Page Type: Customer Story

Dual-Color Voltage ImagingCustomer Stories

Dr. Davide Raccuglia

Institute of Neurophysiology, Charité University Berlin, Germany

Background

Dr. Davide Raccuglia and his team use the model organism Drosophila melanogaster (colloquially known as fruit fly) to study how the brain regulates sleep. Dr. Raccuglia told us more about his research, “What I’m particularly interested in is the functional neural architecture of sensory gates for sleep regulation. We investigate how neural networks interact to create a gate that suppresses sensory processing allowing us to fall and stay asleep. Of course, we also want to understand how sensory information can break such a gate in order to awaken and enable us to react.”

“A great advantage to using Drosophila is that we can target specific neural networks, down to the level of a couple of neurons. We express genetically encoded voltage indicators (GEVIs) in these networks to optically derive the membrane potential of these structures. Using voltage indicators of different colors, we can record simultaneously from different neural networks to study how these networks interact when the flies are tired or rested.”

Figure 1: Dual-color voltage imaging of the Drosophila brain, acquired using a Kinetix22 with a two-way splitter. On the left are the two individual wavelength channels, combined on the right. The green area shows ring neurons expressing the voltage indicator ArcLight, the red area shows the dorsal fan-shaped body expressing the voltage indicator Varnam. Images were acquired using Speed mode at 100 Hz, 40x. The white square on the combined image corresponds to the graph below, indicating the neural activity over time in this area.

Challenge

Voltage imaging is a challenging technique due to the imaging speeds required, as Dr. Raccuglia mentioned, “For neural signaling that we consider slow, we record at 100 Hz. However, we also look into single neuron activity and want to resolve spikes during bursts, which requires recording speeds between 1-2 kHz. So, we need a camera that performs well at high-frequency rates, and is also very light sensitive.”

When imaging at high speeds the camera exposure time is limited, meaning that only very light-sensitive cameras can collect enough signal for each frame, making voltage imaging a combination of speed and sensitivity.

Dr. Raccuglia also images neural activity across different scales, “We measure, for example, the entirety of presynaptic terminals of a neural network, and this compound signal would be comparable to a network-specific local field potential. However, to determine the contribution of single cells we use GEVIs to perform multi-cellular optical electrophysiology.”

This requires a camera with a large field of view and small pixels, in order to be flexible enough to image across large samples and to focus on the single-cell level.

The Kinetix22 is a clear development and improvement on my previous camera solutions, especially for high framerate voltage imaging.

Solution

The Kinetix22 sCMOS camera is a revolution in the field of voltage imaging, due to the combination of extreme imaging speeds, high sensitivity, and a large 22 mm field of view. Dr. Raccuglia explains his experience with the Kinetix22, “The Kinetix22 is exactly what I needed for imaging signaling at high-frequency rates. We mostly use the Kinetix22 in Speed Mode, but we can also achieve 100 Hz in Sensitivity mode which is very useful. We use different modes, either selecting more speed or more sensitivity which is advantageous when combining voltage imaging and optogenetics as they require different frame rates and light conditions. The Kinetix22 is a clear development and improvement on my previous EMCCD devices, especially for high framerate voltage imaging.”

“Another advantage of the Kinetix22 is the smaller pixel size, which improves the resolution, and allows me to increase signal strength by binning the pixel.”

“We found the hardware very simple to install, it’s basically plug and play. We’ve used the camera every day and have not encountered any hardware issues. I especially love how easy it is to crop the sensor in Micro-Manager, and the region can be placed anywhere.”

High-Speed OptogeneticsCustomer Stories

Dr. Issac Kauvar, John Kochalka

Wu Tsai Neurosciences Institute, Stanford University, CA, USA

Background

Dr. Isaac Kauvar is a neuroscientist and engineer, developing tools in order to discover how cortex-spanning neuronal populations support the deployment of internal models during goal-directed behavior.

In order to track and analyze these large-scale activity patterns in the cortex, Dr. Kauvar and graduate student John Kochalka use conventional widefield imaging as well as advanced fluorescent imaging known as ‘cortical observation by synchronous multifocal optical sampling’ (COSMOS) in order to image widespread activity via a transparent window into a mouse brain.

Dr. Kauvar and the team found the need to build a new imaging system for COSMOS due to user demand.

Figure 1: Images of multiple pieces of neuronal tissue taken with the Kinetix sCMOS. The top image shows structural details and vasculature within the tissue, and the bottom image is a still from a video, imaging functional neural activity at high-speed using the COSMOS technique.

