EMCCD Camera Research Articles

Pulsating combustion of ethylene in micro-channels with controlled temperature gradient

ABSTRACTNowadays with the demand for high-efficiency systems, there is a growing development and application of microelectromechanical systems (MEMS devices) which may generate more energy than the modern batteries. In this context, a new experimental device has been developed. It consists of a microtubular reactor with a controlled external temperature profile. The high gas temperature allows the autoignition of the mixture and the flame stabilization in reactors with inner diameter (id) smaller than the ordinary quenching diameter. The temperature profile along the tube is measured continuously using an infrared camera while an EMCCD camera is used to collect the CH* emission from the flame. The present experimental study on C2H4/air mixtures reacting in narrow channels has been undertaken to provide detailed information on flame combustion at microscale. Three kinds of flame behaviors were observed: (i) bright and stable flames under high flow velocity, (ii) flames with repetitive extinction and ignition in the middle flow rates range, and (iii) weak flames at low flow rates. The presence of oscillating transitional regimes was also investigated and a detailed analysis of the unstable regimes (determination of frequencies and characteristic times) was carried out. The effect of the inlet velocity, the equivalence ratio, and the tube id were also investigated. Numerical simulations were also conducted to study the ignition characteristics of unstable flames.

Spatio-temporal dynamics of spontaneous ultra-weak photon emission (autoluminescence) from human hands measured with an EMCCD camera: Dependence on time of day, date and individual subject

Spatio-temporal dynamics of spontaneous ultra-weak photon emission (autoluminescence) from human hands measured with an EMCCD camera: Dependence on time of day, date and individual subject

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.

Pipeline for the Detection of Serendipitous Stellar Occultations by Kuiper Belt Objects with the Colibri Fast-photometry Array**Code for our detection pipeline and Fresnel diffraction modeling program are publicly available at github.com/ekpass/colibri.

We report results from the preliminary trials of Colibri, a dedicated fast-photometry array for the detection of small Kuiper Belt objects (KBOs) through serendipitous stellar occultations. Colibri’s novel data processing pipeline analyzed 4000 star hours with two overlapping-field EMCCD cameras, detecting no KBOs and one false positive occultation event in a high ecliptic latitude field. No occultations would be expected at these latitudes, allowing these results to provide a control sample for the upcoming main Colibri campaign. The empirical false positive rate found by the processing pipeline is consistent with the 0.002% simulation-determined false positive rate. We also describe Colibri’s software design, kernel sets for modeling stellar occultations, and method for retrieving occultation parameters from noisy diffraction curves. Colibri’s main campaign will begin in mid-2018, operating at a 40 Hz sampling rate.

The simulated spectrum of the OGRE X-ray EM-CCD camera system

The X-ray astronomical telescopes in use today, such as Chandra and XMM-Newton, use X-ray grating spectrometers to probe the high energy physics of the Universe. These instruments typically use reflective optics for focussing onto gratings that disperse incident X-rays across a detector, often a Charge-Coupled Device (CCD). The X-ray energy is determined from the position that it was detected on the CCD. Improved technology for the next generation of X-ray grating spectrometers has been developed and will be tested on a sounding rocket experiment known as the Off-plane Grating Rocket Experiment (OGRE). OGRE aims to capture the highest resolution soft X-ray spectrum of Capella, a well-known astronomical X-ray source, during an observation period lasting between 3 and 6 minutes whilst proving the performance and suitability of three key components. These three components consist of a telescope made from silicon mirrors, gold coated silicon X-ray diffraction gratings and a camera that comprises of four Electron-Multiplying (EM)-CCDs that will be arranged to observe the soft X-rays dispersed by the gratings. EM-CCDs have an architecture similar to standard CCDs, with the addition of an EM gain register where the electron signal is amplified so that the effective signal-to-noise ratio of the imager is improved. The devices also have incredibly favourable Quantum Efficiency values for detecting soft X-ray photons. On OGRE, this improved detector performance allows for easier identification of low energy X-rays and fast readouts due to the amplified signal charge making readout noise almost negligible. A simulation that applies the OGRE instrument performance to the Capella soft X-ray spectrum has been developed that allows the distribution of X-rays onto the EM-CCDs to be predicted. A proposed optical model is also discussed which would enable the missions minimum success criteria's photon count requirement to have a high chance of being met with the shortest possible observation time. These results are compared to a Chandra observation to show the overall effectiveness of the new technologies. The current optical module is shown to narrowly meet the minimum success conditions whilst the proposed model comfortably demonstrates the effectiveness of the technologies if a larger effective area is provided.

