Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging.

Over the last decade, single-molecule localization microscopy (SMLM) has developed into a set of powerful techniques that have improved spatial resolution over diffraction-limited microscopy and demonstrated the ability to resolve biological features down to a few tens of nanometers. We introduce a single molecule-based scanning SMLM (scanSMLM) system that enables rapid volume imaging. Along with epi-illumination, the system employs a scanning-based 4f detection for volume imaging. The 4f system comprises a combination of an electrically-tunable lens and high NA detection objective lens. By rapidly changing the aperture (or equivalently the focus) of an electrically-tunable lens (ETL) in a 4f detection system, the selectivity of the axial object plane is achieved, for which the image forms in the image/detector plane. So, in principle, one can scan the object volume by just altering the aperture of ETL. Two schemes were adopted to carry out volume imaging: cyclic scan and conventional scan. The cyclic scheme scans the volume in each scan cycle, whereas plane-wise scanning is performed in the conventional scheme. Hence, the cyclic scan ensures uniform dwell time on each frame during data collection, thereby evenly distributing photobleaching throughout the cell volume. With a minimal change in the system hardware (requiring the addition of an ETL lens and related electronics for step-voltage generation) in the existing SMLM system, volume scanning (along the z-axis) can be achieved. To calibrate and derive critical system parameters, we imaged fluorescent beads embedded in a gel-matrix 3D block as a test sample. Subsequently, scanSMLM is employed to visualize the architecture of actin-filaments and the distribution of Meos-Tom20 molecules on the mitochondrial membrane. The technique is further exploited to understand the clustering of Hemagglutinin (HA) protein single molecules in a transfected cell for studying Influenza-A disease progression. The system, for the first time, enabled 3D visualization of HA distribution that revealed HA cluster formation spanning the entire cell volume, post 24 hrs of transfection. Critical biophysical parameters related to HA clusters (density, the number of HA molecules per cluster, axial span, fraction of clustered molecules, and others) are also determined, giving an unprecedented insight into Influenza-A disease progression at the single-molecule level.

Temporally resolved SMLM (with large PAR shift) enabled visualization of dynamic HA cluster formation and migration in a live cell.

The blinking properties of a single molecule are critical for single-molecule localization microscopy (SMLM). Typically, SMLM techniques involve recording several frames of diffraction-limited bright spots of single-molecules with a detector exposure time close to the blinking period. This sets a limit on the temporal resolution of SMLM to a few tens of milliseconds. Realizing that a substantial fraction of single molecules emit photons for time scales much shorter than the average blinking period, we propose accelerating data collection to capture these fast emitters. Here, we put forward a short exposure-based SMLM (shortSMLM) method powered by sCMOS detector for understanding dynamical events (both at single molecule and ensemble level). The technique is demonstrated on an Influenza-A disease model, where NIH3T3 cells (both fixed and live cells) were transfected by Dendra2-HA plasmid DNA. Analysis shows a 2.76-fold improvement in the temporal resolution that comes with a sacrifice in spatial resolution, and a particle resolution shift PAR-shift (in terms of localization precision) of [Formula: see text] 11.82  nm compared to standard SMLM. We visualized dynamic HA cluster formation in transfected cells post 24 h of DNA transfection. It is noted that a reduction in spatial resolution does not substantially alter cluster characteristics (cluster density, [Formula: see text] molecules/cluster, cluster spread, etc.) and, indeed, preserves critical features. Moreover, the time-lapse imaging reveals the dynamic formation and migration of Hemagglutinin (HA) clusters in a live cell. This suggests that [Formula: see text] using a synchronized high QE sCMOS detector (operated at short exposure times) is excellent for studying temporal dynamics in cellular system.

Lightsheet optical tweezer (LOT) for optical manipulation of microscopic particles and live cells.