Challenge

In order to measure activity across a dense neuronal sample, both a large field of view and high resolution is needed. Imaging across a tissue while trying to identify individual cells requires a large sensor size in order to image efficiently without excessive stitching/ population tiling, and a small pixel size in order to achieve sub-cellular resolution and pick out which cells are active at what time, across the tissue.

The neuronal activity also occurs on a very short timescale and requires fast detectors, whether using optogenetics or calcium/voltage imaging. This means that a suitable detector also needs to operate at a high speed while still retaining the large field of view.

In order to achieve these high speeds and still suitably detect signal, a highly sensitive detector is needed, especially in order to detect weak signals when imaging at high speeds and having a low exposure time.

The Kinetix22 is a clear development and improvement on my previous camera solutions, especially for high framerate voltage imaging.

Solution

The Kinetix sCMOS camera is an ideal solution for both structural and functional neuroscience imaging, featuring a very large imaging area that can acquire high-resolution images at a very high speed. The extremely high speeds across a 10-megapixel sensor, combined with the near-perfect 95% quantum efficiency allow for very high speed and high sensitivity imaging, all with sub-cellular resolution at even low magnifications due to the small pixel size.

John Kochalka told us about his experience with the Kinetix, “Quantitatively we are enjoying the improvements in resolution, the field of view, and speed compared to other sCMOS cameras.”

“The Kinetix seems like it will give us a lot of options down the road, such as voltage imaging, and will let us push the temporal resolution on all the work we’re doing.”

Interferometry and Lithium MappingCustomer Stories

Dr. Matthew Gebbie

Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, USA

Background

Dr. Matthew Gebbie and team are an interfacial science and soft materials lab focusing on molecular interaction forces and self-assembly in soft materials. Dr. Gebbie told us more: “The key theme we are driving at is how ionic self-assembly influences electron transfer and ion transport. This turns out to be a key question, both for energy storage through batteries and capacitors, but also thinking about electrochemical reactivity such as splitting water to generate hydrogen or turning CO2 into CO. All of these areas involve ionic self-assembly at interfaces.”

“For our research, we use interferometry as well as surface-sensitive optical spectroscopy to try and understand what’s happening at interfaces. We are also aiming to use fluorescence microscopy approaches to measure lithium diffusion coefficients in ionic liquids, for next-generation electrolytes and batteries. We want to know how lithium ions move in these materials, even at very low concentrations.”

Figure 1: Image of Interference fringes, used to measure the separation distance between two mica surfaces. Different electrolytes can be confined between these surfaces to determine how ion size, concentration, and chemical properties influence electric double-layer formation. The distance resolution in that image is approaching the size of a water molecule.

Figure 2: A video of darkfield particle tracking, illustrative of the types of single particle tracking measurements in development to evaluate electric field-driven dynamics in ionic liquids.

Challenge

Dr. Gebbie is using a range of different microscopy and spectroscopy techniques to interrogate surfaces and materials, mainly interferometry, optical spectroscopy and highly-sensitive fluorescence spectroscopy, similar to single-molecule imaging methods. Each of these techniques comes with its own challenges, as Dr. Gebbie explained.

“For both interferometry and optical spectroscopy, signal sensitivity is a challenge we have to think about. The more sensitive we can be, the higher framerates we can access. We are aiming for these short acquisition times in order to do high frame rate interferometry.”

“Frame size is also very important for interferometry; we need to be able to image across the full width of the sensor to utilize all our diffraction gratings and the fringe splitting they produce. The number of pixels between two adjacent fringes is very important, as with interferometry we are targeting resolutions down to 3 angstroms (Å), this is approaching the size of a water molecule.”

“With our fluorescence microscopy methods, we need high-performance detectors in order to be confident about the fluorescence shifts that we see. We want to map lithium mobility in ionic liquids, and the detector is vital to see how low a concentration of lithium we can detect. We want to put in the minimal amount of fluorophore and remain highly sensitive to optical shifts.”

The Kinetix22 is a clear development and improvement on my previous camera solutions, especially for high framerate voltage imaging.

Solution

The Prime 95B sCMOS is the ultimate answer for sensitivity, combining a large 11 μm pixel with near-perfect 95% quantum efficiency at peak. Featuring a large sensor and the ability to image at high frame rates, the Prime 95B is an ideal solution for these microscopy and spectroscopy challenges.

