Development of a Charge-Multiplication CMOS Image Sensor Based on Capacitive Trench for Low-Light-Level Imaging.

This paper presents an electron multiplication charge coupled device (EMCCD) based on capacitive deep trench isolation (CDTI) and developed using complementary metal oxide semiconductor (CMOS) technology. The CDTI transfer register offers a charge transfer inefficiency lower than 10-4 and a low dark current o 0.11nA/cm2 at room temperature. In this work, the timing diagram is adapted to use this CDTI transfer register in an electron multiplication mode. The results highlight some limitations of this device in such an EM configuration: for instance, an unexpected increase in the dark current is observed. A design modification is then proposed to overcome these limitations and rely on the addition of an electrode on the top of the register. Thus, this new device preserves the good transfer performance of the register while adding an electron multiplication function. Technology computer-aided design (TCAD) simulations in 2D and 3D are performed with this new design and reveal a very promising structure.

Recent Enhancements to Interline and Electron Multiplying CCD Image Sensors.

This paper describes recent process modifications made to enhance the performance of interline and electron-multiplying charge-coupled-device (EMCCD) image sensors. By use of MeV ion implantation, quantum efficiency in the NIR region of the spectrum was increased by 2×, and image smear was reduced by 6 dB. By reducing the depth of the shallow photodiode (PD) implants, the photodiode-to-vertical-charge-coupled-device (VCCD) transfer gate voltage required for no-lag operation was reduced by 3 V, and the electronic shutter voltage was reduced by 9 V. The thinner, surface pinning layer also resulted in a reduction of smear by 4 dB in the blue portion of the visible spectrum. For EMCCDs, gain aging was eliminated by providing an oxide-only dielectric under its multiplication phase, while retaining the oxide-nitride-oxide (ONO) gate dielectrics elsewhere in the device.

Single Photon Counting UV Solar-Blind Detectors Using Silicon and III-Nitride Materials.

Ultraviolet (UV) studies in astronomy, cosmology, planetary studies, biological and medical applications often require precision detection of faint objects and in many cases require photon-counting detection. We present an overview of two approaches for achieving photon counting in the UV. The first approach involves UV enhancement of photon-counting silicon detectors, including electron multiplying charge-coupled devices and avalanche photodiodes. The approach used here employs molecular beam epitaxy for delta doping and superlattice doping for surface passivation and high UV quantum efficiency. Additional UV enhancements include antireflection (AR) and solar-blind UV bandpass coatings prepared by atomic layer deposition. Quantum efficiency (QE) measurements show QE > 50% in the 100-300 nm range for detectors with simple AR coatings, and QE ≅ 80% at ~206 nm has been shown when more complex AR coatings are used. The second approach is based on avalanche photodiodes in III-nitride materials with high QE and intrinsic solar blindness.

A mutation associated with centronuclear myopathy enhances the size and stability of dynamin 2 complexes in cells.

BACKGROUND: Dynamin 2 (Dyn2) is a ~100kDa GTPase that assembles around the necks of nascent endocytic and Golgi vesicles and catalyzes membrane scission. Mutations in Dyn2 that cause centronuclear myopathy (CNM) have been shown to stabilize Dyn2 polymers against GTP-dependent disassembly in vitro. Precisely timed regulation of assembly and disassembly is believed to be critical for Dyn2 function in membrane vesiculation, and the CNM mutations interfere with this regulation by shifting the equilibrium toward the assembled state. METHODS: In this study we use two fluorescence fluctuation spectroscopy (FFS) approaches to show that a CNM mutant form of Dyn2 also has a greater propensity to self-assemble in the cytosol and on the plasma membrane of living cells. RESULTS: Results obtained using brightness analysis indicate that unassembled wild-type Dyn2 is predominantly tetrameric in the cytosol, although different oligomeric species are observed, depending on the concentration of expressed protein. In contrast, an R369W mutant identified in CNM patients forms higher-order oligomers at concentrations above 1µM. Investigation of Dyn2-R369W by Total Internal Reflection Fluorescence (TIRF) FFS reveals that this mutant forms larger and more stable clathrin-containing structures on the plasma membrane than wild-type Dyn2. CONCLUSIONS AND GENERAL SIGNIFICANCE: These observations may explain defects in membrane trafficking reported in CNM patient cells and in heterologous systems expressing CNM-associated Dyn2 mutants.

A comparative study of EM-CCD and CMOS cameras for particle ion trajectory imaging.

