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.

TLR-mediated aggresome-like induced structures comprise antimicrobial peptides and attenuate intracellular bacterial survival.

Immune cells employ diverse mechanisms for host defense. Macrophages, in response to TLR activation, assemble aggresome-like induced structures (ALIS). Our group has shown TLR4-signaling transcriptionally upregulates p62/sequestome1, which assembles ALIS. We have demonstrated that TLR4-mediated autophagy is, in fact, selective-autophagy of ALIS. We hypothesize that TLR-mediated autophagy and ALIS contribute to host-defense. Here we show that ALIS are assembled in macrophages upon exposure to different bacteria. These structures are associated with pathogen-containing phagosomes. Importantly, we present evidence of increased bacterial burden, where ALIS assembly is prevented with p62-specific siRNA. We have employed 3D-super-resolution structured illumination microscopy (3D-SR-SIM) and mass-spectrometric (MS) analyses to gain insight into the assembly of ALIS. Ultra-structural analyses of known constituents of ALIS (p62, ubiquitin, LC3) reveal that ALIS are organized structures with distinct patterns of alignment. Furthermore, MS-analyses of ALIS identified, among others, several proteins of known antimicrobial properties. We have validated MS data by testing the association of some of these molecules (Bst2, IFITM2, IFITM3) with ALIS and the phagocytosed-bacteria. We surmise that AMPs enrichment in ALIS leads to their delivery to bacteria-containing phagosomes and restricts the bacteria. Our findings in this paper support hitherto unknown functions of ALIS in host-defense.