seen in mature erythrocytes is not observed until circulating erythrocytes are formed, we suggest that membrane maturation involves a gradual and cooperative assembly process that is not triggered by the synthesis of any single protein.

JTD Keywords: Band 3 diffusion, Erythrocyte, Progenitor cells, Single particle tracking, Streptavidin quantum dot