Ground-based instruments of the PWING project to investigate dynamics of the inner magnetosphere at subauroral latitudes as a part of the ERG-ground coordinated observation network

The plasmas (electrons and ions) in the inner magnetosphere have wide energy ranges from electron volts to mega-electron volts (MeV). These plasmas rotate around the Earth longitudinally due to the gradient and curvature of the geomagnetic field and by the co-rotation motion with timescales from several tens of hours to less than 10 min. They interact with plasma waves at frequencies of mHz to kHz mainly in the equatorial plane of the magnetosphere, obtain energies up to MeV, and are lost into the ionosphere. In order to provide the global distribution and quantitative evaluation of the dynamical variation of these plasmas and waves in the inner magnetosphere, the PWING project (study of dynamical variation of particles and waves in the inner magnetosphere using ground-based network observations, http://www.isee.nagoya-u.ac.jp/dimr/PWING/) has been carried out since April 2016. This paper describes the stations and instrumentation of the PWING project. We operate all-sky airglow/aurora imagers, 64-Hz sampling induction magnetometers, 40-kHz sampling loop antennas, and 64-Hz sampling riometers at eight stations at subauroral latitudes (~ 60° geomagnetic latitude) in the northern hemisphere, as well as 100-Hz sampling EMCCD cameras at three stations. These stations are distributed longitudinally in Canada, Iceland, Finland, Russia, and Alaska to obtain the longitudinal distribution of plasmas and waves in the inner magnetosphere. This PWING longitudinal network has been developed as a part of the ERG (Arase)-ground coordinated observation network. The ERG (Arase) satellite was launched on December 20, 2016, and has been in full operation since March 2017. We will combine these ground network observations with the ERG (Arase) satellite and global modeling studies. These comprehensive datasets will contribute to the investigation of dynamical variation of particles and waves in the inner magnetosphere, which is one of the most important research topics in recent space physics, and the outcome of our research will improve safe and secure use of geospace around the Earth.

Quantitative performance evaluation of a back-illuminated sCMOS camera with 95% QE for super-resolution localization microscopy.

Scientific Complementary Metal Oxide Semiconductor (sCMOS) cameras were introduced into the market in 2009 and are now becoming a major type of commercial cameras for low-light imaging. sCMOS cameras provide simultaneously low read noise, high readout speed, and large pixel array; however, the relatively low quantum efficiency (QE) of sCMOS cameras has been a major limitation for its application in single molecule imaging, especially super-resolution localization microscopy which requires high detection sensitivity. Here we report the imaging performance of a newly released back-illuminated sCMOS camera (called Dhyana 95 from Tucsen) which is claimed to be the world's first 95% QE sCMOS camera. The imaging performance evaluation is based on a new methodology which is designed to provide paired images from two tested cameras under almost identical experimental conditions. We verified that this new 95% QE sCMOS camera is able to provide superior imaging performance over a representative front-illuminated sCMOS camera (Hamamatsu Flash 4.0 V2) and a popular back-illuminated EMCCD camera (Andor iXon 897 Ultra) in a wide signal range. We hope this study will inspire more studies on using sCMOS cameras in super-resolution localization microscopy, or even single molecule imaging. © 2017 International Society for Advancement of Cytometry.

