bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.05.02.591710

TRapping and IMaging (TRIMing) of Cells/Multicellular Organisms in Free Living Environment Enabled by Adaptive Lightsheet Optical Tweezer (aLOT)

Baro, N.·Basumatary, J.·Pant, N.·Mondal, P. P.
View original source ↗Export metadata ↓

Abstract

To be able to trap and image in a live cell / organism on the go is an incredible feat and paves the way for immobilization-free interrogation. This is a step towards the interrogation of cells / live species in their natural environment. To facilitate, a TRIMing technique primarily based on an adaptive lightsheet optical tweezer (aLOT) system is proposed. The TRIMing technique combines the benefits of touch-free optical tweezing and high-resolution imaging. The entire system is built on a single platform for rapid interrogation of freely moving live biological specimens. The trapping system combines an electrical-tunable lens (ETL), cylindrical lens, and an objective lens to generate adaptive PSF. The ETL (in the beam-expander) adaptively changes the beam cross-section (to either a parallel beam or converging point-beam) entering the back-aperture of cylindrical lens, resulting in a point or a line spot at the focus. An objective lens placed at the focus of a cylindrical lens converts the spot to a tightly focused diffraction-limited lightsheet or point PSF. Depending on the object type (spherical or elongated), the system can flip between point and sheet PSF at a rate of 200 Hz. The system is integrated to a separate fluorescence arm to enable the imaging of trapped objects (cells or organisms). The TRIMing system operates in a brightfield mode to optically trap using point / sheet PSF and subsequently switched to fluorescence mode for imaging. The potential of the system is demonstrated by trapping live specimens (HeLa cells and C. elegans labelled with Bodipy dye) and imaging them in a freely moving environment. Characterization shows a point and sheet PSF size of, 43.42 m2 and 70.5 x 4.9m2 with a trap stiffness of 1.15 x 10-3 pN/nm and 0.46 x 10-3 pN/nm, respectively. Fluorescently-labelled live specimens were investigated that showed the random distribution of organelles (lipid droplets) both in cells and C. elegans. The TRIMing system demonstrated a resolution of < 0.7m, a contrast of {approx} 0.84, a SNR of {approx} 11 dB. This allows a good combination of rapid trapping and high-quality imaging. In addition, the system allows near real-time determination of critical biophysical parameters, such as organelle size of 1.01 m (in cells) and 1.29 m (in C. elegans) with a density of 0.021#/m2 and 0.039#/m2, respectively. The number of lipid droplets are found to be nearly double for C. elegans as compared to HeLa cells. These parameters are directly linked to the physiological state of live biological species. Overall, the developed TRIMing system allows high-quality imaging of live specimens in a free living environment. Statement of SignificanceThe ability to image live specimens in a free-living environment is phenomenal. The existing techniques often constrain/fix/anesthetize these organisms to image their physiological state. This comes with a lot of conditioning and directly affects the physiological state or developmental process in biological species, especially the brain undergoing neuronal activity. The proposed TRIMing technique elevates this requirement by optically trapping the moving object and simultaneously imaging the internal organelles with high resolution in a free environment. The technique is expected to have widespread applications in diverse disciplines ranging from fundamental cell biology to optical physics.

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Baro, N., Basumatary, J., Pant, N., Mondal, P. P.. 2024-05-05. TRapping and IMaging (TRIMing) of Cells/Multicellular Organisms in Free Living Environment Enabled by Adaptive Lightsheet Optical Tweezer (aLOT). https://doi.org/10.1101/2024.05.02.591710

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Autonomous Homeostatic Synthetic Cells via Self-Gating DNA Nanopores

Homeostasis is a fundamental hallmark of living organisms, arising from the complex interplay between biochemical reactions and regulatory feedback systems. Reconstituting such self-regulating behaviour in minimal synthetic cells enables continuous, persistent operation of biochemical reactions for extended amount of time. In this work, we demonstrate a minimal homeostatic synthetic cell capable of autonomous flux regulation using DNA nanotechnology and bottom-up synthetic biology. Our homeostatic architecture consists of Giant Unilamellar Vesicles (GUVs) equipped with gated DNA nanopores, encapsulated in vitro transcription (IVT) machinery, and an RNA degradation system. We achieve homeostasis under varying external chemical stimuli specifically varying concentrations of rNTPs by implementing a negative feedback loop between rNTP influx and RNA production. In our system, DNA nanopores facilitate the influx of rNTPs from the external environment, driving internal transcription. Crucially, the transcription process generates RNA "blockers" designed to bind and gate the DNA nanopores, thereby attenuating further rNTP influx. Our system is dynamic as encapsulated RNases slowly degrade the RNA blockers, allowing the pores to reopen as blocker concentration goes down. We first characterise the functionality and gating efficiency of the DNA nanopores using both pre-synthesised and in situ produced DNA and RNA blockers. We then demonstrate that rNTP flux through these pores is sufficient to drive IVT within the GUVs. Finally, by integrating these modules, we demonstrate robust homeostasis: the system maintains a steady-state level of RNA production for up to 16 hours. By harnessing the controllability of negative feedback loop, we demonstrate thresholding of the homeostasis level using single-stranded regulator DNA. This work establishes a versatile framework for engineering adaptive and self-sustaining responsive nanomaterials and synthetic cell chassis.