Dr. Gebbie shared his experience with the Prime 95B, “We have two Prime 95Bs, one we are using in a surface forces system for interferometry, with plans to also do optical spectroscopy in situ. The other camera is used to track single fluorescent particles, looking at dynamics and electrolytes. The 95B has also played a big role in our studies to use fluorophores to study lithium diffusion. The resolution and sensitivity are clearly a big step up from what I was seeing previously, with the Prime 95B we can see on the order of a fraction of a mol percent, and I don’t think we would have been successful with cheaper, more conventional detectors. I’m pretty convinced we can measure more lithium diffusion coefficients in a few months than people have measured in the prior 15 years.”

“On the surface forces side the Prime 95B opens the door to trying laser-based optical spectroscopy such as Raman or IR in situ in these nanoconfined electrochemical interfaces. We could not pull this off with previous detectors, and from what we’ve seen the 95B makes this possible. This is a uniquely powerful instrument for us, as we didn’t want to deal with something with issues like an EMCCD. With the Prime 95B, we can continually push the limits with lower and lower intensity signals at higher and higher framerates, and that’s exactly what we need for both the interferometry as well as the fluorescence mapping.”

diSPIM Light-SheetCustomer Stories

Prof. Matthias Weiss, Ivana Jeremic

Physics of Living Matter, University of Bayreuth, Germany

Background

Prof. Matthias Weiss and PhD student Ivana Jeremic research challenging problems at the interface of physics and biology, focusing on understanding self-organization processes in living organisms. Prof. Weiss told us about his latest project, “We have built a new light sheet microscopy system for imaging dynamic processes within large samples, with Ivana’s project focusing on the embryogenesis of transgenic nematode Caenorhabditis elegans until gastrulation. Early C. elegans embryos seem to work on autopilot in terms of self-organization, and we have been successful already in monitoring mechanical cues that drive cell positions until gastrulation. With these data, we were able to even predict cell positions and migration paths via a computational model.”

“Now we want to dive a bit deeper and get information on how cells structure themselves internally before undergoing division, so we can learn more about individual steps during embryogenesis that has been missing in our analytical predictions so far.” Prof. Weiss and team are using an inverted SPIM (iSPIM) light sheet imaging system in order to observe cell behavior within C. elegans samples throughout development.

Figure 1: Images taken from a 3D stack of a C. elegans embryo in the two-cell state, acquired with the Kinetix sCMOS. High-intensity areas represent the actomyosin cortex that exerts chiral forces during cell division. The numbers on each image represent the z-position within the stack.

Challenge

While light-sheet microscopy is well-suited to imaging large samples with relatively low illumination levels, the light sheet itself requires fine-tuning for the best results. Prof. Weiss told us more about his imaging challenges, “We want a light sheet that is long and thin enough so that we can get the full volume of the worm embryo, which is about 50 μm in diameter. Bleaching is an issue as excess light can poison the embryo. If we used something like laser scanning confocal, the embryo would never go beyond the four-cell stage and would die, so the light sheet with low laser illumination is ideal as we can even observe hatching.”

“Some of the fluorescent protein constructs within the sample change their expression during embryogenesis so therefore we have to be able to capture signals from low to high intensity without having to change camera modes, so we need a high dynamic range.”

Alongside a sensitive camera with a high dynamic range, this project is also best suited to a detector with a large sensor and a small pixel in order to capture the maximum resolution across the largest field of view.

The Kinetix22 is a clear development and improvement on my previous camera solutions, especially for high framerate voltage imaging.

Solution

The Kinetix sCMOS is an ideal solution for this application as well as light-sheet microscopy in general, combining a balanced 6.5 μm pixel with a huge 10-megapixel array across a 29 mm sensor, resulting in high resolution across the whole C. elegans embryo even at lower magnifications.

Prof. Weiss told us about his experience with the Kinetix, “Typically we can take 50 images through the volume of the worm embryo in around 5 seconds, but due to the improved signal-to-noise ratio with the Kinetix we can further tune our experiments and image faster with shorter exposure times so we can also look at more rapid features within the sample.”

“Also, with the greater quantum efficiency of the Kinetix, we can tune down the laser intensity so that we can be gentler and keep it more in the native state. In the long term, we intend to also image and rate man-made bio fabricates over days, so we really need the sensitivity in order not to perturb the samples’ development.”

“We will use dynamic range mode for the good signal-to-noise ratio at the decent speed and ability to image weak and intense signals, we were impressed by the signal-to-noise ratio we got with the Kinetix.”