Spectral characteristics of voltage-sensitive indocyanine green fluorescence in the heart

Indocyanine green (ICG) fluorescent dye has been approved by the FDA for use in medical diagnostics. Recently, we demonstrated that ICG dye has voltage-sensitive properties with a dual-component (fast and slow) response in the Langendorff-perfused rabbit heart. Here, we extended our studies by showing the different spectral properties of both components for analysis of the fractional change in ICG fluorescence in response to voltage changes. We used light from four LEDs to obtain excitation; emission was measured using an EMCCD camera with band-pass filters and a spectrometer. We applied a graphical model with Gaussian functions to construct and evaluate the individual emission curves and calculated the voltage-sensitive portion of each component of the ICG fluorescence in the rabbit heart. The results revealed that each isolated component (fast and slow) emanates from a unique ICG pool in a different environment within the cell membrane and that each component is also composed of two constituents (ICG-monomeric and ICG-aggregated). We propose the existence of different voltage-sensitive mechanisms for the components: (I) electrochromism and field-induced reorientation for the fast component; and (II) field-induced dye squeezing that amplifies intermolecular interactions, resulting in self-quenching of the dye fluorescence, for the slow component.

Rainbow correlation imaging with macroscopic twin beam

We present the implementation of a correlation-imaging protocol that exploits both the spatial and spectral correlations of macroscopic twin-beam states generated by parametric downconversion. In particular, the spectral resolution of an imaging spectrometer coupled to an EMCCD camera is used in a proof-of-principle experiment to encrypt and decrypt a simple code to be transmitted between two parties. In order to optimize the trade-off between visibility and resolution, we provide the characterization of the correlation images as a function of the spatio-spectral properties of twin beams generated at different pump power values.

Acquisition of multiple photon pairs with an EMCCD camera

The detection and characterization of quantum states of light plays an important role in quantum science. Traditional methods use single-photon detectors, but these are generally limited to point measurements; consequently, multi-pixel devices are now being utilized in quantum measurements, especially in the field of quantum imaging. Here, we demonstrate the capability of an EMCCD camera to record multiple coincidence events originating from parametric downconversion where the mean photon number per pixel is much greater than unity. The multi-pixel nature of the camera enables us to record correlations ranging from to 10 000 coincidences per frame. This approach to quantum measurements provide mechanisms for recording quantum signatures for bright correlated photon sources.

An all-sky camera for recording three auroral emissions

The EKA-3/180-11 emission camera creates a snapshot of the celestial hemisphere in the selected auroral emissions of 427.8, 557.7, and 630.0 nm, and makes it possible to determine the spatial distribution of the intensity of these emissions. The instrument is based on an EMCCD camera; the spectral selection is carried out by interference filters. Scanning in spectral channels is carried out by an electromechanical filter wheel with a frame rate of 0.1 Hz. The instrument is designed for regular ground observations and acts as both a narrow-angle emission photometer and an all-sky camera.

A New Method to Detect Intra- and Extracellular Conformational Changes in Ion Channels with High Temporal Resolution

Ligand-gated ion channels (LGICs) and voltage-gated ion channels (VGICs) are membrane proteins that represent important drug targets. While electrophysiological techniques allow the distinction between open, closed and desensitized/inactivated states, it is not capable to directly detect conformational changes that might precede, accompany or follow the gating or ligand binding events of an ion channel. The simultaneous detection of current and emission from a fluorescent label in voltage-clamp fluorometry (VCF) has proven a powerful technique to overcome this limitation by allowing to monitor conformational rearrangements at the channel gate and in a protein domain of interest. However, ligand application in VCF is slow and limited to extracellular domains. Here, we developed a novel, ultrafast patch-clamp fluorometry (ufPCF) setup for use with both LGICs and VGICs. A combination of a digital mirror device, an emCCD camera and a piezo-driven perfusion tool enable us to achieve a time resolution of around 1 ms for ligand application and fluorescence detection. Importantly, ufPCF can be used in both outside-out and inside-out patches, irrespective of the position of fluorescent dye within the protein. For example, ufPCF allows easy access to the intracellular side of extracellularly labeled channels in inside-out patches, enabling the modulation of intracellular conditions while monitoring rearrangements in extracellular domains (and vice versa). Further, our ufPCF can be used with fluorescent unnatural amino acids, which can be introduced anywhere in the protein. Overall, we find this method to be highly versatile and to generally result in larger fluorescence changes compared to VCF. We anticipate ufPCF to significantly aid our understanding of the pharmacology and molecular function of both LGICs and VGICs and we intend to extend its use to studies using transporters and pumps.

