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 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.

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 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.

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 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.

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 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.

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 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.

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 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.

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.”

Live Cardiac 3D Spinning-DiskCustomer Stories

Prof. Francesco Pasqualini

Synthetic Physiology Lab, University of Pavia, Italy

Background

Prof. Francesco Pasqualini is a Harvard-trained bioengineer leading the synthetic physiology laboratory at the University of Pavia, currently researching cardiac development using engineered cell culture platforms. By optimizing this platform with cell lines and then moving to human induced pluripotent stem cells, Prof. Pasqualini can get a unique perspective of the developing heart.

Prof. Pasqualini told us more, “We are studying the mechanobiology of the heart as well as investigating the use of various extracellular matrix components to control tissue and organ-level behavior. We are particularly interested in recapitulating the early phases of human heart development using defined extracellular matrix and cardiac cell types. All using live-cell microscopy, of course.”

This study uses a Nikon Ti2 microscope and a modular spinning-disk confocal module, the X Light V3 from CrestOptics.

Figure 1: An image acquired with the Kinetix sCMOS on a spinning-disk confocal, showing XY, YZ, and XZ projections of live HaCaT cells stained with actin (grey), tubulin (green) and two nuclear stains (cyan and magenta). The bottom XZ projection and side YZ projection show the 3D organization of tubulin microtubule networks on flat cells, and mitotic spindle in the rounded dividing cells. Data from Dr. Di Sante, Ms. Pezzotti, and Ms. Torchia in the Pasqualini lab.

Challenge

Cardiac research involves imaging of both structural and functional aspects of cardiac cells. While structural imaging requires a camera with a large field of view and a small pixel to get high spatial resolution at the desired magnification, functional imaging requires a camera sensitive enough to operate at high speeds to capture calcium and voltage activity across cells and tissues.

When working with multiple cell types and multiple timescales, it is vital to have a flexible yet powerful imaging system that can meet the needs of each experiment, whether imaging the morphology of large groups of cells or imaging small sub-populations with high speeds to observe activity.

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.

Solution

The Kinetix is a proven solution when paired with the Nikon Ti2 and CrestOptics X-Light V3, resulting in a powerful imaging system. This allows for flexible imaging both now, and in the future when bigger, faster, more sensitive experiments are planned to image ultra-low voltage signal levels in developing heart tissues in vitro.

Prof. Pasqualini described his experience with the Kinetix, “I like the different Kinetix modes, we can image lots of cells at ~100 fps, and if we need to, we can look at another type of cell at ~1000 fps for calcium imaging. I also like Sub-Electron mode for images that don’t need to be fast, the lower read noise makes a big difference.”

DeepSIM Super-ResolutionCustomer Stories

Dr. Alessandra Scarpellini, Dr. Maria Giubettini

CrestOptics S.p.A., Via di Torre Rossa, Rome, Italy

Background

CrestOptics is a leading company in the development and manufacture of advanced systems for fluorescence microscopy, featuring products such as the X-Light series of spinning-disk confocal modules. CrestOptics has also recently launched DeepSIM, a super-resolution module for 3D samples. We discussed DeepSIM with the Head of Sales and Marketing Dr. Scarpellini, and Application Specialist Dr. Giubettini.

Dr. Scarpellini told us more, “We noticed an increase in demand for higher resolution to see more of biological samples, but many options for super-resolution imaging were not accessible due to high cost, specialized sample preparation, or incompatibility with live sample imaging. This is why we developed DeepSIM, which makes super-resolution accessible, similar to how we made spinning-disk accessible. You can work with the same sample you’d typically use, but in a variety of configurations, such as combining DeepSIM with a spinning disk confocal or as a standalone, on upright or inverted microscopes, and able to work with a full range of objectives.”

Dr. Giubettini expanded on the possible imaging configurations, “It’s super easy to change between three modalities: widefield, spinning disk, and DeepSIM, you can switch to SIM to go into more detail on a sample, all while working with the same sample preparation.”