High-resolution and real-time imaging of particle ion trajectories is essential in nuclear medicine and nuclear engineering. One potential method to achieve high-resolution real-time trajectory imaging of particle ions involves utilizing an imaging system that integrates a scintillator plate with a magnifying unit and a cooled electron multiplying charge-coupled device (EM-CCD) camera. However, acquiring an EM-CCD camera might prove challenging due to the discontinuation of CCD sensor manufacturing by vendors. As an alternative imaging approach, a low-noise, high-sensitivity camera utilizing a cooled complementary metal-oxide-semiconductor (CMOS) sensor offers a promising solution for imaging particle ion trajectories. Yet, it remains uncertain whether CMOS-based cameras can perform as effectively as CCD-based cameras in capturing particle ion trajectories. To address these concerns, we conducted a comparative analysis of the imaging performance between a CMOS-based system and an EM-CCD-based system for capturing alpha particle trajectories. The results revealed that both systems could image the trajectories of alpha particle, but the spatial resolution with the CMOS-based camera exceeded that of the EM-CCD-based camera, primarily due to the smaller pixel size of the sensor. While the signal-to-noise ratio (SNR) of the trajectory image from the CMOS-based camera initially lagged behind that from the EM-CCD-based camera, this disparity was mitigated by implementing binning techniques on the CMOS-based camera images. In conclusion, our findings suggest that a cooled CMOS camera could serve as a viable alternative for imaging particle ion trajectories.

Automated pancreatic islet viability assessment for transplantation using bright-field deep morphological signature.

Islets transplanted for type-1 diabetes have their viability reduced by warm ischemia, dimethyloxalylglycine (DMOG; hypoxia model), oxidative stress and cytokine injury. This results in frequent transplant failures and the major burden of patients having to undergo multiple rounds of treatment for insulin independence. Presently there is no reliable measure to assess islet preparation viability prior to clinical transplantation. We investigated deep morphological signatures (DMS) for detecting the exposure of islets to viability compromising insults from brightfield images. Accuracies ranged from 98 % to 68 % for; ROS damage, pro-inflammatory cytokines, warm ischemia and DMOG. When islets were disaggregated to single cells to enable higher throughput data collection, good accuracy was still obtained (83-71 %). Encapsulation of islets reduced accuracy for cytokine exposure, but it was still high (78 %). Unsupervised modelling of the DMS for islet preparations transplanted into a syngeneic mouse model was able to predict whether or not they would restore glucose control with 100 % accuracy. Our strategy for constructing DMS' is effective for the assessment of islet pre-transplant viability. If translated into the clinic, standard equipment could be used to prospectively identify non-functional islet preparations unable to contribute to the restoration of glucose control and reduce the burden of unsuccessful treatments.

MicroFPGA: An affordable FPGA platform for microscope control.

Modern microscopy relies increasingly on microscope automation to improve throughput, ensure reproducibility or observe rare events. Automation requires computer control of the important elements of the microscope. Furthermore, optical elements that are usually fixed or manually movable can be placed on electronically-controllable elements. In most cases, a central electronics board is necessary to generate the control signals they require and to communicate with the computer. For such tasks, Arduino microcontrollers are widely used due to their low cost and programming entry barrier. However, they are limiting in their performance for applications that require high-speed or multiple parallel processes. Field programmable gate arrays (FPGA) are the perfect technology for high-speed microscope control, as they are capable of processing signals in parallel and with high temporal precision. While plummeting prices made the technology available to consumers, a major hurdle remaining is the complex languages used to configure them. In this work, we used an affordable FPGA, delivered with an open-source and friendly-to-use programming language, to create a versatile microscope control platform called MicroFPGA. It is capable of synchronously triggering cameras and multiple lasers following complex patterns, as well as generating various signals used to control microscope elements such as filter wheels, servomotor stages, flip-mirrors, laser power or acousto-optic modulators. MicroFPGA is open-source and we provide online Micro-Manager, Java, Python and LabVIEW libraries, together with blueprints and tutorials.

Super-Resolution Radial Fluctuations (SRRF) Microscopy.

Super-resolution Radial Fluctuations (SRRF) imaging is a computational approach to fixed and live-cell super-resolution microscopy that is highly accessible to life science researchers since it uses common microscopes and open-source software plugins for ImageJ. This allows users to generate super-resolution images using the same equipment, fluorophores, fluorescent proteins and methods they routinely employ for their studies without specialized sample preparations or reagents. Here, we discuss a step-by-step workflow for acquiring and analyzing images using the NanoJ-SRRF software developed by the Ricardo Henriques group, with a focus on imaging chromatin. Increased accessibility of affordable super-resolution imaging techniques is an important step in extending the reach of this revolution in cellular imaging to a greater number of laboratories.