High-Speed Voltage ImagingCustomer Stories

Prof. Zhenyu Gao, Prof. Daan Brinks

Department of Neuroscience, Erasmus University Medical Center, The Netherlands

Background

The lab of Prof. Zhenyu Gao at the Erasmus University Medical Center studies how the brain controls motion, learning, and memory. In order to study these functions, Prof. Gao’s lab uses in vivo methods to detect electric signals in the brains of mice models. This is achieved by either utilizing electrophysiological methods or fluorescent optical methods, or a combination of both.

To visualize activity in the cortex of the mouse brain, voltage imaging can be used. This imaging technique provides an optical readout from fluorescent voltage indicators, which is an incredibly direct method of determining neuronal activities. Voltage imaging experiments are refined in the lab of Prof. Daan Brinks who collaborates tightly with Prof. Gao’s lab.

Figure 1: The Kinetix sCMOS connected to an imaging system within an electrophysiology cage, set up for electrophysiology and/or voltage imaging experiments.

Challenge

In order to detect voltage signals in neurons, some key criteria need to be fulfilled. As imaging frequency needs to be in the range of 1-2 kHz (1000-2000 fps) in order to be able to precisely describe individual action potentials, a camera is required which is capable of this recording speed. Because of the high frame rate required, signal levels per frame will be very low, which requires a camera that has a very high sensitivity from the quantum efficiency point of view and also a low enough noise level to reliably detect even minute changes in the signal. Only by maximizing signal collection and minimizing noise contributions can a camera detect signals at low enough exposures (less than 1 ms) to operate at 1 kHz or more.

Signal levels of the currently used Archon voltage indicator reports signals from the soma (cell body) of neurons. While signal levels in electrophysiological methods can resolve even very low signal levels with high temporal resolution, voltage indicators work on the basis that their reported signals sometimes are only encoded in 1-10% increases (or decreases) in their baseline signal. These small fluctuations in signal need to be accurately collected and analyzed in order to determine neural function from voltage imaging data.

The Kinetix22 is a clear development and improvement on my previous camera solutions, especially for high framerate voltage imaging.

Solution

The Kinetix sCMOS presents a groundbreaking combination of both speed and sensitivity, making it a proven and ideal solution for demanding applications like voltage imaging.

The Kinetix Speed Mode images at 500 fps across the full 29 mm field of view, increasing to 1000-2000 fps at smaller regions, even to over 100,000 fps for extreme speed applications. This kind of speed is only possible thanks to the low read noise and near-perfect 95% quantum efficiency of the Kinetix.

Prof. Gao told us that “the Kinetix is the perfect solution for our requirements to detect voltage signals in vivo because it is the optimal solution – namely being fast and sensitive”. In particular, another benefit of the Kinetix is that it can image very fast and still provide a much larger field of view at the same time than previous camera solutions. This enables Prof. Gao’s lab to image many neurons at once at high speeds, putting the neuronal activities in context with each other, eventually allowing a correlation between sensory stimuli, cortical activity, and behavioral consequences.

Plant Calcium ImagingCustomer Stories

Prof. Zhen-Ming Pei

Department of Biology, Duke University, North Carolina, US

Background

The lab of Prof. Zhen-Ming Pei is interested in the early signalling events by which plants sense environmental signals and decode them to give the appropriate responses. Upon perception of external signals, cell surface receptors trigger an increase in cytosolic free calcium concentration, which is mediated by ion channels. Prof. Pei’s long-term goals are to identify these receptors and ion channels, isolate their interacting components, and assign molecular functions to them.

An example of Prof. Pei’s research comes from a recent Science publication, concerning the plant immune response surveillance system consisting of intracellular nucleotide–binding leucine-rich repeat receptors (NLRs) capable of triggering immunity in response to pathogen activity, leading to activation of plant defences.

The lab currently uses a multidisciplinary approach including biophysics, biochemistry, cell biology, molecular genetics, and function genomics, in order to dissect the signalling cascades of external calcium as well as nitric oxide in the model plant organism Arabidopsis.

Figure 1: Prime 95B sCMOS used for fluorescent Fura-2 calcium imaging of HeLa cells. HeLa cells contained a mutation in the N terminal RNL motif on intracellular [Ca2+] in NRG1.1 D485V and ADR1-expressing HeLa cells, as visualized with Fura-2, before or 2 minutes after CaCl2 addition. Calcium activity scaled to the pseudo-color bar.

