Due to its sectioning capability, large field of view, and minimal light exposure, selective plane illumination microscopy has become the preferred choice for 3D time lapse imaging. Single cells in a dish can be conveniently imaged using an upright/inverted configuration. However, for measurements on long time scales (hours to days), mechanical drift is a problem; especially for studies of mammalian cells that typically require heating to 37°C which causes a thermal gradient across the instrument. Since the light sheet diverges towards the edges of the field of view, such a drift leads to a decrease in axial resolution over time. Or, even worse, the specimen could move out of the imaging volume. Here, we present a simple, cost-effective way to stabilize the axial position using the microscope camera to track the sample position. Thereby, sample loss is prevented and an optimal axial resolution is maintained by keeping the sample at the position where the light sheet is at its thinnest. We demonstrate the virtue of our approach by measurements of the light sheet thickness and 3D time lapse imaging of a cell monolayer at physiological conditions.

Light sheet microscopy is a powerful technique for rapid, three-dimensional fluorescence imaging of large specimen such as drosophila and zebrafish embryos. Yet, beam divergence results in a loss of axial resolution at the periphery of the light sheet. Here, we demonstrate how an electrically tunable lens can be utilized to maintain the minimal, diffraction-limited thickness of the light sheet over a wide field of view (>600 µm) at high frame rates (40 fps). This mode of operation is necessary for the application of fluorescence fluctuation spectroscopy in images. Microsc. Res. Tech. 81:924-928, 2018. © 2016 Wiley Periodicals, Inc.

The ability to measure biomolecular dynamics within cells and tissues is very important to understand fundamental physiological processes including cell adhesion, signalling, movement, division or metabolism. Usually, such information is obtained using particle tracking methods or single point fluctuation spectroscopy. We show that image mean square displacement analysis, applied to single plane illumination microscopy data, is a faster and more efficient way of unravelling rapid, three-dimensional molecular transport and interaction within living cells. From a stack of camera images recorded in seconds, the type of dynamics such as free diffusion, flow or binding can be identified and quantified without being limited by current camera frame rates. Also, light exposure levels are very low and the image mean square displacement method does not require calibration of the microscope point spread function. To demonstrate the advantages of our approach, we quantified the dynamics of several different proteins in the cyto- and nucleoplasm of living cells. For example, from a single measurement, we were able to determine the diffusion coefficient of free clathrin molecules as well as the transport velocity of clathrin-coated vesicles involved in endocytosis. Used in conjunction with dual view detection, we further show how protein-protein interactions can be quantified.

Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein.

Fluorescence-based single particle counting devices have become very powerful tools for human health-related applications such as the detection of blood-borne pathogens. Instead of passing the sample fluid through a thin tube or microfluidic chip, as it is commonly practiced in flow cytometers and sorter devices, single particle counters scan the fluid volume by rotation and translation of the sample container. Hence, single particle counters are not limited by the fluid flow friction and can scan a large volume in a short timeframe while maintaining high sensitivity. A single particle can be detected in a milliliter of the fluid sample within minutes, and diagnostics are being developed using this principle. Until now, signal detection with particle counters has been based on signal intensity and signal separation into multiple wavelength bands coupled with multiple detectors, which limits the number of species that can be resolved. In this paper, we applied fluorescence lifetime detection to single particle counting to increase specificity and enable multiplexing with a single detector. We demonstrate how this principle can be used for diagnostic assays based on fluorescence quenching.

Educated natural killer (NK) cells have inhibitory receptors specific for self major histocompatibility complex (MHC) class I molecules and kill cancer cells more efficiently than do NK cells that do not have such receptors (hyporesponsive NK cells). The mechanism behind this functional empowerment through education has so far not been fully described. In addition, distinctive phenotypic markers of educated NK cells at the single-cell level are lacking. We developed a refined version of the image mean square displacement (iMSD) method (called iMSD carpet analysis) and used it in combination with single-particle tracking to characterize the dynamics of the activating receptor NKp46 and the inhibitory receptor Ly49A on resting educated versus hyporesponsive murine NK cells. Most of the NKp46 and Ly49A molecules were restricted to microdomains; however, individual NKp46 molecules resided in these domains for shorter periods and diffused faster on the surface of educated, compared to hyporesponsive, NK cells. In contrast, the movement of Ly49A was more constrained in educated NK cells compared to hyporesponsive NK cells. Either disrupting the actin cytoskeleton or adding cholesterol to the cells prohibited activating signaling, suggesting that the dynamics of receptor movements within the cell membrane are critical for the proper activation of NK cells. The faster and more dynamic movement of NKp46 in educated NK cells may facilitate a swifter response to interactions with target cells.

Despite recent advances in optical super-resolution, we lack a method that can visualize the path followed by diffusing molecules in the cytoplasm or in the nucleus of cells. Fluorescence correlation spectroscopy (FCS) provides molecular dynamics at the single molecule level by averaging the behavior of many molecules over time at a single spot, thus achieving very good statistics but at only one point in the cell. Earlier image-based methods including raster-scan and spatiotemporal image correlation need spatial averaging over relatively large areas, thus compromising spatial resolution. Here, we use spatial pair-cross-correlation in two dimensions (2D-pCF) to obtain relatively high resolution images of molecular diffusion dynamics and transport in live cells. The 2D-pCF method measures the time for a particle to go from one location to another by cross-correlating the intensity fluctuations at specific points in an image. Hence, a visual map of the average path followed by molecules is created.

Selective plane illumination microscopy (SPIM), or light sheet microscopy, is a powerful imaging approach. However, access to and interfacing microscopes with microfluidics have remained challenging. Complex interfacing with microfluidics has limited the SPIM's utility for studying the hydrodynamics of freely moving multicellular organisms. We developed SPIM-Flow, an inexpensive light sheet platform that enables easy integration with microfluidics. We used SPIM-Flow to investigate the hydrodynamics of a freely moving Hydra polyp via particle tracking in millimeter-sized chambers. Initial experiments across multiple animals, feeding on a chip (Artemia franciscana nauplii used as food), and baseline behaviors (tentacle swaying, elongation, and bending) indicated the organisms' health inside the system. Fluidics were used to investigate Hydra's response to flow. The results suggested that the animals responded to an established flow by bending and swaying their tentacles in the flow direction. Finally, using SPIM-Flow in a proof-of-concept experiment, the shear stress required to detach an animal from a surface was demonstrated. Our results demonstrated SPIM-Flow's utility for investigating the hydrodynamics of freely moving animals.