Biology subjects

Millett-Sikking, A.

Publications and source records attributed to Millett-Sikking, A..

8 recordsLinked to original sources

bioRxiv2026

Triplet tumbling microscopy enables in situ quantification of protein complex assembly and dynamics

Protein-protein interactions (PPIs) mediate diverse cellular processes, but PPIs are typically characterized using reconstituted in vitro biochemical and biophysical approaches. Current approaches for PPI detection in living cells are limited in the scope of interactions they can capture and often require prior knowledge of the interacting partners. To close this gap, we developed triplet tumbling microscopy (TTM), which reveals the interactions of a tagged protein of interest in cells in real time. TTM reports protein complex size from rotational diffusion ("tumbling") by leveraging infrared-triggerable emission from triplet states to track tumbling over nanoseconds to hundreds of microseconds. These long-lived triplets overcome the size limitations of existing rotational diffusion-based approaches, enabling TTM to measure species from small protein complexes to organelle-scale beads. In living cells, we apply TTM to detect PPIs, quantify fraction bound, and distinguish protein complexes by size. We measure diverse types of interactions, including rapamycin-induced dimerization, p53 homo-oligomerization, and binding of the E3-ligase E6AP to the human papilloma virus 16 E6 protein. The required hardware is compatible with most fluorescent microscopes, making TTM a versatile way to extract molecular insights from the complex context of living cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/723557v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@c77bc1org.highwire.dtl.DTLVardef@8de44forg.highwire.dtl.DTLVardef@18803f2org.highwire.dtl.DTLVardef@bb3afe_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Multi-immersion Oblique Plane Microscope (miOPM): A reconfigurable platform for high-resolution Light-Sheet Fluorescence Microscopy.

Light-Sheet Fluorescence Microscopy (LSFM) enables gentle, rapid, and efficient volumetric imaging of biological specimens. Despite its potential, LSFM has fragmented into multiple variants, each narrowly optimized for specific samples or imaging regimes, limiting its broad applicability. To overcome this barrier, we introduce the multi-immersion oblique plane microscope (miOPM), an adaptable LSFM platform for high-resolution imaging ranging from subcellular dynamics to whole organisms and cleared tissues. miOPM uniquely supports seamless interchangeability of oil, water, and air objectives, and maintains diffraction limited performance across a refractive index range of 1.33 - 1.51. It is compatible with standard sample mounting, current clearing protocols and high-throughput 3D imaging. We leverage miOPM to image diverse biological specimens at multiple spatial scales. By greatly expanding the adaptability and ease of use of LSFM, miOPM is poised to democratize advanced three-dimensional imaging.

bioengineering↗

bioRxiv2024

The biophysical mechanism of mitochondrial pearling

Mitochondrial networks exhibit remarkable dynamics that are driven in part by fission and fusion events. However, there are other reorganizations of the network that do not involve fission and fusion. One such exception is the elusive, "beads-on-a-string" morphological transition of mitochondria. During such transitions, the cylindrical tubes of the mitochondrial membrane transiently undergo shape changes to a string of "pearls" connected along thin tubes. These dynamics have been anecdotally observed in many contexts and given disparate explanations. Here we unify these observations by proposing a common underlying mechanism based on the biophysical properties of tubular fluid membranes for which it is known that, under particular regimes of tension and pressure, membranes reach an instability and undergo a shape transition to a string of connected pearls. First, we use high-speed light-sheet microscopy to show that transient, short-lived pearling events occur spontaneously in the mitochondrial network in every cell type we have examined, including primary fibroblasts, T-cells, neurons, and budding yeast. We present evidence that transient mitochondrial pearling occurs during important biological events, particularly during T cell activation, neuronal firing, and replicative senescence. Using our high-temporal resolution data, we identify two distinct categories of spontaneous pearling, i) internal pressure-driven pearling generated by ionic flux, and ii) external tension-driven pearling generated by the cytoskeleton. By applying live-cell STED and FIB-SEM imaging we document the structural reorganization of inner cristae membranes during mitochondrial pearling and the role of the MICOS complex in regulating the frequency of pearling events. We then establish numerous methods for inducing pearling, including the ability to induce these dynamics with single mitochondrion precision. These methods include ionophores, channel activators, osmotic shock, detergents, laser stimulation, membrane intercalating molecules, chemical fixation, and micro-needle force. These disparate inducers establish three main physical causes of pearling, i) ionic flux producing internal osmotic pressure, ii) membrane packing lowering bending elasticity, and iii) external mechanical force increasing membrane tension. Pearling dynamics thereby reveal a fundamental biophysical facet of mitochondrial biology. We suggest that pearling should take its place beside fission and fusion as a key process of mitochondrial dynamics, with implications for physiology, disease, and aging.

biophysics↗

bioRxiv2024

Active Remote Focus Stabilization in Oblique Plane Microscopy

Light-sheet fluorescence microscopy (LSFM) has demonstrated great potential in the life sciences owing to its efficient volumetric imaging capabilities. For long term imaging, the light-sheet typically needs to be stabilized to the detection focal plane for the best imaging results. Current light-sheet stabilization methods rely on fluorescence emission from the sample, which may interrupt the scientific imaging and add to sample photobleaching. Here we show that for oblique plane microscopes (OPM), a subset of LSFM where a single primary objective is used for illumination and detection, light-sheet stabilization can be achieved without expending sample fluorescence. Our method achieves ~43nm axial precision and maintains the light-sheet well within the depth of focus of the detection system for hour-long acquisition runs in a lab environment that would otherwise detune the system. We demonstrate subcellular imaging of the actin skeleton in melanoma cancer cells with a stabilized OPM.

biophysics↗

bioRxiv2022

Increasing the Field-of-View in Oblique Plane Microscopy via optical tiling

Fast volumetric imaging of large fluorescent samples with high-resolution is required for many biological applications. Oblique plane microscopy (OPM) provides high spatiotemporal resolution, but the field of view is typically limited by its optical train and the pixel number of the camera. Mechanically scanning the sample or decreasing the overall magnification of the imaging system can partially address this challenge, albeit by reducing the volumetric imaging speed or spatial sampling, respectively. In this Letter, we introduce a novel dual-axis scan unit for OPM that enables rapid and high-resolution volumetric imaging throughout a volume of 800 x 500 x 200 microns. This enables imaging of model organisms, such as zebrafish embryos, with subcellular resolution. Furthermore, we combined this microscope with a real-time and multi-perspective projection imaging technique to increase the volumetric interrogation rate to more than 10 Hz.

bioengineering↗

bioRxiv2022

The Yeast Lifespan Machine: a microfluidic platform for automated replicative lifespan measurements

The budding yeast, Saccharomyces cerevisiae, has emerged as a model system for studying the aging processes in eukaryotic cells. However, the full complement of tools available in this organism has not been fully applied, in part because of limitations in throughput that restrict the ability to carry out detailed analyses. Recent advances in microfluidics have provided direct longitudinal observation of entire yeast lifespans, but have not yet achieved the normal scale of operation possible in this model system. Here we present a microfluidic platform, called the Yeast Lifespan Machine, where we combine improvements in microfluidics, image acquisition, and image analysis tools to increase robustness and throughput of lifespan measurements in aging yeast cells. We demonstrate the platforms ability to measure the lifespan of large populations of cells and distinguish long- and short-lived mutants, all with minimal involvement of the experimenter. We also show that environmental pH is capable of significantly modulating lifespan depending on the growth media, highlighting how microfluidic technologies reveal determinants of lifespan that are otherwise difficult to ascertain.

cell biology↗

bioRxiv2020

High-Resolution, Large Field-of-View, and Multi-View Single Objective Light-Sheet Microscopy

Recent developments in Oblique Plane Microscopy (OPM) have shown that it can achieve high spatio-temporal resolution. Here we describe a single objective light-sheet microscope based on oblique plane illumination that achieves: (i) large field of view and high-resolution imaging via a custom remote focusing objective; (ii) fast volumetric imaging by means of light-sheet stabilised stage scanning - a novel scanning modality that extends the imaging volume without compromising imaging speed nor quality; (iii) multi-view imaging by alternating the orientation of light-sheet illumination and detection to improve the image quality on large samples; (iv) simpler design and ergonomics by remote placement of coverslips to allow inverted imaging, enabling imaging across scales in a high-throughput format. Overall, we achieved a resolution of 450 nm laterally and 2 m axially and a field of view of 3000 m x 800 m x 300 m. We demonstrate the speed, field of view, resolution and versatility of our novel instrument by imaging various systems, including zebrafish whole brain activity, Drosophila egg chamber development, and zebrafish development - up to nine embryos simultaneously.

developmental biology↗

bioRxiv2020

A Single-Objective Light-Sheet Microscope with 200 nm-Scale Resolution.

We present an Oblique Plane Microscope that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of Lattice Light-Sheet Microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

biophysics↗