Challenge

Calcium imaging and live-cell imaging both come with their own challenges, requiring a camera that is sensitive enough to obtain a signal while also maintaining a fast imaging rate. In order to acquire images quickly enough to observe calcium activity a short exposure time is necessary, which in turn reduces the time available to collect signal, resulting in a low signal level. Cameras for this application would need to maximize signal collection and minimize noise levels in order to get a high signal-to-noise ratio while imaging at speed.

For Prof. Pei’s calcium imaging experiments, Fura-2 fluorescence imaging was performed using a Zeiss Axiovert microscope equipped with two filter wheels and an sCMOS camera. With excitation at ~350 nm and emission at ~500 nm, another challenge is using a camera with high sensitivity at a wide range of different wavelengths of light.

The Kinetix22 is a clear development and improvement on my previous camera solutions, especially for high framerate voltage imaging.

Solution

The Prime 95B camera represents the ultimate in sCMOS sensitivity, featuring a large 11 μm pixel optimized for Nyquist at high magnifications and for low signal, high sensitivity imaging. The Prime 95B also operates at up to 80 fps across the full frame, allowing for easy capture of fast calcium signals while maintaining high sensitivity and a large field of view to fit in as many cells as possible.

Prof. Pei made use of the Prime 95B in their recent Science publication, imaging HeLa cells with the Fura-2 calcium indicator. Prof. Pei gave us his opinion on the Prime 95B sCMOS, saying “The camera is very good and we have not yet pushed it to the limit, we hope to use them more in the future.”

Reference

Jacob, P., Kim, N. H., Wu, F., El-Kasmi, F., Chi, Y., Walton, W. G., Furzer, O. J., Lietzan, A. D., Sunil, S., Kempthorn, K., Redinbo, M. R., Pei, Z. M., Wan, L., & Dangl, J. L. (2021). Plant “helper” immune receptors are Ca2+-permeable nonselective cation channels. Science (New York), 373(6553), 420–425. https://doi.org/10.1126/science.abg7917

High-Speed Calcium ImagingCustomer Stories

Prof. Kirill Volynski

Institute of Neurology, University College London, UK

Background

The Volynski lab, led by Kirill Volynski, Professor of Neuroscience at University College London (UCL), is primarily interested in understanding the regulation of neurotransmitter release which forms the basis of communication among neurons in the brain.

As explained by Prof. Volynski, “Synapses between neurons are critical sites of modulation and plasticity, both in health and in disease. Therefore detailed knowledge of the cellular mechanisms that regulate synaptic transmission at the level of individual synapses is a prerequisite for understanding the operation of complex neuronal circuits.”

“We have recently developed new imaging methods which, for the first time, allow us to study the relationship between Ca2+ entry and vesicular exocytosis, and to probe presynaptic ion channel function in individual small presynaptic terminals. This is based on using fluorescence microscopy to image rapid changes in the concentration and rate of vesicle discharge of Ca2+ ions; and on the use of super-resolution scanning ion conductance microscopy for patch-clamp recordings from small presynaptic boutons.”

“Using these methods we investigate how different channels that mediate Ca2+ influx into the terminal control the release of vesicles, how they influence synaptic plasticity, and how synapses are influenced by other modulatory neurotransmitters acting upon presynaptic terminals.”

Figure 1: Axonal arbor of a hippocampal neuron in culture expressing glutamate sniffer SF-iGluSnFR probe, acquired with the Kinetix sCMOS.

Challenge

High-speed Ca2+ imaging requires both sensitivity and speed. Previously Prof. Volynski’s lab was using a Prime 95B 25mm to maximize sensitivity and field of view while achieving high speeds of acquisition. This camera provided a considerable upgrade in terms of speed, the field of view (FOV), and stability to an earlier EMCCD solution. But the speed of the camera was still a limiting factor both for keeping up with the high-speed dynamics in the sample, but also for light acquisition, due to the necessity to use a ‘pseudo-global shutter’ trigger to control the light source.

In a rolling shutter camera such as the Prime 95B, the acquisition of a frame starts at the top of the sensor and very quickly sweeps down to the bottom. Although the time difference between the top and bottom of the sensor is very small, it can introduce distortions into highly precise high-speed experiments, so a ‘pseudo-global shutter’ must be used in order to capture the entire sensor at once. This works by using advanced hardware triggering to begin acquisition of an image only when all of the camera sensor rows are acquiring, then deactivating until the next frame. This concept is outlined in a timing diagram in Figure 2.