Figure 1: Comparison of widefield (WF), X-Light V3 spinning-disk confocal (CF), and DeepSIM super-resolution (SR) images, all acquired with the Kinetix sCMOS. The top row is HeLa cells at 60x (GFP-alpha tubulin in green, lysosomes in red), the middle row is human brain organoids at 20x (CTIP2-positive deep layer cortical neurons in green, pan-neuronal MAP2 in red), and the bottom row is 60 μm thick 3D volumes of mouse brain tissue. Thanks to the Consiglio Nazionale delle Ricerche (CNR) for HeLa samples, and Istituto Italiano di Technologie for brain organoid samples.

Challenge

Dr. Scarpellini told us what DeepSIM needs from a camera, “a pixel size of 6.5 μm, good hardware triggering options, and as much speed as we can get, because we need to acquire multiple frames to get one super-resolved image. So low readout time and high speed is important to us, along with high sensitivity.”

DeepSIM requires a camera that can image at high speeds while maintaining good signal-to-noise ratio at low exposure, along with a 6.5 μm pixel. In addition, when the DeepSIM is combined with the X-Light V3 spinning disk, the maximum field of view increases to 25 mm, requiring a camera with a suitably large sensor.

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.
Figure 2: A full suite of products mention in this customer story. Here we see a Kinetix sCMOS and Prime 95B sCMOS attached to a X-Light V3 spinning disk module, in turn attached to a DeepSIM module, which is attached to a photoport on a Nikon Ti-2 microscope.

Solution

The Kinetix sCMOS is an ideal solution for CrestOptics’ advanced imaging systems such as the X-Light V3 spinning disk and DeepSIM modules, thanks to the unique combination of high acquisition speed, high sensitivity, and 6.5 μm pixels across a 29 mm sensor. For an example of live cell imaging with the Kinetix and DeepSIM, please refer to application notes from the CrestOptics website.

Dr. Scarpellini described her experience with the Kinetix, “The Kinetix was already a camera that we liked a lot with our X-Light V3 spinning-disk systems, because of the speed and wide field of view. This also makes the Kinetix a very good choice when combining the spinning-disk with DeepSIM.”

“For the standalone DeepSIM our recommendation for users who need the highest possible speed is to use the Kinetix22, but some users might also be happy with the Prime BSI Express as well.”

Links

https://crestoptics.com/deepsim/

https://crestoptics.com/x-light-v3/

High Content ImagingCustomer Stories

Dr. Christopher Toepfer

Radcliffe Department of Medicine, University of Oxford, UK

Background

Dr. Chris Toepfer is a principal investigator interested in understanding how cardiac physiology changes in inherited cardiovascular conditions. The lab uses fluorescence microscopy and calcium imaging to observe how cellular contractility is affected across different heart conditions, such as those seen in professional sports.

Using human stem-cell-derived cardiomyocytes with a known genome, they then use CRISPR Cas9 to insert patient-specific mutations and GFP tags into this cell model and observe the effects; such as whether the cells beat harder or faster than normal. This is done with calcium imaging to monitor calcium flux of a cell, and imaging of the GFP-labelled sarcomeres themselves, enabling Dr. Toepfer and team to measure contractility on the fundamental unit of muscle contraction in real-time.

Figure 1: Two Kinetix sCMOS cameras connected to a Nikon Ti2 using a Cairn TwinCam for high-content imaging.

Challenge

The sarcomere is a challenging sample to image, as described by Dr. Toepfer: “There are often hundreds of sarcomeres and we need to track each individual one in every single cell, and we do this across hundreds of cells so this is high throughput… Unfortunately, the GFP-tagged protein is only seen a couple of times in each sarcomere, and a sarcomere is only 2.1 µm in size, so we are limited by the availability of light.”

“The cells beat once or twice a second and the contraction is very fast, so we need a high framerate to see and track the movement… the initial contraction happens over 50-100 ms and we want to capture as many frames in that short period of time as we can, to really characterize how the contraction and relaxation occur.”

The requirement for capturing many rapid events over a large imaging area makes this a very demanding experiment; one that is often limited by the hardware being used. The previous microscope had a 22mm FOV, and the EMCCD cameras on the system were limited to an even smaller imaging area.

In addition, imaging calcium with RFP and sarcomeres with GFP requires the use of multiple channels, meaning each acquisition takes longer. Dr. Toepfer mention his previous system for imaging these samples, saying “With our previous system we could only do around 30 fps in a single channel”, which was due to the speed limitations of EMCCD technology.