Bioluminescence Resonance Energy Transfer (BRET) Imaging in Living Cells: Image Acquisition and Quantification.

Bioluminescence resonance energy transfer (BRET) is an energy transfer phenomenon from a luciferase donor to a fluorescence acceptor and serves as an indicator of protein-protein interaction or protein proximity. BRET imaging is a powerful tool in the investigation of signaling proteins because it enables spatial analysis of such protein interactions. Here, we describe a method exerting high-resolution BRET imaging by combining bright-light output luciferases, such as NanoLuc , photon-counting EM-CCD, and unique algorithms for image correction and denoising.

Investigation of the Intrinsic Spatial Resolution of an Intensified EMCCD Scintillation Camera.

In this paper, we present an experimental and Monte Carlo investigation of the intrinsic spatial resolution that can be achieved with the intensified electron-multiplying charge-coupled device (I-EMCCD) gamma camera [1]-[4]. This detector has a very low readout noise, an ultra-high spatial resolution and a large active area of ~ 80 mm diameter, which is well-suited for small animal imaging applications. The intrinsic detector resolutions achieved with different scintillators and under different experimental conditions were compared. In this study, the simple centroiding method was compared with two model-fitting approaches for finding the locations of gamma ray interactions. The results from Monte Carlo simulation have demonstrated that with an appropriate detector configuration, it is possible to achieve an intrinsic resolution of ~ 30 µm FWHM for detecting 27-35 keV gamma rays. The I-EMCCD scintillation camera offers a promising candidate for future ultra-high resolution SPECT imaging applications.

An Intensified EMCCD Camera for Low Energy Gamma Ray Imaging Applications.

This paper presents the design and feasibility study of a very-high resolution gamma camera for detecting 27-35 keV X and gamma rays emitted by I-125 labelled radiotracers. This detector consists of a newly developed Electron-multiplying CCD (EMCCD) sensor and a de-magnifier tube coupled to a thin layer of scintillator. A prototype detector was developed and experimentally evaluated. This detector has a detection area of ~ 5 cm2. It provided an intrinsic spatial resolution of < 60 µm FWHM and a high signal-to-noise ratio for detecting the 27-35 keV photons, which ensures an excellent counting efficiency. This detector will be used as the key component for a single photon emission microscope (SPEM) system that is under development.

Investigation of signal thresholding to reduce the effects of instrument noise of an EMCCD based micro-CT system.

This project investigated the signal thresholding effectiveness at reducing the instrument noise of an electron multiplying charged coupled device (EMCCD) based micro-CT system at low x-ray exposure levels. Scans of a mouse spine and an iodine phantom were taken using an EMCCD detector coupled with a micro-CT system. An iodine filter of 4 mg/cm2 area density was placed in the beam. The output signal was thresholded using some multiple of the inherent background noise. For each threshold, 100, 200, and 300 frames were summed for each projection to evaluate the effect on the reconstructed image. The projection images from the scans were compared using line profiles and their SNR. Our results indicate that, as the threshold was increased, the line profiles of the projection images showed less statistical variation, but also lower signal levels, so that the SNR of the projection images decreased as the threshold increased. When the line profile of a projection image obtained using a signal threshold is compared with one obtained using energy integrating mode, the profile obtained using thresholding had less variation than that obtained using energy integration, which indicates less instrument noise. The SNR at the edges of the scan object is higher in the thresholded images when compared with the energy integrated projection images. We conclude that thresholding the output signal from an EMCCD detector at low x-ray exposure levels is an effective method to reduce the instrument noise of an EMCCD detector.

Investigation of Noise and Contrast Sensitivity of an Electron Multiplying Charge-Coupled Device (EMCCD) based Cone Beam Micro-CT System.

A small animal micro-CT system was built using an EMCCD detectors having complex pre-digitization amplification technology, high-resolution, high-sensitivity and low-noise. Noise in CBCT reconstructed images when using pre-digitization amplification behaves differently than commonly used detectors and warrants a detailed investigation. In this study, noise power and contrast sensitivity were estimated for the newly built system. Noise analysis was performed by scanning a water phantom. Tube voltage was lowered to minimum delivered by the tube (20 kVp and 0.5 mA) and detector gain was varied. Contrast sensitivity was analyzed by using a phantom containing different iodine contrast solutions (20% to 70%) filled in six different tubes. First, we scanned the phantom using various x-ray exposures at 40 kVp while changing the gain to maintain the background air value of the projection images constant. Next, the exposure was varied while the detector gain was maintained constant. Radial NPS plots show that noise power level increases as gain increases. Contrast sensitivity was analyzed by calculating ratio of signal-to-noise ratios (SNR) for increased gain with those of low constant gain at each exposure. The SNR value at low constant gain was always lower than SNR of high detector gain at all x-ray settings and iodine contrast. The largest increase of SNR approached 1.3 for low contrast feature for an iodine concentration of 20%. Despite an increase in noise level as gain increases, the SNR improvement shows that signal level also increases because of the unique on-chip gain of the detector.

