Real-time study of spatio-temporal dynamics (4D) of physiological activities in alive biological specimens with different FOVs and resolutions simultaneously.

This article reports the development of a microscopy imaging system that gives feasibility for studying spatio-temporal dynamics of physiological activities of alive biological specimens (over entire volume not only for a particular section, i.e., in 4D). The imaging technology facilitates to obtain two image frames of a section of the larger specimen ([Formula: see text]) with different FOVs at different resolutions or magnifications simultaneously in real-time (in addition to recovery of 3D (volume) information). Again, this imaging system addresses the longstanding challenges of housing multiple light sources (6 at the maximum till date) in microscopy (in general) and light sheet fluorescence microscopy (LSFM) (in particular), by using a tuneable pulsed laser source (with an operating wavelength in the range [Formula: see text]-670 nm) in contrast to the conventional CW laser source being adopted for inducing photo-excitation of tagged fluorophores. In the present study, we employ four wavelengths ([Formula: see text] 488 nm, 585 nm, 590 nm, and 594 nm). Our study also demonstrates quantitative characterization of spatio-temporal dynamics (velocity-both amplitude and direction) of organelles (mitochondria) and their mutual correlationships. Mitochondria close to the nucleus (or in clustered cells) are observed to possess a lower degree of freedom in comparison to that at the cellular periphery (or isolated cells). In addition, the study demonstrates real-time observation and recording of the development and growth of all tracheal branches during the entire period ([Formula: see text] min) of embryonic development (Drosophila). The experimental results-with experiments being conducted in various and diversified biological specimens (Drosophila melanogaster, mouse embryo, and HeLa cells)-demonstrate that the study is of great scientific impact both from the aspects of technology and biological sciences.

Lint, a transmembrane serine protease, regulates growth and metabolism in Drosophila.

Insulin signaling in Drosophila has a significant role in regulating growth, metabolism, fecundity, stress response, and longevity. The molecular mechanism by which insulin signaling regulates these vital processes is dependent on the nutrient status and oxygen availability of the organism. In a genetic screen to identify novel genes that regulate Drosophila insulin signaling, we discovered lumens interrupted (lint), a gene that has previously been shown to act in tracheal development. The knockdown of lint gene expression using a Dilp2Gal4 driver which expresses in the neuronal insulin producing cells (IPCs), led to defects in systemic insulin signaling, metabolic status and growth. However, our analysis of lint knockdown phenotypes revealed that downregulation of lint in the trachea and not IPCs was responsible for the growth phenotypes, as the Gal4 driver is also expressed in the tracheal system. We found various tracheal terminal branch defects, including reduction in the length as well as number of branches in the lint knockdown background. Our study reveals that substantial effects of lint downregulation arose because of tracheal defects, which induced tissue hypoxia, altered systemic insulin/TOR signaling, and resulted in effects on developmental growth regulation.