Figure 2: The timing of rolling shutter cameras, and using triggering of the light source to achieve global behavior.

The time the camera must wait until exposure begins is known as the ‘dead time’ or ‘frame time’ and is directly determined by the camera frame rate. With a faster camera, more global data could be acquired.

The Kinetix22 is a clear development and improvement on my previous camera solutions, especially for high framerate voltage imaging.

Solution

The Kinetix is a groundbreaking sCMOS camera that provides the same near-perfect 95% quantum efficiency as the Prime 95B while measuring signals more accurately thanks to the lower read noise of its ‘Sensitivity’ mode. Other improvements come in both frame rate and sensor size. The Kinetix is a 10 Megapixel camera with an enormous 29.4 mm diagonal sensor – in its ‘Sensitivity’ mode, this entire field of view can be read out at 88 frames per second, but the Kinetix also has a ‘Speed’ mode, where the entire 10 MP sensor is read at an astonishing 500 frames per second.

What improvements will the Kinetix ‘Sensitivity’ and ‘Speed’ modes have for pseudo-global shutter imaging?

Sensitivity Mode: More Exposure Time

Something vital for Prof. Volynski is extending the effective exposure time, namely the ‘frame time’ plus the ‘trigger on’ time. The Kinetix has a much faster frame time than the Prime 95B, does this result in greater effective exposure time if we compare a similar 200-row region of interest (ROI) between the Kinetix in Sensitivity mode and the Prime 95B?

With a 200-row ROI both cameras have a speed of 300 fps leaving 3.3 ms per frame. The difference is the frame time: 2.08 ms for Prime 95B leaving 1.25 ms for the light source to be on and photons to be collected; and 0.71 ms for the Kinetix in Sensitivity mode leaving 2.6 ms for the light source to be on, more than double that of Prime 95B. In this manner, the shorter frame time of the Kinetix allows for a longer effective exposure at the same frame rate, as outlined in Figure 3.

Figure 3: Timing diagram for Pseudo-Global Shutter mode for the Prime 95B and Kinetix (Sensitivity). Each camera runs at 300 fps across a 200 row ROI. Due to the shorter frame time, the Kinetix in its ‘Sensitivity’ mode is able to achieve a significantly longer ‘Trigger On’ time during which light can be collected.

Speed Mode: More Exposure Time and Higher Speeds

As well as a greater effective exposure time, Prof. Volynski also looks for high speeds in order to capture dynamic calcium activity. This is where the Kinetix Speed mode comes in, operating at 500 fps across the whole sensor and allowing for capture of ultra-fast features.

If we look at the same example as the previous comparison but with the Kinetix in Speed mode, the frame time is so short (0.13 ms) that there is time for a 200-row acquisition, resulting in an overall framerate of 600 fps. Even with this doubling of the acquisition speed, the trigger on time is still 20% longer than the Prime 95B at 1.54ms, providing both more speed and more illumination time as shown in Figure 4.

Figure 4: Timing diagram for Pseudo-Global Shutter mode for Prime 95B and Kinetix (Speed). Same target of 300 fps and 200-row ROI as previous. In its Speed mode, the frame time of the Kinetix is so much faster that twice the number of frames can be collected in the same time period, but also maintaining a 20% longer ‘Trigger On’ time per frame during which light can be collected.

Summary

The Kinetix is a groundbreaking combination of speed and sensitivity, offering ultra-high speeds across a huge sensor with ultra-low noise contributions. As well as being powerful, the Kinetix is also highly flexible, allowing for fine control over readout using advanced hardware triggering and readout modes such as Pseudo-Global Shutter.

The speed increase the Kinetix provides over previous generation CMOS cameras can lead to a significant increase in effective exposure time for increased light collection in pseudo-global shutter applications with its ‘Sensitivity’ mode. Furthermore, the incredibly fast frame time of the ‘Speed’ mode can provide speed increase combined with effective exposure time increases.

The Kinetix also does this while delivering an 18% larger horizontal field of view, due to the larger width of the Kinetix sensor.

Intravital NIR ImagingCustomer Stories

Dr. Epameinondas Gousopoulos (MD/Ph.D.), Dr. Stefan Wolf

Division of Plastic and Hand Surgery, University Hospital Zürich, Switzerland

Background

The group of Prof. Gousopoulos at the University Hospital Zurich is focused on researching lymphedema, a condition where lymphatic system dysfunction results in swelling in parts of the body. The group has established a mouse model in order to investigate this disease.

