HyU: Hybrid Unmixing for longitudinal in vivo imaging of low signal to noise fluorescence

The expanded application of fluorescence imaging in biomedical and biological research towards more complex systems and geometries requires tools that can analyze a multitude of components at widely varying time- and length-scales. The major challenge in such complex imaging experiments is to cleanly separate multiple fluorescent labels with overlapping spectra from one another and background autofluorescence, without perturbing the sample with high levels of light. Thus, there is a requirement for efficient and robust analysis tools capable of quantitatively separating these signals. In response, we have combined multispectral fluorescence microscopy with hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). Here we demonstrate its capabilities in the dynamic imaging of multiple fluorescent labels in live, developing zebrafish embryos. HyU is more sensitive to low light levels of fluorescence compared to conventional linear unmixing approaches, permitting better multiplexed volumetric imaging over time, with less bleaching. HyU can also simultaneously image both bright exogenous and dim endogenous labels because of its high dynamic range. This allows studies of cellular behaviors, tagged components, and cell metabolism within the same specimen, offering a powerful window into the orchestrated complexity of biological systems.

O-BODIPYs as fluorescent labels for sugars: glucose, xylose and ribose

Sugar-O-BODIPY conjugates, in which the fluorescent dye O-BODIPY is linked to sugars directly through B-O-C bonds, have been synthesized via an acid-catalyzed transesterification procedure. Conjugates of glucose, xylose and ribose...

Dual Color Triads: Synthesis, Photophysics and Application in Live Cell Imaging

Fluorescent labels and probes constitute an important class of organic materials used in the development of sensor systems and imaging platforms for various chemical and molecular biology applications. In the...

Light-induced engagement of microglia to focally remodel synapses in the adult brain

Microglia continuously monitor synapses, but active synaptic remodeling by microglia in mature healthy brains is rarely directly observed. We performed targeted photoablation of single synapses in mature transgenic mice expressing fluorescent labels in neurons and microglia. The photodamage focally increased the duration of microglia-neuron contacts, and dramatically exacerbated both the turnover of dendritic spines and presynaptic boutons as well as the generation of new filopodia originating from spine heads or boutons. The results of microglia depletion confirmed that elevated spine turnover and the generation of presynaptic filopodia are microglia-dependent processes.