Optical trapping and patterning cells or microscopic particles is fascinating. We developed a light sheet-based optical tweezer to trap dielectric particles and live HeLa cells. The technique requires the generation of a tightly focussed diffraction-limited light-sheet realized by a combination of cylindrical lens and high NA objective lens. The resultant field is a focussed line (along x-axis) perpendicular to the beam propagation direction (z-axis). This is unlike traditional optical tweezers that are fundamentally point-traps and can trap one particle at a time. Several spherical beads undergoing Brownian motion in the solution are trapped by the lightsheet gradient potential, and the time (to reach trap-centre) is estimated from the video captured at 230 frames/s. High-speed imaging of beads with increasing laser power shows a steady increase in trap stiffness with a maximum of 0.00118 pN/nm at 52.5 mW. This is order less than the traditional point-traps, and hence may be suitable for applications requiring delicate optical forces. On the brighter side, light sheet tweezer (LOT) can simultaneously trap multiple objects with the distinct ability to manipulate them in the transverse (xy) plane via translation and rotation. However, the trapped beads displayed free movement along the light-sheet axis (x-axis), exhibiting a single degree of freedom. Furthermore, the tweezer is used to trap and pattern live HeLa cells in various shapes and structures. Subsequently, the cells were cultured for a prolonged period of time (> 18 h), and cell viability was ascertained. We anticipate that LOT can be used to study constrained dynamics of microscopic particles and help understand the patterned cell growth that has implications in optical imaging, microscopy, and cell biology.

Light sheet based volume flow cytometry (VFC) for rapid volume reconstruction and parameter estimation on the go.

Optical imaging is paramount for disease diagnosis and to access its progression over time. The proposed optical flow imaging (VFC/iLIFE) is a powerful technique that adds new capabilities (3D volume visualization, organelle-level resolution, and multi-organelle screening) to the existing system. Unlike state-of-the-art point-illumination-based biomedical imaging techniques, the sheet-based VFC technique is capable of single-shot sectional visualization, high throughput interrogation, real-time parameter estimation, and instant volume reconstruction with organelle-level resolution of live specimens. The specimen flow system was realized on a multichannel (Y-type) microfluidic chip that enables visualization of organelle distribution in several cells in-parallel at a relatively high flow-rate (2000 nl/min). The calibration of VFC system requires the study of point emitters (fluorescent beads) at physiologically relevant flow-rates (500-2000 nl/min) for determining flow-induced optical aberration in the system point spread function (PSF). Subsequently, the recorded raw images and volumes were computationally deconvolved with flow-variant PSF to reconstruct the cell volume. High throughput investigation of the mitochondrial network in HeLa cancer cell was carried out at sub-cellular resolution in real-time and critical parameters (mitochondria count and size distribution, morphology, entropy, and cell strain statistics) were determined on-the-go. These parameters determine the physiological state of cells, and the changes over-time, revealing the metastatic progression of diseases. Overall, the developed VFC system enables real-time monitoring of sub-cellular organelle organization at a high-throughput with high-content capacity.

Fluorescence based rapid optical volume screening system (OVSS) for interrogating multicellular organisms.

Continuous monitoring of large specimens for long durations requires fast volume imaging. This is essential for understanding the processes occurring during the developmental stages of multicellular organisms. One of the key obstacles of fluorescence based prolonged monitoring and data collection is photobleaching. To capture the biological processes and simultaneously overcome the effect of bleaching, we developed single- and multi-color lightsheet based OVSS imaging technique that enables rapid screening of multiple tissues in an organism. Our approach based on OVSS imaging employs quantized step rotation of the specimen to record 2D angular data that reduces data acquisition time when compared to the existing light sheet imaging system (SPIM). A co-planar multicolor light sheet PSF is introduced to illuminate the tissues labelled with spectrally-separated fluorescent probes. The detection is carried out using a dual-channel sub-system that can simultaneously record spectrally separate volume stacks of the target organ. Arduino-based control systems were employed to automatize and control the volume data acquisition process. To illustrate the advantages of our approach, we have noninvasively imaged the Drosophila larvae and Zebrafish embryo. Dynamic studies of multiple organs (muscle and yolk-sac) in Zebrafish for a prolonged duration (5 days) were carried out to understand muscle structuring (Dystrophin, microfibers), primitive Macrophages (in yolk-sac) and inter-dependent lipid and protein-based metabolism. The volume-based study, intensity line-plots and inter-dependence ratio analysis allowed us to understand the transition from lipid-based metabolism to protein-based metabolism during early development (Pharyngula period with a critical transition time, [Formula: see text] h post-fertilization) in Zebrafish. The advantage of multicolor lightsheet illumination, fast volume scanning, simultaneous visualization of multiple organs and an order-less photobleaching makes OVSS imaging the system of choice for rapid monitoring and real-time assessment of macroscopic biological organisms with microscopic resolution.