Characterization of Intracellular Sodium Homeostasis in Murine Atrial Myocytes

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+ and provides insight into the activation of the sarcolemmal sodium calcium exchanger (NCX) and the behavior of both Na+ channels and the Na+,K+-ATPase. In atrial diseases such as atrial fibrillation (AF), intracellular Ca2+ signaling is profoundly altered. While the mechanisms underlying altered intracellular Ca2+ homeostasis are well characterized, the role that [Na+]i may play and its dysregulation in disease in atrial myocytes is less well understood. For example, we have previously shown that high rate (10 Hz) atrial activation for 5 days in a rabbit model leads to an expected but significant reduction in [Na+]i (Greiser et al, JCI, 2014; 124: 4759-72).Here we characterize intracellular Na+ homeostasis in murine atrial myocytes using the Na+ indicator SBFI (Molecular Probes) in quiescent cells. Dual UV excitation (340 nm and 380 nm) was provided by a rapid switching Sutter DG5-plus illuminator using a 300 W xenon light source with widefield imaging by an EMCCD camera (Princeton Instruments). Freshly isolated atrial myocytes were loaded with 10 μmol/l SBFI-AM for 90 min in the presence of the non-ionic surfactant Pluronic F-127 (0.05 %w/v). After washout of the extra cellular dye, SBFI-AM de-esterification was allowed for 20 minutes before proceeding with [Na+]i measurements. Fluorescence images (F340 and F380) were collected at 510 ± 40 nm. The calibration of [Na+]i was performed in each cell separately after the measurements of quiescent [Na+]i by exposing the SBFI-loaded myocytes to various extracellular [Na+] ([Na+]o) in the presence of 10 μmol/l gramicidin D and 100 μmol/l-strophanthidin. Resting [Na+]i in mouse atrial myocytes was 8.2 ± 0.6 mM. External field stimulation (4 ms, 20 V, 1 Hz) increased [Na+]i by 3.1 ± 0.2 mM. These data show that [Na+]i in murine atrial myocytes is lower than in murine ventricular myocytes (12 ± 1 mM, Correll et al, Circ Res, 2014;114:249-56). These data and more will provide a broad understanding of atrial Na+ homeostasis and its implication for atrial myocyte Ca2+ signaling.

Mechanosensetive ATP Release Involves a Non-Conductive Pathway: Evidence from Large Field of View Real-Time Imaging

Cell swelling is a potent ATP secretion stimulus. While the sources of ATP are known, it's mechanisms of secretion in epithelial cells remain unclear. Two secretion pathways have been proposed: exocytosis of nucleotide-containing vesicles or conductive release through pore-forming channels, like pannexins or connexins. In this project, we have tested whether ATP is released or not through a conductive pathway in A549 lung epithelial cells. Real-time ATP secretion from glass-adherent A549 cells was imaged with a custom-designed lens system, combining a large field of view and a high light-gathering power, mounted directly on a Photometrics® EvolveTM 512 EMCCD camera. ATP was detected with a light-emitting luciferin-luciferase reaction. Secretion was stimulated by acute cell swelling or exposure to ionomycin (10 µM). Plasma membrane permeability to large molecules was assessed in parallel by propidium iodide (PI) uptake with epifluorescence Zeiss Axio Observer microscope. Cells were grown in 10% (control cells) or 0.1% FBS (fragile cells). Our novel imaging system revealed a strong homogeneous ATP secretion lasting several minutes from control A549 cells stimulated with 50% hypotonic shock. Yet, no PI-stained cells were detected under these conditions. In contrast, cells rendered fragile, and thus prone to cell lysis, showed an appreciable amount of PI-stained cells post-hypotonic shock and a punctuate pattern of ATP secretion co-localizing with PI-positive cells. Sudden intracellular calcium elevation with ionomycin provoked a rapid, albeit small, ATP secretion. Based on the fact that hypotonically-induced ATP release in normal conditions was not accompanied with detectable permeation of PI, we conclude that ATP in A549 cells is released by a non-conductive pathway.

Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions

Tracking cells and proteins' phenotypic changes in deep suspensions is critical for the direct imaging of blood-related phenomena in in vitro replica of cardiovascular systems and blood-handling devices. This paper introduces fluorescence imaging techniques for space and time resolved detection of platelet activation, von Willebrand factor (VWF) conformational changes, and VWF-platelet interaction in deep suspensions. Labeled VWF, platelets, and VWF-platelet strands are suspended in deep cuvettes, illuminated, and imaged with a high-sensitivity EM-CCD camera, allowing detection using an exposure time of 1 ms. In-house postprocessing algorithms identify and track the moving signals. Recombinant VWF-eGFP (rVWF-eGFP) and VWF labeled with an FITC-conjugated polyclonal antibody are employed. Anti-P-Selectin FITC-conjugated antibodies and the calcium-sensitive probe Indo-1 are used to detect activated platelets. A positive correlation between the mean number of platelets detected per image and the percentage of activated platelets determined through flow cytometry is obtained, validating the technique. An increase in the number of rVWF-eGFP signals upon exposure to shear stress demonstrates the technique's ability to detect breakup of self-aggregates. VWF globular and unfolded conformations and self-aggregation are also observed. The ability to track the size and shape of VWF-platelet strands in space and time provides means to detect pro- and antithrombotic processes.

On the efficiency of techniques for the reduction of impulsive noise in astronomical images

The impulsive noise in astronomical images originates from various sources. It develops as a result of thermal generation in pixels, collision of cosmic rays with image sensor or may be induced by high readout voltage in Electron Multiplying CCD (EMCCD). It is usually efficiently removed by employing the dark frames or by averaging several exposures. Unfortunately, there are some circumstances, when either the observed objects or positions of impulsive pixels evolve and therefore each obtained image has to be filtered independently. In this article we present an overview of impulsive noise filtering methods and compare their efficiency for the purpose of astronomical image enhancement. The employed set of noise templates consists of dark frames obtained from CCD and EMCCD cameras working on ground and in space. The experiments conducted on synthetic and real images, allowed for drawing numerous conclusions about the usefulness of several filtering methods for various: (1) widths of stellar profiles, (2) signal to noise ratios, (3) noise distributions and (4) applied imaging techniques. The results of presented evaluation are especially valuable for selection of the most efficient filtering schema in astronomical image processing pipelines.

(Invited) Electrochemical Luminescent Imaging of Activated Neutrophils Using Multi-Microwell Array Electrode

Vasculitis is an autoimmune disease leads to various sicknesses (e.g. arteriosclerosis). Mechanisms of vasculitis have not been accurately understood, and early detection and diagnosis of vasculitis remain constant challenges. One of the causes of vasculitis is neutrophil-derived reactive oxygen species (ROS) that attack blood vessels. Therefore, detection of ROS derived from activated neutrophils could be important for early diagnosis of vasculitis. Herein, we developed a device for detection of ROS from activated neutrophil at single-cell resolution using electrochemiluminescence (ECL) method. ECL is it’s a highly sensitive method, which is suitable for detection of ROS from a single activated neutrophil. Results & discussion We constructed a multi-microwell array electrode device that enables to detect the generation of ROS from activated neutrophils. Microwell array was fabricated using PDMS and put on an electrode. By sealing neutrophils into the microwells, we could prevent diffusion of ROS, and keep its concentration high. Luminescence was generated by the reaction between luminol and ROS, and its intensity was quantified using EM-CCD camera. Neutrophils were isolated from healthy human blood, and were activated by adding PMA. The activated neutrophils are recognized as a model cells for vasculitis researches. The activated cells and non-activated cells were trapped in microwells and their luminescence was quantified by the EM-CCD camera. As a result, we succeeded in detecting ROS from activated neutrophils. This device may have potential to diagnose vasculitis by comparing the number and condition of activated neutrophils between healthy people and vasculitis patients.

