sCMOS Camera Research Articles

Fast 3-D Imaging of Brain Organoids With a New Single-Objective Planar-Illumination Two-Photon Microscope.

Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies (EBs) enlarge the scope of investigations to multi-cellular phenomena. The highest level of complexity, brain organoids that—in many aspects—recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that uses planar two-photon (2P) excitation. Its particularity is that—unlike two- or three-lens light-sheet microscopes—it uses a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petráň spinning-disk scheme for achieving wide-field excitation. However, unlike the Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This new design, advantageous for 2P excitation, circumvents problems arising with the tandem disk from the large wavelength difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited by the light sheet is collected with the same objective and imaged onto a fast sCMOS camera. We demonstrate 3-D imaging of TO-PRO3-stained EBs and of brain organoids, uncleared and after rapid partial transparisation with triethanolamine formamide (RTF) and we compare the performance of our instrument to that of a confocal laser-scanning microscope (CLSM) having a similar numerical aperture. Our large-field 2P-spinning disk microscope permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.

Bioluminescent flashes drive nighttime schooling behavior and synchronized swimming dynamics in flashlight fish

Schooling fishes, like flocking birds and swarming insects, display remarkable behavioral coordination. While over 25% of fish species exhibit schooling behavior, nighttime schooling has rarely been observed or reported. This is due to vision being the primary modality for schooling, which is corroborated by the fact that most fish schools disperse at critically low light levels. Here we report on a large aggregation of the bioluminescent flashlight fish Anomalops katoptron that exhibited nighttime schooling behavior during multiple moon phases, including the new moon. Data were recorded with a suite of low-light imaging devices, including a high-speed, high-resolution scientific complementary metal-oxide-semiconductor (sCMOS) camera. Image analysis revealed nighttime schooling using synchronized bioluminescent flashing displays, and demonstrated that school motion synchrony exhibits correlation with relative swim speed. A computer model of flashlight fish schooling behavior shows that only a small percentage of individuals need to exhibit bioluminescence in order for school cohesion to be maintained. Flashlight fish schooling is unique among fishes, in that bioluminescence enables schooling in conditions of no ambient light. In addition, some members can still partake in the school while not actively exhibiting their bioluminescence. Image analysis of our field data and model demonstrate that if a small percentage of fish become motivated to change direction, the rest of the school follows. The use of bioluminescence by flashlight fish to enable schooling in shallow water adds an additional ecological application to bioluminescence and suggests that schooling behavior in mesopelagic bioluminescent fishes may be also mediated by luminescent displays.

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules.

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera andwide-fieldilluminationmicrotubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.

Abstract 462: High Speed Imaging of Single Cardiomyocyte Action Potentials Using a Far-red Genetically Encoded Voltage Sensor

The ability to image cardiac membrane potentials allows for the observation of cellular communication and electrical activity, both of which are important to maintain cardiac syncytium; these can be altered in diseases (e.g. Long QT Syndrome). Traditionally voltage dyes such as Di-8-ANEPPS have been used to optically measure action potentials (APs). However, these dyes express transiently, have poor signal to noise ratios, and are toxic. More recently, genetically encoded voltage indicators (GEVIs) have been developed to replace state-of-the-art voltage dyes, but sensors currently used within the cardiac field exhibit poor kinetics and/or low signal to noise ratios (SNR). Recently, Archon1, a new genetically encoded voltage sensor, was developed in the neuroscience field; this sensor exhibits excellent membrane localization, temporal sensitivity, and SNR, enabling the optical detection of individual spikes in neurons. Here we use Archon1 for the first time in cardiac cells, to monitor single cell cardiac APs in 2D and 3D in vitro systems in response to different environmental stimuli. Human induced pluripotent stem cell-derived cardiomyocytes were infected with Archon1 and imaged using a one-photon fluorescence microscope equipped with a high speed sCMOS camera to demonstrate cardiac AP tracings. The kinetics and SNR of Archon1 are compared to traditional electrophysiology and Di-8-ANEPPS measurements. Additionally, E-4031 (K+ Channel Blocker) and Nifedipine (Ca2+ Channel Blocker) were used to demonstrate the sensitivity of this sensor in a drug dosage study. To study the APs of single cells within a 3D engineered microtissue, cardiomyocytes expressing Archon1 were seeded into a force transducing micro-pillar device and the APs for optically isolated cells were recorded. Demonstration of this new genetically encoded voltage sensor in cardiac cells enables the monitoring of single and multi-cell APs in 2D and 3D applications and can be extended to in vivo. This tool, newly applied to cardiac biology and tissue engineering will allow for better and more accurate observation of cardiac electrical activity to probe human cardiovascular disease.