Postdoc Dr. Stefan Wolf told us more about his research, “We use surgery to remove lymphatic vessels in the mouse tail, this introduces lymphedema and causes swelling in the mouse tail. We then image the lymphatic vessels within the tail using an intravital microscope setup.”

“We inject a lymphatic-specific near-infrared (NIR) fluorescent tracer dye into the tail tip and image the flow down the tail, visualizing the capillaries and lymphatic collectors. This helps us understand the underlying mechanisms, as well as screen for new pharmacological compounds which may influence the onset of lymphedema or protect the lymphatic vessels.”

Figure 1: Flow of a NIR dye through a section of a mouse tail, imaged with the Prime BSI Express. Hairs, capillaries, and lymphatic vessels are all visible within the tail.

Challenge

Intravital imaging involves imaging large live organisms, in this case, mice. Breathing and other small movements can decrease image quality, and small magnifications are needed to capture large areas of the sample. In this case, imaging uses magnifications between 4x and 12x, requiring a camera with a small pixel in order to best match Nyquist sampling and get good resolution.

Low magnifications also allow for a large field of view, so a camera with a large sensor is also suitable, which can also image at a video rate in order to capture dynamic movements of the dye through the lymphatic system within the tail.

In addition, as the lymph networks are beneath the skin, NIR wavelengths are used to penetrate below the tail surface and image the dye within. This requires a camera with high sensitivity and quantum efficiency in NIR wavelengths (>700 nm), in order to best capture the signal.

The Kinetix22 is a clear development and improvement on my previous camera solutions, especially for high framerate voltage imaging.

Solution

The Prime BSI Express is a compact yet powerful sCMOS camera with a small pixel, large sensor and sensitivity far into the NIR range, making it an ideal solution for this application.

Dr. Wolf told us more about his experience with the Prime BSI Express, “We combined the camera with a Zeiss microscope for intravital imaging, it worked really well with Zen imaging software, and the camera instantly connected and we had no problems at all.”

“We definitely needed the sensitivity of the sensor in order to capture even faint traces of the dye and have a much better range of data. I’ve never seen a camera before that can image such a tiny signal with hardly any noise, it’s everything we wished for!”

“We might also need the speed of the Prime BSI Express for other projects, such as imaging blood flow within capillaries at wound sites. We wanted the perfect solution for our experiments now and for the future.”

High-Speed Starscape AstronomyCustomer Stories

Prof. Richard Gomer

Interdisciplinary Life Sciences Building, Texas A&M University, TX, US

Background

Prof. Richard Gomer at Texas A&M University is involved with astronomy research beyond the reaches of the solar system. Prof. Gomer told us more about his research, “A simple question in astronomy is whether or not there is material associated with the solar system well out past the orbit of Pluto. There is good evidence of something called a Kuiper belt, but way out further past that, halfway to the nearest star, there might be a spherical collection of icy, rocky objects called the Oort cloud, but nobody has been able to detect it as these objects are so small and so far away.”

“One way we can detect objects from the Oort cloud is if they pass in front of a star, we would see light from that star blink off and blink back on again.”

Prof. Gomer and his colleague James Hitchcock use telescopes located in western Texas to look at rich clusters of stars and try to observe events that may prove the existence of the Oort cloud.

Figure 1: Image of a starfield taken by the Kinetix sCMOS. The image shows the full 3200×3200 pixel sensor (indicated by axis labels) and includes an intensity scale to the right. Stars in the image are identified by black squares and colored depending on the light intensity, with the red squares used for background correction.

Challenge

This demanding application requires gathering light intensity data from distant stars, a process complicated by the Earth’s atmosphere, orbit, and the limited amount of imaging time available each night. Prof. Gomer further explained the challenges involved with his research, “Due to the small size of these objects, and the fact that the Earth is moving in its orbit, what you end up with is the light of the star blinks off for just a few milliseconds, and these events are really very rare.”

“We could use detectors that are just one pixel and can detect rapid changes in light, but we’d only be able to look at one star. The ideal thing would be to look at a field of stars, so you’d have many chances to observe an event.”

The more stars in the camera’s field of view, the more opportunities to observe an event, and the more reference points available to compare these events to. This requires a camera with a large field of view that can also image at a high speed in order to capture these millisecond-scale events.

The Kinetix22 is a clear development and improvement on my previous camera solutions, especially for high framerate voltage imaging.