biophysics↗
bioRxiv2026

A Generic Numbering Scheme for TMEM16 Scramblases

The TMEM16 family of calcium-activated phospholipid scramblases (CaPLSs) and chloride channels (CaCCs) performs diverse physiological functions that include regulation of blood coagulation and apoptotic signaling, through a shared ten-transmembrane-helix (TM) architecture organized around a hydrophilic lipid-translocating groove. Mechanistic studies of TMEM16 family members have been hampered by the absence of a unified positional reference framework that would permit direct comparison of structurally equivalent residues across paralogs with different sequence numbering systems. Here we introduce a generic numbering scheme for TMEM16 scramblases (GNS-TMEM16), modeled on the Ballesteros & Weinstein system established for class A G protein-coupled receptors. A reference alignment (TMEM16-RA) was constructed from twelve human and mouse TMEM16 scramblases (TMEM16C/D/E/F/G/J) using structure-based ClustalW alignment of the ten TM helices. From this alignment, a TM-specific reference residue (TsRR) was identified for each helix by hierarchical application of three criteria: (1) 100% conservation in the core TMEM16-RA; (2) conservation in an augmented reference alignment (TMEM16-ARA) incorporating a group of phylogenetically more distant homologs composed of nhTMEM16, afTMEM16, TMEM16K, TMEM16A, and TMEM16B; and (3) structural and functional considerations, including helix-perturbing character, groove localization, conserved motif membership, and central TM position. The resulting ten TsRRs are Y1.50, W2.50, R3.50, E4.50, F5.50, P6.50, E7.50, D8.50, W9.50, and E10.50, and are illustrated in mTMEM16F. Each residue is assigned the identifier N.m(k), where N is the TM number, m is the position relative to the TsRR (for which m = 50), and k is the absolute sequence number. Loop residues receive dual identifiers referenced to the TsRRs of both flanking helices. Application of the GNS-TMEM16 is illustrated with the comparisons of the groove-opening measurements using pairwise distances between residues identified by their N.m indices to be corresponding across mTMEM16F, afTMEM16, and nhTMEM16. The results bring to light the advantages of corresponding residues identification in different TMEM16 proteins and show that the mammalian scramblase undergoes substantially larger separation at the extracellular groove entrance than either fungal homolog. Comparison of mutagenesis data guided by N.m correspondence shows at the conserved (E3.55,R6.26) salt-bridge locus, Ala substitution reduces activity more than 100-fold in nhTMEM16 but less than 2-fold in afTMEM16, illustrating that the GNS identifies structural equivalence of position without implying functional equivalence of the residue, which is a distinct advantage of GNS in providing mechanistic interpretation across paralogs. Also described is a protocol for extending the GNS-TMEM16 to uncharacterized protein sequences, including AlphaFold-predicted models, using structural superposition to mTMEM16F. Thus, the presented GNS-TMEM16 provides a stable positional reference for the integration and comparative analysis of structural, computational, and functional data across the TMEM16 family, utilizing a construction strategy applicable to yet other polytopic membrane protein families sharing a common transmembrane fold.

biophysics↗
bioRxiv2026

An agent-based 3D model of non-genetic adaptation in cancer tissues under electrical, mechanical, and hypoxic stress

Non-genetic adaptation enables cancer cells to alter their phenotype under stress without requiring new mutations. However, the mechanisms by which electrical, mechanical, and hypoxic cues combine to shape this process in 3D tissues remain poorly understood. This work presents an agent-based tumor model that integrates vascular oxygen supply, a globally imposed electric field, mechanically mediated crowding and compression cues, phenotype transitions, cell growth, mitosis, death, and inheritance of adaptive memory across division. The simulated tumors exhibit a three-stage trajectory consisting of necrosis onset, transient collapse of live mass, and partial regrowth accompanied by progressive accumulation of adapted cells. Continuous electrical stimulation produces a dose-dependent reduction in live mass while markedly increasing the adapted fraction, with comparatively limited changes in final necrotic burden. This response is strongly conditioned by mechanics and reshapes (and is reshaped by) adaptive capacity. Pulsed stimulation further shows that, in the model, electric field amplitude and temporal schedule jointly determine memory phenomena, phenotypic diversification, and growth recovery. These results show that coupling local oxygen availability, mechanical constraints, electrical forcing, and history-dependent phenotype transitions can generate distinct tissue-level patterns of phenotypic heterogeneity. Both stimulus magnitude and temporal protocol influenced the resulting population structure, suggesting that the history of physical stress may be an important determinant of adaptive dynamics in spatially organized tumor models.

biophysics↗