Evaluation of scientific complementary metal–oxide–semiconductor sensors for sky survey applications

AbstractScientific complementary metal–oxide–semiconductor (CMOS) image sensors are a modern alternative for typical charge‐coupled device detectors, as they offer low read‐out noise, large sensitive area, and high frame rates. All these make them promising devices for a modern wide‐field sky surveys. However, the peculiarities of CMOS technology have to be properly taken into account when analyzing the data. In order to characterize these, we performed extensive laboratory testing of Andor Marana scientific CMOS (sCMOS) camera. Here, we report its results, especially on the temporal stability and linearity, and compare it with the previous versions of Andor sCMOS cameras. We also present the results of an on‐sky testing of this sensor connected to a wide‐field lens and discuss its applications for astronomical sky surveys.

3D dosimetry for proton therapy

We have been developing novel 3-dimensional (3D) detector systems using organic plastic and liquid scintillators to measure and image the dose distribution from proton therapy beams in near-real time. Proof-of-concept and initial feasibility studies using a single charge-coupled device camera have already been conducted. Our recent studies focused on the characterization of scanning proton beams used for patient treatments using a 3D liquid scintillator-based detector system with a set of scientific-complementary metal-oxide-semiconductor (sCMOS) cameras. The basic concept consists of using a large volume of a solid or liquid scintillator to measure or image the dose distributions from proton beams in 3D. We recently developed a large liquid scintillator-based detector system consisting of a 20 × 20 × 20cm transparent acrylic tank filled with a water-equivalent, commercially available liquid scintillator that generates scintillation light when irradiated with protons. To track rapid spatial and dose variations in spot-scanned proton beams, we used 3 high-speed sCMOS cameras to image the scintillation light signals from 3 orthogonal projections in cine mode. Furthermore, we developed a new image acquisition approach that synchronized camera imaging times with dynamic pencil-beam deliveries to efficiently capture the dose and therefore enable accurate dosimetric calculations. This system was fully developed and characterized at the Proton Therapy Center at The University of Texas MD Anderson Cancer Center. We show that such systems can provide fast and accurate measurements of the range, lateral profile, and lateral position of scanning proton beams with excellent spatial resolution (0.21 mm). We also demonstrate that such detectors can rapidly measure proton beam characteristics and intensities at multiple energies, which makes them an ideal tool for scanned proton-beam systems, beam quality assurance studies, and verification of patient treatment delivery.

Using a bio-inspired copper complex to investigate reactive mass transfer around an oxygen bubble rising freely in a thin-gap cell

The present study describes an original colorimetric method to visualize and quantify the local oxygen mass transfer around a rising bubble in reactive media. This method is based on the use of a colorless bio-inspired copper complex, Cu(btmgp)I, specially tailored for the study, which, dissolved in acetonitrile, oxidizes into an orange copper-complex [Cu2O2(btmgp)2]I2. The latter complex, unstable at ambient temperature, decays quite fast into two Cu(II) complexes, leaving a permanent pale-green color as final products. The flow investigated consists in a pure oxygen single bubble rising freely in a confined thin-gap cell (400 × 200 × 1 mm). A wide range of motion regimes for the bubbles are observed as the Archimedes number ranges from 860<Ar<18,800 and the Reynolds number from 580<Re<8200. A high-resolution 16-bit sCMOS camera, combined with specific filters, is used to capture images from a region-of-interest of the cell, illuminated by a white LED backlight panel. An ad hoc calibration protocol is developed to correlate the grey-levels from the colored signal to the equivalent oxygen concentrations. This procedure then allows to measure indirectly the amount of oxygen transferred to the liquid phase. The series of images are also treated to identify the bubble motion and properties. Thanks to this method, equivalent oxygen concentration fields, gap-averaged and time-averaged, can be reached with high precision in the far-field wake of the bubbles, enabling thus to deeply characterize the mass transfer mechanisms under reactive conditions in such confined configuration, and to establish a dimensionless representation in terms of Sherwood number versus Peclet number. At last, thanks to the knowledge of the kinetic rate of the reaction and of the diffusion coefficients, the Hatta number and the enhancement factor are estimated, and thus the intrinsic Sherwood numbers; these results demonstrate that the enhancement of the mass transfer by the reactions involved with the copper-complexes is not negligible (almost 12–15%).

