Biology subjects

Daugird, T. A.

Publications and source records attributed to Daugird, T. A..

6 recordsLinked to original sources

bioRxiv2025

A Multimodal Adaptive Optical Microscope For In Vivo Imaging from Molecules to Organisms

Understanding biological systems requires observing features and processes across vast spatial and temporal scales, spanning nanometers to centimeters and milliseconds to days, often using multiple imaging modalities within complex native microenvironments. Yet, achieving this comprehensive view is challenging because microscopes optimized for specific tasks typically lack versatility due to inherent optical and sample handling trade-offs, and frequently suffer performance degradation from sample-induced optical aberrations in multicellular contexts. Here, we present MOSAIC, a reconfigurable microscope that integrates multiple advanced imaging techniques including light-sheet, label-free, super-resolution, and multi-photon, all equipped with adaptive optics. MOSAIC enables non-invasive imaging of subcellular dynamics in both cultured cells and live multicellular organisms, nanoscale mapping of molecular architectures across millimeter-scale expanded tissues, and structural/functional neural imaging within live mice. MOSAIC facilitates correlative studies across biological scales within the same specimen, providing an integrated platform for broad biological investigation.

cell biology↗

bioRxiv2024

Mapping the nuclear landscape with multiplexed super-resolution fluorescence microscopy

The nucleus coordinates many different processes. Visualizing how these are spatially organized requires imaging protein complexes, epigenetic marks, and DNA across scales from single molecules to the whole nucleus. To accomplish this, we developed a multiplexed imaging protocol to localize 13 different nuclear targets with nanometer precision in single cells. We show that nuclear specification into active and repressive states exists along a spectrum of length scales, emerging below one micron and becoming strengthened at the nanoscale with unique organizational principles in both heterochromatin and euchromatin. HP1- was positively correlated with DNA at the microscale but uncorrelated at the nanoscale. RNA Polymerase II, p300, and CDK9 were positively correlated at the microscale but became partitioned below 300 nm. Perturbing histone acetylation or transcription disrupted nanoscale organization but had less effect at the microscale. We envision that our imaging and analysis pipeline will be useful to reveal the organizational principles not only of the cell nucleus but also other cellular compartments.

cell biology↗

bioRxiv2024

A posterior approach to correct for focal plane offsets in lattice light sheet structured illumination microscopy

SignificanceLattice light sheet structured illumination microscopy (latticeSIM) has proven highly effective in producing 3D images with super resolution rapidly and with minimal photobleaching. However, due to the use of two separate objectives, sample-induced aberrations can result in an offset between the planes of excitation and detection, causing artifacts in the reconstructed images. AimWe introduce a posterior approach to detect and correct for the axial offset between the excitation and detection focal planes in latticeSIM and provide a method to minimize artifacts in the reconstructed images. ApproachWe utilized the residual phase information within the overlap regions of the laterally shifted structured illumination microscopy (SIM) information components in frequency space to retrieve the axial offset between the excitation and the detection focal planes in latticeSIM. ResultsWe validated our technique through simulations and experiments, encompassing a range of samples from fluorescent beads to subcellular structures of adherent cells. We also show utilizing transfer functions with the same axial offset as that which was present during the data acquisition results in reconstructed images with minimal artifacts and salvages otherwise unusable data. ConclusionWe envision that our method will be a valuable addition to restore image quality in latticeSIM datasets even for those acquired under non-ideal experimental conditions.

biophysics↗

bioRxiv2024

A series of spontaneously blinking dyes for super-resolution microscopy

Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.

cell biology↗

bioRxiv2023

Correlative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment

In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we developed an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrated that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacted nucleosome diffusive properties in a manner that was dependent on local chromatin density and supportive of a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Our results reveal that nuclear heterogeneity arises from both active and passive process and highlights the need to account for different organizational principals when modeling different chromatin environments.

biophysics↗

bioRxiv2023

Smart Lattice Light Sheet Microscopy for imaging rare and complex cellular events

Light sheet microscopes enable rapid, high-resolution imaging of biological specimens; however, biological processes span a variety of spatiotemporal scales. Moreover, long-term phenotypes are often instigated by rare or fleeting biological events that are difficult to capture with a single imaging modality and constant imaging parameters. To overcome this limitation, we present smartLLSM, a microscope that incorporates AI-based instrument control to autonomously switch between epifluorescent inverted imaging and lattice light sheet microscopy. We apply this technology to two major scenarios. First, we demonstrate that the instrument provides population-level statistics of cell cycle states across thousands of cells on a coverslip. Second, we show that by using real-time image feedback to switch between imaging modes, the instrument autonomously captures multicolor 3D datasets or 4D time-lapse movies of dividing cells at rates that dramatically exceed human capabilities. Quantitative image analysis on high-content + high-throughput datasets reveal kinetochore and chromosome dynamics in dividing cells and determine the effects of drug perturbation on cells in specific mitotic stages. This new methodology enables efficient detection of rare events within a heterogeneous cell population and records these processes with high spatiotemporal 4D imaging over statistically significant replicates.

biophysics↗