"Calibration-on-the-spot": How to calibrate an EMCCD camera from its images.

In order to count photons with a camera, the camera must be calibrated. Photon counting is necessary, e.g., to determine the precision of localization-based super-resolution microscopy. Here we present a protocol that calibrates an EMCCD camera from information contained in isolated, diffraction-limited spots in any image taken by the camera, thus making dedicated calibration procedures redundant by enabling calibration post festum, from images filed without calibration information.

TH-AB-209-03: Overcoming Resolution Limitations of Diffuse Optical Signals in X-Ray Induced Luminescence (XIL) Imaging Via Selective Plane Illumination and 2D Deconvolution

Purpose: To evaluate the resolution and sensitivity of XIL imaging using a surface radiance simulation based on optical diffusion and maximum likelihood expectation maximization (MLEM) image reconstruction. XIL imaging seeks to determine the distribution of luminescent nanophosphors, which could be used as nanodosimeters or radiosensitizers. Methods: The XIL simulation generated a homogeneous slab with optical properties similar to tissue. X-ray activated nanophosphors were placed at 1.0 cm depth in the tissue in concentrations of 10−4 g/mL in two volumes of 10 mm3 with varying separations between each other. An analytical optical diffusion model determined the surface radiance from the photon distributions generated at depth in the tissue by the nanophosphors. The simulation then determined the detected luminescent signal collected with a f/1.0 aperture lens and back-illuminated EMCCD camera. The surface radiance was deconvolved using a MLEM algorithm to estimate the nanophosphors distribution and the resolution. To account for both Poisson and Gaussian noise, a shifted Poisson imaging model was used in the deconvolution. The deconvolved distributions were fitted to a Gaussian after radial averaging to measure the full width at half maximum (FWHM) and the peak to peak distance between distributions was measured to determine the resolving power. Results: Simulated surface radiances for doses from 1mGy to 100 cGy were computed. Each image was deconvolved using 1000 iterations. At 1mGy, deconvolution reduced the FWHM of the nanophosphors distribution by 65% and had a resolving power is 3.84 mm. Decreasing the dose from 100 cGy to 1 mGy increased the FWHM by 22% but allowed for a dose reduction of a factor of 1000. Conclusion: Deconvolving the detected surface radiance allows for dose reduction while maintaining the resolution of the nanophosphors. It proves to be a useful technique in overcoming the resolution limitations of diffuse optical imaging in tissue. C. S. acknowledges support from the NIH National Institute of General Medical Sciences (Award number R25GM109439, Project Title: University of Chicago Initiative for Maximizing Student Development, IMSD). B. Q. and P. L. acknowledge support from NIH grant R01EB017293.

Absolute calibration of an EMCCD camera by quantum correlation, linking photon counting to the analog regime.

We show how the same setup and procedure, exploiting spatially multimode quantum correlations, allows the absolute calibration of an electron-multiplying charge-coupled (EMCCD) camera from the analog regime down to the single-photon-counting level, just by adjusting the brightness of the quantum source. At the single-photon level, an EMCCD can be operated as an on-off detector, where quantum efficiency depends on the discriminating threshold. We develop a simple model to explain the connection of the two different regimes demonstrating that the efficiency estimated in the analog (bright) regime allows us to accurately predict the detector behavior in the photocounting regime and vice versa. This work establishes a bridge between two regions of the optical measurements that up to now have been based on completely different standards, detectors, and measurement techniques.