A Teensy microcontroller-based interface for optical imaging camera control during behavioral experiments

BackgroundSystems neuroscience experiments often require the integration of precisely timed data acquisition and behavioral monitoring. While specialized commercial systems have been designed to meet various needs of data acquisition and device control, they often fail to offer flexibility to interface with new instruments and variable behavioral experimental designs. New methodWe developed a Teensy 3.2 microcontroller-based interface that is easily programmable, and offers high-speed, precisely timed behavioral data acquisition and digital and analog outputs for controlling sCMOS cameras and other devices. ResultsWe demonstrate the flexibility and the temporal precision of the Teensy interface in two experimental settings. In one example, we used the Teensy interface to record an animal’s directional movement on a spherical treadmill, while delivering repeated digital pulses that can be used to control image acquisition from a sCMOS camera. In another example, we used the Teensy interface to deliver an auditory stimulus and a gentle eye puff at precise times in a trace conditioning eye blink behavioral paradigm, while delivering repeated digital pulses to trigger camera image acquisition. Comparison with existing methodsThis interface allows high-speed and temporally precise digital data acquisition and device control during diverse behavioral experiments. ConclusionThe Teensy interface, consisting of a Teensy 3.2 and custom software functions, provides a temporally precise, low-cost, and flexible platform to integrate sCMOS camera control into behavioral experiments.

In-depth analysis of high-speed, cold neutron imaging of air-water two-phase flows

We have recently published first results of high frame rate cold neutron radiography of air-water two-phase flows in a bubbler. We have demonstrated imaging rates up to 800 frames per second by using a high-efficiency scintillator screen in combination with the highest available flux on the ICON beam line of the SINQ continuous spallation source and an imaging detector based on a high-speed sCMOS camera. Here we present an in-depth analysis of the experiments taken at several air injection rates and at different imaging rates. The dynamically resolved gas fraction distribution is investigated together with the bubble size and the instantaneous gas interfacial velocity distributions. The latter is evaluated using the optical flow (OF) method. The analysis presented here can be useful not only for analyzing dynamic neutron imaging sequences of multi-phase flows but of multi-component, mixing systems in general.

Spontaneous parametric down-conversion of photons at 660 nm to the terahertz and sub-terahertz frequency range.

We report on spontaneous parametric down-conversion (SPDC) in periodically poled lithium niobate (PPLN) using 660 nm pump wavelength and the type 0 phase-matching condition to the terahertz and even sub-terahertz frequency range. Detection of the frequency-shifted signal photons is achieved by using highly efficient and narrowband volume Bragg gratings and an uncooled sCMOS camera. The acquired frequency-angular spectrum shows backward and forward generation of terahertz and sub-terahertz photons by SPDC, as well as up-conversion and higher order quasi phase-matching (QPM). The frequency-angular spectrum is theoretically calculated using a Monte-Carlo integration scheme showing a high agreement with the measurement. This work is one important step toward quantum sensing and imaging in the terahertz and sub-terahertz frequency range.

Photon-Free Calibration of CMOS Cameras for Precise Single Molecule Localization Microscopy Reconstruction

Scientific-grade CMOS (sCMOS) cameras have evolved into a preferred detector type for single molecule localization microscopy (SMLM). Compared to EM-CCDs, sCMOS cameras do not only feature potentially increased localization precisions and framerates, but also come at typically lower cost. Transitioning to industry-grade CMOS cameras can decrease the cost much further, while in principle maintaining the advantages of sCMOS detectors. Consequently, such highly affordable cameras are recently gaining popularity in the community. However, considerable pixel-to-pixel variations in terms of noise, offset and gain which are well-known for sCMOS cameras can be even pronounced for CMOS cameras and impair the localization uncertainties. Explicit consideration during the reconstruction procedure is able to largely circumvent this effect if the detector is characterized accordingly. To this end, we present a novel approach to characterize CMOS cameras. It relies on thermally generated electrons only, but allows for similar data processing as in the traditional approach of using photoelectrons. Going without the need of external stimuli, this makes in-situ camera characterization possible. We show that industry-grade CMOS cameras which have been characterized by this approach can efficiently applied to common SMLM schemes of PALM, dSTORM and DNA-PAINT.

