Functional in vivo imaging using fluorescence lifetime light-sheet microscopy.

Light-sheet microscopy has become an indispensable tool for fast, low phototoxicity volumetric imaging of biological samples, predominantly providing structural or analyte concentration data in its standard format. Fluorescence lifetime imaging microscopy (FLIM) provides functional contrast, but often at limited acquisition speeds and with complex implementation. Therefore, we incorporate a dedicated frequency domain CMOS FLIM camera and intensity-modulated laser into a light-sheet setup to add fluorescence lifetime imaging functionality, allowing the rapid acquisition of volumetric data with concentration independent contrast. We then apply the system to image live transgenic zebrafish, demonstrating the capacity to rapidly collect volumetric FLIM data from an in vivo sample.

Tools to Image Germplasm Dynamics During Early Zebrafish Development.

During the first day of zebrafish development, ribonucleoprotein (RNP) complexes called germplasm form large aggregates that initially segregate asymmetrically during cleavage stages. After zygotic genome activation, the granules break into smaller fragments that associate with the nuclear membrane as perinuclear (germ) granules toward the end of gastrulation. The mechanisms underlying the highly dynamic behavior of germ granules are not well studied but thought to be facilitated by the cytoskeleton. Here, we present efficient mounting strategies using 3d-printed tools that generate wells on agarose-coated sample holders to allow high-resolution imaging of multiplexed embryos that are less than one day post-fertilization (dpf) on inverted (spinning disk confocal) as well as upright (lattice light-sheet and diSPIM) microscopes. In particular, our tools and methodology allow water dipping lenses to have direct access to mounted embryos, with no obstructions to the light path (e.g., through low melting agarose or methyl cellulose). Moreover, the multiplexed tight arrays of wells generated by our tools facilitate efficient mounting of early embryos (including cleavage stages) for live imaging. These methods and tools, together with new transgenic reporter lines, can facilitate the study of germ granule dynamics throughout their lifetime in detail, at high resolution and throughput, using live imaging technologies.

The Structure and Measurement of Unusual Sensory Experiences in Different Modalities: The Multi-Modality Unusual Sensory Experiences Questionnaire (MUSEQ).

Hallucinations and other unusual sensory experiences (USE) can occur in all modalities in the general population. Yet, the existing literature is dominated by investigations into auditory hallucinations ("voices"), while other modalities remain under-researched. Furthermore, there is a paucity of measures which can systematically assess different modalities, which limits our ability to detect individual and group differences across modalities. The current study explored such differences using a new scale, the Multi-Modality Unusual Sensory Experiences Questionnaire (MUSEQ). The MUSEQ is a 43-item self-report measure which assesses USE in six modalities: auditory, visual, olfactory, gustatory, bodily sensations, and sensed presence. Scale development and validation involved a total of 1,300 participants, which included: 513 students and community members for initial development, 32 individuals with schizophrenia spectrum disorder or bipolar disorder for validation, 659 students for factor replication, and 96 students for test-retest reliability. Confirmatory factor analyses showed that a correlated-factors model and bifactor model yielded acceptable model fit, while a unidimensional model fitted poorly. These findings were confirmed in the replication sample. Results showed contributions from a general common factor, as well as modality-specific factors. The latter accounted for less variance than the general factor, but could still detect theoretically meaningful group differences. The MUSEQ showed good reliability, construct validity, and could discriminate non-clinical and clinical groups. The MUSEQ offers a reliable means of measuring hallucinations and other USE in six different modalities.

Femtosecond optoinjection of intact tobacco BY-2 cells using a reconfigurable photoporation platform.

A tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant ("diffraction-free") Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells.