Miniaturized head-mounted device for whole cortex mesoscale imaging in freely behaving mice

ABSTRACTThe advent of genetically encoded calcium indicators, along with surgical preparations such as thinned skulls or refractive index matched skulls, have enabled mesoscale cortical activity imaging in head-fixed mice. Such imaging studies have revealed complex patterns of coordinated activity across the cortex during spontaneous behaviors, goal-directed behavior, locomotion, motor learning, and perceptual decision making. However, neural activity during unrestrained behavior significantly differs from neural activity in head-fixed animals. Whole-cortex imaging in freely behaving mice will enable the study of neural activity in a larger, more complex repertoire of behaviors not possible in head-fixed animals. Here we present the “Mesoscope,” a wide-field miniaturized, head-mounted fluorescence microscope compatible with transparent polymer skulls recently developed by our group. With a field of view of 8 mm x 10 mm and weighing less than 4 g, the Mesoscope can image most of the mouse dorsal cortex with resolution ranging from 39 to 56 µm. Stroboscopic illumination with blue and green LEDs allows for the measurement of both fluorescence changes due to calcium activity and reflectance signals to capture hemodynamic changes. We have used the Mesoscope to successfully record mesoscale calcium activity across the dorsal cortex during sensory-evoked stimuli, open field behaviors, and social interactions. Finally, combining the mesoscale imaging with electrophysiology enabled us to measure dynamics in extracellular glutamate release in the cortex during the transition from wakefulness to natural sleep.

Ultra-High-Throughput Multi-Parametric Imaging Flow Cytometry

I will present a microfluidic imaging flow cytometer incorporating stroboscopic illumination, for blur-free cellular analysis at throughputs exceeding 100,000 cells per second. By combining passive (inertial or viscoelastic) focusing of cells in parallel microchannels with stroboscopic illumination, such chip-based cytometers are able to extract multi-colour fluorescence and bright-field images of single cells moving at high linear velocities. This in turn allows accurate sizing of individual cells, intracellular localization and analysis of heterogeneous cell suspensions. The method is showcased through the rapid enumeration of apoptotic cells, high-throughput discrimination cell cycle phases and localization of p-bodies.

Magneto-Optic Eddy Current Introscopy Based on Garnet Films

Magneto-optic eddy current introscopy device based on garnet films with stroboscopic illumination is described. Experiment was done with liquid-phase epitaxy garnet films with composition (Bi,Eu,Lu)3(Fe,Ga,Al)5O12 and (Bi,Tm)3(Fe,Ga)5O12, crystallographic orientation (111).

A caged progesterone analog alters intracellular Ca2+ and flagellar bending in human sperm

Progesterone is a physiological agonist for mammalian sperm, modulating its flagellar movement and facilitating the acrosome reaction. To study the initial action of progesterone, we developed a caged analog with a photosensitive group: nitrophenylethanediol, at position 20. Using this compound combined with stroboscopic illumination, we performed Ca2+imaging of human spermatozoa and analyzed the effects of progesterone on the intracellular Ca2+concentration ([Ca2+]i) of beating flagella for the first time. We observed a transient [Ca2+]iincrease in the head and the flagellum upon photolysis of the caged progesterone and an increase in flagellar curvature. Detailed kinetic analysis revealed that progesterone elicits an increase in the [Ca2+]iimmediately in the flagellum (mid-piece and principal piece), thereafter in the head with a short time lag. This observation is different from the progesterone-induced Ca2+mobilization in mouse spermatozoa, where the Ca2+rise initiates at the base of the sperm head. Our finding is mostly consistent with the recent discovery that progesterone activates CatSper channels in human spermatozoa, but not in mouse spermatozoa.