Feasibility of using spatial frequency-domain imaging intraoperatively during tumor resection.

Mapping the optical absorption and scattering properties of tissues using spatial frequency-domain imaging (SFDI) enhances quantitative fluorescence imaging of protoporphyrin IX (PpIX) in gliomas in the preclinical setting. The feasibility of using SFDI in the operating room was investigated here. A benchtop SFDI system was modified to mount directly to a commercial operating microscope. A digital light processing module imposed a selectable spatial light pattern from a broad-band xenon arc lamp to illuminate the surgical field. White light excitation and a liquid crystal-tunable filter allowed the diffuse reflectance images to be recorded at discrete wavelengths from 450 to 720nm on a sCMOS camera. The performance was first tested in tissue-simulating phantoms, and data were then acquired intraoperatively during brain tumor resection surgery. The optical absorption and transport scattering coefficients could be estimated with average errors of 3.2% and 4.5% for the benchtop and clinical systems, respectively, with spatial resolution of better than 0.7mm. These findings suggest that SFDI can be implemented in a clinically relevant configuration to achieve accurate mapping of the optical properties in the surgical field that can then be applied to achieve quantitative imaging of the fluorophore.

Optical identification and quantification of single neutron detection in 6LiF/ZnS scintillators using a CMOS camera

The possibility of observing the light from single neutron detection in scintillation materials by highly pixelated silicon sensors coupled with a demagnification optical lens could lead to a new generation of position-sensitive neutron detectors with relatively large area coverage and competitive spatial resolution. Several ZnS/6LiF-based scintillators were tested at the SINQ Beamline for Neutron Optics (BOA) using a neutron radiography setup mounting a sCMOS camera with an f/0.95 commercial objective. Single neutrons were identified and distinguished from the camera noise and particles detected directly in the silicon sensor in all the tested scintillators, with exposures starting from 1 ms. Linearity between neutron counting and exposure times was obtained from 10 ms up to 200 ms.

Hessian single-molecule localization microscopy using sCMOS camera

Single-molecule localization microscopy (SMLM) has the highest spatial resolution among the existing super-resolution imaging techniques, but its temporal resolution needs further improvement. An sCMOS camera can effectively increase the imaging rate due to its large field of view and fast imaging speed. Using an sCMOS camera for SMLM imaging can significantly improve the imaging time resolution, but the unique single-pixel-dependent readout noise of sCMOS cameras severely limits their application in SMLM imaging. This paper develops a Hessian-based SMLM (Hessian-SMLM) method that can correct the variance, gain, and offset of a single pixel of a camera and effectively eliminate the pixel-dependent readout noise of sCMOS cameras, especially when the signal-to-noise ratio is low. Using Hessian-SMLM to image mEos3.2-labeled actin was able to significantly reduce the artifacts due to camera noise.

Temporal multiplexing of the scientific grade camera for hyper-frame-rate imaging.

In order to maximize the spatio-temporal resolution of the scientific grade camera at width-limited ROI, this paper proposes a new hyper-frame-rate imaging method by temporal multiplexing the sub-region of the image sensor. In the system, a dual-axis scanning galvanometer is localized at the relay pupil plane and a high quality scan lens is utilized to form an image-side telecentric path. Following this path can overcome bandwidth waste in the conventional exposure and readout mode, and maintain other performances of image sensors. As a result, the sCMOS camera has performed 432fps over 820 × 700 pixel arrays to record the dynamic heartbeat of zebrafish larvae.

Design of SCMOS Camera System

In order to meet the demands of high dynamic range for CMOS camera under the mode of global shutter, a camera system is designed based on GSENSE2020S SCMOS image sensor device. By analyzing the readout structure characteristics of pixel of GSENSE2020S chip and the driving timing under the mode of global shutter, we choose ZYNQ with DDR3 as the processing structure and complete the design of SOPC including the control of imaging parameters, driving timing of SCMOS, capturing the image, image preprocessing and SOPC on, and introduces the function of every module. The designed camera realizes the imaging output of 90 frames of 2048×2048 pixel and 12 bit depth with high sensitivity, high dynamic range per second by the imaging test, and it reach the requirement of scientific imaging.

