Scott E Fraser

Fluorescent biosensors revolutionized biomedical science by enabling the direct measurement of signaling activities in living cells, yet the current technology is limited in resolution and dimensionality. Here, we introduce highly sensitive chemigenetic kinase activity biosensors that combine the genetically encodable self-labeling protein tag HaloTag7 with bright far-red-emitting synthetic fluorophores. This technology enables five-color biosensor multiplexing, 4D activity imaging, and functional super-resolution imaging via stimulated emission depletion (STED) microscopy.

Fourier light field microscopy (FLFM) captures a sample 3D volume in a single snapshot, providing game-changing imaging speed for various bio-imaging applications. Existing FLFM platforms have often been designed as single-purpose implementations, with a fixed performance in resolution and field of view, restricting widespread applications. Here, to democratize FLFM toward broader adoption for biomedical research, we describe a multi-purpose implementation of FLFM that enables synchronous volumetric imaging across a wide range of resolution and field of view. With our single instrument, we demonstrate a variety of bio-imaging applications across scales, including sub-cellular dynamics, tissue cellular dynamics, and whole-brain neural activity.

Insulin vesicle heterogeneity within the pancreatic β cell has been previously observed, but the extent of this heterogeneity has not been determined due to limitations in organelle separation techniques. There has previously been a lack of unbiased methods for studying differences in organelles, such as the insulin vesicle. Direct current insulator-based dielectrophoresis (DC-iDEP) separates particles based on various biophysical properties without requiring fluorescent labels, characterizing them by their electrokinetic mobility ratio (EKMr). This method has been applied to insulin vesicles isolated from mammalian insulin-secreting cell lines in order to investigate the degree of insulin vesicle heterogeneity, as well as how these subpopulations fluctuate under various cell conditions. There were significant differences in the distribution of insulin vesicles between conditions, suggesting variations in subpopulation …

Understanding the complexity of life requires that it be studied with as many dimensions as possible in each experiment. STELLARIS 8 DIVE multiphoton system enables flexible spectral detection and the combination of lifetime-based information with deep imaging beyond 1 mm.

Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative divisions that generate new tissue patterns with proliferative divisions that promote growth is poorly understood. SHORTROOT (SHR) and SCARECROW (SCR) are transcription factors that are required for formative divisions in the stem cell niche of Arabidopsis roots 1, 2. Here we show that levels of SHR and SCR early in the cell cycle determine the orientation of the division plane, resulting in either formative or proliferative cell division. We used 4D quantitative, long-term and frequent (every 15 min for up to 48 h) light sheet and confocal microscopy to probe the dynamics of SHR and SCR in tandem within single cells of living roots. Directly controlling their dynamics with an SHR induction system enabled us to challenge an existing bistable model 3 of the SHR …

For generations researchers have been observing the dynamic processes of life through the lens of a microscope. This has offered tremendous insights into biological phenomena that span multiple orders of time- and length-scales ranging from the pure magic of molecular reorganization at the membrane of immune cells, to cell migration and differentiation during development or wound healing. Standard fluorescence microscopy techniques offer glimpses at such processes in vitro, however, when applied in intact systems, they are challenged by reduced signal strengths and signal-to-noise ratios that result from deeper imaging. As a remedy, two-photon excitation (TPE) microscopy takes a special place, because it allows us to investigate processes in vivo, in their natural environment, even in a living animal. Here, we review the fundamental principles underlying TPE aimed at basic and advanced microscopy …

During development, cells in multicellular organisms undergo a series of heavily regulated steps that lead to their differentiation into specific cell types. When this process goes wrong–it can lead to diseases like cancer and developmental malformations. Unlike animals, where differentiation stops primarily after embryogenesis, plants grow continuously and have differentiating cells throughout their lifespan. One major differentiation process occurs when a specific stem cell divides formatively into the endodermis and cortex. In the model system-Arabidopsis thaliana, SHORTROOT (SHR) and SCARECROW (SCR) are transcription factors (TFs) that regulate the decision-making process of whether to divide formatively or proliferatively. Using an inducible SHR system, we followed the dynamics of these TFs and observed the cell outcome in long timelapses with high temporal resolution of more than 1000 cells. We …

This invention relates to a hyperspectral imaging system for denoising and/or color unmixing multiple overlapping spectra in a low signal-to-noise regime with a fast analysis time. This system may carry out Hyper-Spectral Phasors (HySP) calculations to effectively analyze hyper-spectral time-lapse data. For example, this system may carry out Hyper-Spectral Phasors (HySP) calculations to effectively analyze five-dimensional (5D) hyper-spectral time-lapse data. Advantages of this imaging system may include:(a) fast computational speed,(b) the ease of phasor analysis, and (c) a denoising algorithm to obtain the minimally-acceptable signal-to-noise ratio (SNR). An unmixed color image of a target may be generated. These images may be used in diagnosis of a health condition, which may enhance a patient's clinical outcome and evolution of the patient's health.