Solution

The Kinetix is the ideal combination of speed and field of view, able to acquire images extremely fast across a large 10 megapixel, 29 mm sensor. The Kinetix has the temporal resolution to capture these rapid events, the spatial resolution to differentiate different star clusters (due to the small pixel), the sensitivity to determine small fluctuations in light intensity from each star, and the sensor size to image an entire star field, maximizing the chances of recording an event.

Prof. Gomer told us about his experience with the Kinetix, “Up until 2020 we weren’t able to find imagers that could do 500 frames a second across a large format sensor, but then along came the Kinetix, and it was just perfect. There’s really nothing like the Kinetix, nothing that can run as fast. This wouldn’t have been possible without the amazing Teledyne Photometrics staff, especially Angela Mills and Rachit Mohindra.”

“We captured data in both the Speed mode, which is just insanely fast, and the Sensitivity mode, which has really good noise levels. We are using custom software that takes in a Kinetix image, subtracts the background and adds up the data values of each star, we can then plot the brightness of each star over time.”

Quantum CommunicationCustomer Stories

Dr. Tim Schröder, Mr. Maarten van der Hoeven

Integrated Quantum Photonics Lab, Humboldt-University of Berlin, Germany

Background

The Integrated Quantum Photonics lab of Dr. Tim Schröder at the Humboldt-University of Berlin is interested in understanding, controlling, and developing use cases for quantum research. In this particular project, Maarten van der Hoeven is characterizing and studying the behavior of color centers in diamond nanostructures. These color centers are extremely stable single photon sources that can be utilized to build quantum sensors or quantum communication devices with high communication rates. To achieve this, Maarten searches for ways to couple those quantum systems to collect transmitted photons as effectively as possible.

For color centers in diamond, the fabrication of nanostructures that contain emitters is a well-known method for enhancing the extraction of photons. These nanostructures can be used for single-mode fiber coupling of the color center’s emission, as it is a requirement for high photon collection efficiency and a necessity for integrated systems.

Figure 1: A widefield image taken with the Prime BSI sCMOS camera, pixel array shown in axis labels. Most of the bright spots on the image are tin-vacancy centers, intensity scaled to the scale on the right of the image.

Challenge

In order to detect individual point sources, which are low in signal down to a few photons per millisecond, a camera with a very high quantum efficiency and very low read noise is required. A confocal raster scanning method was previously used, which was much slower than simultaneously locating many tens or hundreds of color centers.

A well-controlled scientific camera sensor gives reliable access to a quantifiable number of detected photons. Other, less optimized camera solutions do not easily allow for quantitative analysis but require frequent calibration and/or contain patterned artifacts in offset and image leading to worse results. Moreover, for some experiments, it is crucial to have a large enough full well capacity to obtain recordings of various signal levels from very dim to very bright – only possible with a high bit-depth and well capacity.

Lastly, the color centers are at random locations and require locating in respect to landmarks on the diamond so further processing can be performed. A large field of view sensor would be truly beneficial as it speeds up the entire process and makes it very repeatable.

The Kinetix22 is a clear development and improvement on my previous camera solutions, especially for high framerate voltage imaging.

Solution

Maarten told us that the Prime BSI sCMOS camera is a very good solution for their imaging needs, as individual color centers can be reliably identified, reproducibly located and characterized. The Prime BSI has become an established solution of choice on the color center setup, and this group has recently purchased a second camera for a new system.

As well as using a Teledyne Photometrics Prime BSI sCMOS camera for widefield acquisition, this self-built setup also includes a Teledyne Princeton Instruments SpectraPro HRS 500 spectrometer with a ProEM EMCCD. As spectroscopy data can only be revealed at the locations where events are situated, a very precise localization needs to take place based on the camera images and relayed with a high precision XY-stage in order for the spectroscope to measure at the intended positions requiring a delicate interaction between all components controlled by a single software interface. The fast and accurate combination of the Prime BSI and SpectraPro enables reliable identification of the nature of individual color centers. With its astigmatism-corrected light path and the flexibility to switch and interchange grating turrets, the SpectraPro HRS is the ideal partner for the Prime BSI on this system.

Overall, Maarten said that “[we] can carry out experiments with our self-built setup which we could not perform before. Further efforts will be made to improve the entire system even more in order to gain additional insight into color centers, their behavior and eventually lead to technological progress in our understanding and use of them in the scope of the groups research focus.”