Scanless multitarget-matching multiphoton excitation fluorescence microscopy

Using the combination of a reflective blazed grating and a reflective phase-only diffractive spatial light modulator (SLM), scanless multitarget-matching multiphoton excitation fluorescence microscopy (SMTM-MPM) was achieved. The SLM shaped an incoming mode-locked, near-infrared Ti:sapphire laser beam into an excitation pattern with addressable shapes and sizes that matched the samples of interest in the field of view. Temporal and spatial focusing were simultaneously realized by combining an objective lens and a blazed grating. The fluorescence signal from illuminated areas was recorded by a two-dimensional sCMOS camera. Compared with a conventional temporal focusing multiphoton microscope, our microscope achieved effective use of the laser power and decreased photodamage with higher axial resolution.

Wide-Field Monitoring of Single Fluorescent Molecules and Nanoparticles without Immobilization

Surface-based wide-field imaging (“surface-WIFI”) of single fluorescent molecules has transformed our understanding of many biological mechanisms by directly accessing molecular heterogeneity and real-time reaction trajectories. By monitoring the function of hundreds of immobilised molecules simultaneously, surface-WIFI achieves high throughput, outperforming its confocal microscopy counterpart that relies on point detection. However, surface-WIFI relies on surface tethering of molecules, which requires the preparation of modified surfaces and insertion of biomolecule modifiers; further, the molecules of interest (e.g., proteins and their complexes) may stick to surfaces non-specifically and lose their function. These issues significantly limit the appeal of surface-WIFI. To address these challenges, we introduce solution-based wide-field imaging (“solution-WIFI”), a method that combines wide-field ratiometric imaging and tracking of diffusing fluorescent molecules. Solution-WIFI requires no surface attachment, is immune to surface interactions, and also reports on molecular size. To implement solution-WIFI, we used oblique illumination and ratiometric imaging of diffusing molecules on a sCMOS camera with a temporal resolution down to 2.5 ms. We used custom software to localize and track particles, extract their intensities and associated ratios (e.g., Förster energy resonance transfer (FRET), spectral signatures, molecular stoichiometries), and characterize thousands of individual molecules per minute of imaging. For validation, we tracked doubly labelled double-stranded DNA that serve as FRET standards, and recovered FRET distributions similar to those for surface measurements. Our system was capable of tracking tiny fluorescent molecules, such as single Cy3B dyes (with characteristic size of ∼1 nm and diffusion coefficients reaching 50 µm2/s in 50% glycerol). We also determined the size distribution of fluorescently labelled nanodiamonds, as well as the fraction of nanodiamond population that contain fluorescent nitrogen vacancy (NV) centers. Solution-WIFI is a general method for high-throughput analysis of structure, dynamics, and diffusion of many biomolecules without immobilization.

Cylindrical microlensing for enhanced collection efficiency of small pixel SPAD arrays in single-molecule localisation microscopy.

Single-photon avalanche photodiode (SPAD) image sensors offer time-gated photon counting, at high binary frame rates of >100 kFPS and with no readout noise. This makes them well-suited to a range of scientific applications, including microscopy, sensing and quantum optics. However, due to the complex electronics required, the fill factor tends to be significantly lower (< 10%) than that of EMCCD and sCMOS cameras (>90%), whilst the pixel size is typically larger, impacting the sensitivity and practicalities of the SPAD devices. This paper presents the first characterisation of a cylindrical-shaped microlens array applied to a small, 8 micron, pixel SPAD imager. The enhanced fill factor, ≈50% for collimated light, is the highest reported value amongst SPAD sensors with comparable resolution and pixel pitch. We demonstrate the impact of the increased sensitivity in single-molecule localisation microscopy, obtaining a resolution of below 40nm, the best reported figure for a SPAD sensor.

A flexible, on-line magnetic spectrometer for ultra-intense laser produced fast electron measurement

We have developed an on-line magnetic spectrometer to measure energy distributions of fast electrons generated from ultra-intense laser-solid interactions. The spectrometer consists of a sheet of plastic scintillator, a bundle of non-scintillating plastic fibers, and an sCMOS camera recording system. The design advantages include on-line capturing ability, versatility of detection arrangement, and resistance to harsh in-chamber environment. The validity of the instrument was tested experimentally. This spectrometer can be applied to the characterization of fast electron source for understanding fundamental laser-plasma interaction physics and to the optimization of high-repetition-rate laser-driven applications.