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.

Solution

The new system provides the lab with the capabilities to overcome all of the previous limitations: a larger imaging area, higher acquisition speeds and overall a higher throughput of data. The combination of the Kinetix and the Nikon Ti2 means that the entire 25 mm can be imaged at ~600 fps which is 20x more data than the previous system.

Dr. Toepfer told us about his experience with the Kinetix, “The Kinetix is great, the field of view being larger is very helpful for us so we can capture multiple cells now, our acquisitions are a lot quicker so we can have a lot more experiments done in the same amount of time and get a lot more data, which is also of a higher quality.”

“The Kinetix is allowing us to look at rapid events… now we are running at 100 fps compared to the previous 30 fps, and if we shrink the chip we can go to over 1000 fps, which now rivals other techniques used to image calcium such as photomultiplier tubes, but on a microscope.” Dr. Toepfer also wishes to measure and correlate the action potentials that trigger calcium release upon contraction, using fluorophores for voltage imaging. These voltage events occur far faster than the contraction cycle, and would require even higher speeds to image, easy obtainable with the Kinetix.

The lab is currently using two Kinetix cameras in combination with the TwinCam emission splitter from Cairn Research. In addition to the increased throughout provided by a new microscope and new camera, the TwinCam means that they can now image across multiple fluorescent channels in real-time, allowing for the capture of both calcium waves and sarcomere contractions and correlation
between these two channels.

Live Cell Microfluidic PlatformCustomer Stories

Dr. Yu Ting Chow, Dr. Amir Tahmasebipour

Mekonos Inc., San Francisco, California, US

Background

Mekonos Inc. is a start-up company developing a biomedical microelectromechanical system (bio-MEMS) platform based on semiconductor and microfluidic technologies, to enable single-cell transfection with high viability and a scalable workflow.

We spoke with Dr. Amir Tahmasebipour, a Senior Scientist at Mekonos Inc., to learn more, “We do a lot of experiments: prototyping, development, designing, iterating and troubleshooting devices, and as this is used for single-cell transfection we work on very small scales. Our microfluidic platform is high-throughput and uses high velocities, so we really rely on good imaging devices to be able to characterize our system, for both microfluidic and MEMS key technologies.”

“Our devices allow us to flow substances over attached cells, with the aim to manipulate and transfect single cells on several different timescales. We use imaging systems to measure the quality and quantity of transfection, as well as track where particles and cells are going.

Figure 1: Live cells within the Mekonos Inc. microfluidic device under flow. The video stack was acquired with the Prime BSI Express sCMOS.

Challenge

These devices involve both very small components and dynamically moving parts, requiring both high resolution and high image quality at a high speed. Dr. Tahmasebipour told us more, “We rely on videos more than pictures, as everything has a time component to it, with signals changing or fluids changing, as well as objects in the microfluidic devices move through the field of view very quickly. Our systems require enough light to have a short timescale of exposure, as we try to get as high a framerate as possible at as high a quality as possible, for tens of thousands of particles that we can scan through.”

“Resolution is also very important in order to pinpoint what is a cell and what is debris, we really like getting as high quality of a picture as we can. We also want to couple the camera with a Nikon microscope and use a variety of magnifications for characterization, up to 50x or 100x for tiny MEMS components close to the diffraction limit.”

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.

Solution

The team at Mekonos Inc. are using the Prime BSI Express sCMOS, which features both a high imaging speed and a high-resolution sensor thanks to the small pixel size. With a single-sensor design and low noise overall, the Prime BSI Express produces images of very high quality.

Dr. Tahmasebipour described his experiences using the camera, “The Prime BSI Express camera is our number one source of generating images, especially for validating our designs. It’s one of the higher-end bang for your buck options for us and that’s the main reason we chose it, and we are really happy with it.”

“We wanted a robust camera that produces clean images with not a lot of noise, that can go down to low millisecond exposure without losing too much brightness. The Prime BSI Express does it all and is one of the better pieces of equipment that we work with as far as imaging goes. Very reliable, we like how it talks to the Nikon software and our in-house software for exporting data, the software engineers are very happy with the ease of use with this camera.”