Live Cell Bioluminescence Imaging in Temporal Reaction of G Protein-Coupled Receptor for High-Throughput Screening and Analysis.

G protein-coupled receptors (GPCRs) are notable targets of basic therapeutics. Many screening methods have been established to identify novel agents for GPCR signaling in a high-throughput manner. However, information related to the temporal reaction of GPCR with specific ligands remains poor. We recently developed a bioluminescence method for the quantitative detection of the interaction between GPCR and ß-arrestin using split luciferase complementation. To monitor time-course variation of the interactions, a new imaging system contributes to the accurate evaluation of drugs for GPCRs in a high-throughput manner.

Assessing camera performance for quantitative microscopy.

Charge-coupled device and, increasingly, scientific complementary metal oxide semiconductor cameras are the most common digital detectors used for quantitative microscopy applications. Manufacturers provide technical specification data on the average or expected performance characteristics for each model of camera. However, the performance of individual cameras may vary, and many of the characteristics that are important for quantitation can be easily measured. Though it may seem obvious, it is important to remember that the digitized image you collect is merely a representation of the sample itself--and no camera can capture a perfect representation of an optical image. A clear understanding and characterization of the sources of noise and imprecision in your camera are important for rigorous quantitative analysis of digital images. In this chapter, we review the camera performance characteristics that are most critical for generating accurate and precise quantitative data and provide a step-by-step protocol for measuring these characteristics in your camera.

Spinning-disk confocal microscopy: present technology and future trends.

Live-cell imaging requires not only high temporal resolution but also illumination powers low enough to minimize photodamage. Traditional single-point laser scanning confocal microscopy (LSCM) is generally limited by both the relatively slow speed at which it can acquire optical sections by serial raster scanning (a few Hz) and the higher potential for phototoxicity. These limitations have driven the development of rapid, parallel forms of confocal microscopy, the most popular of which is the spinning-disk confocal microscope (SDCM). Here, we briefly introduce the SDCM technique, discuss its strengths and weaknesses against LSCM, and update the reader on some recent developments in SDCM technology that improve its performance and expand its utility for life science research now and in the future.

Camera technologies for low light imaging: overview and relative advantages.

"Camera Technologies for Low Light Imaging" is designed to offer the reader a summary of the current camera choices for low light imaging with special emphasis on the practical aspects related to each. Four major camera technologies, cooled charge-coupled device, cooled sCMOS, intensified cameras, and electron multiplier cameras, are discussed and compared. Supporting information about noise characteristics of photoelectrons and camera noise are provided and used for illuminating the practical aspects of using these cameras.

Graphical User Interface for a Dual-Module EMCCD X-ray Detector Array.

A new Graphical User Interface (GUI) was developed using Laboratory Virtual Instrumentation Engineering Workbench (LabVIEW) for a high-resolution, high-sensitivity Solid State X-ray Image Intensifier (SSXII), which is a new x-ray detector for radiographic and fluoroscopic imaging, consisting of an array of Electron-Multiplying CCDs (EMCCDs) each having a variable on-chip electron-multiplication gain of up to 2000× to reduce the effect of readout noise. To enlarge the field-of-view (FOV), each EMCCD sensor is coupled to an x-ray phosphor through a fiberoptic taper. Two EMCCD camera modules are used in our prototype to form a computer-controlled array; however, larger arrays are under development. The new GUI provides patient registration, EMCCD module control, image acquisition, and patient image review. Images from the array are stitched into a 2k×1k pixel image that can be acquired and saved at a rate of 17 Hz (faster with pixel binning). When reviewing the patient's data, the operator can select images from the patient's directory tree listed by the GUI and cycle through the images using a slider bar. Commonly used camera parameters including exposure time, trigger mode, and individual EMCCD gain can be easily adjusted using the GUI. The GUI is designed to accommodate expansion of the EMCCD array to even larger FOVs with more modules. The high-resolution, high-sensitivity EMCCD modular-array SSXII imager with the new user-friendly GUI should enable angiographers and interventionalists to visualize smaller vessels and endovascular devices, helping them to make more accurate diagnoses and to perform more precise image-guided interventions.