Page Type: Customer Story

High-Speed Voltage ImagingCustomer Stories

Prof. Xue Han and Dr. Eric Lowet

Biomedical Engineering Department, College of Engineering, Boston University

Background

The lab of Prof. Xue Han uses optical methods to probe neural circuits in awake animals. With a background in optogenetics, Prof. Han has developed many key molecular tools for optogenetics, including SomArchon, an archaerhodopsin-based voltage sensor. With a combination of voltage imaging using SomArchon, calcium imaging, and optogenetics, the Han Lab can optically interface with neural circuits.

Prof. Han told us about her experience with voltage imaging, “By using genetically encoded voltage sensors (GEVIs) we can record from many neurons in vivo, with their genetic identity and morphology. We can also observe sub-threshold dynamics separate from the firing of action potentials, we can probe the dynamics of individual neurons or a whole neuronal population. We can really see the voltage inputs now, which we haven’t been able to see over the past century.”

Through voltage imaging, Prof. Xue Han and postdoc Dr. Eric Lowet can investigate functional behavior from neural samples.

Figure 1: Voltage Imaging with the Kinetix sCMOS from the Han Lab. The top image shows voltage data from a single neuron at the center of the Kinetix field of view, imaging the mouse visual cortex at 500 Hz, 40x mag. The three bottom traces show visual cortex neurons L1/2 imaged at 5 kHz; each trace below magnified from the orange box above. Data courtesy of Dr. Eric Lowet, Han Lab.

Challenge

The challenge of voltage imaging is speed, as described by Prof. Han, “How can you image a large area at high speeds? For voltage imaging we want kilohertz, 10s of kilohertz. A single action potential has a duration of one or two milliseconds, we are not going to be happy to record at one kilohertz, that gives us one data point for a millisecond event, and we may even miss it. The key challenge here is to image a large field of view at super high speed, as fast as the camera can go, as high as the computer can handle.”

At such high imaging speeds very low exposure times are necessary, resulting in low signal levels. As well as having a large field of view and extreme imaging speeds, the camera is also required to be highly sensitive with low noise levels, in order to record as much relevant data as possible over short time-frames of activity in neural samples.

We can see high signal-to-noise voltage from hundreds of neurons at once, the Kinetix is the first camera that was able to do this. This will definitely be a game changer in the voltage imaging field.

Solution

The Kinetix features the extreme speeds, large-format sensor, and high sensitivity required for voltage imaging, and removes the camera as a bottleneck for the imaging system. With the 8-bit Speed mode, the Kinetix can record at 500 Hz across all 10 megapixels, with far higher speeds at smaller regions, easily into the kilohertz and beyond.

Dr. Eric Lowet described a recent experiment with the Kinetix for voltage imaging, “We are imaging the visual cortex of an active, awake mouse at 40x, using the full field of the Kinetix. We can focus our laser point on a single neuron and see high signal-to-noise voltage spikes, which showed us that this 8-bit Speed mode does work, and I think this is awesome.”

“The most surprising thing for us was that we are able to record full field at 500 Hz and we were able to see single spikes and good signal to noise membrane voltage of neurons, which is very promising. Compared to other cameras we tested [the Kinetix] is the first camera that was able to do this, and with high quality.”

“We are able to record many hundreds of neurons at once or record few neurons at very, very high sampling rates. This opens up a lot of new opportunities, recording from many neurons will definitely be a game-changer in the field.”

Whole Tissue Calcium ImagingCustomer Stories

Dr. Marcel Hörning

Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Germany

Background

Dr. Marcel Hörning is a physicist and bioengineer, the Principal Investigator of the Biobased Materials Group, led by Prof. Ingrid Weiss at the University of Stuttgart. Dr. Hörning recently obtained funding from the DFG for research into electro-mechanical wave formations in cardiac tissue.

Dr. Hörning described his recent work, “We have a model cardiac system involving re-engineered ex vivo primary tissue cultures of the heart, we grow these cardiac tissues in the lab and observe the electro-mechanical waves across the tissue. We have action potentials, calcium signaling, and mechanical contraction, all synchronized and in patterns such as spiral waves and alternans.”

“We found a simple method using Fourier transformation to visualize these patterns in real-time, with this Fourier transformation imaging (FFI) we can identify complex patterns of alternans using calcium imaging, membrane potential, and contraction patterns.”

Figure 1: Spiral calcium waves across a piece of in vitro cardiac tissue, taken with the Kinetix. The image shows a 1.5 cm diameter piece of tissue, imaged with a 2x lens and background (minimum intensity) subtraction. The grey section contains no cells. Recorded by Julia Erhardt.

Challenge

Imaging functional activity over a large piece of tissue requires a camera with a high spatial and temporal resolution, high speed for functional activity recordings (calcium and membrane potential), and high spatial resolution for imaging morphology at a cellular level within the tissues.

Dr. Hörning explained his imaging challenges, “We want to image over a large field of view at a 2x magnification with sub-cellular resolution. We also need to capture at a high speed in order to resolve the signals. Essentially, we need a combination of high spatial and temporal resolution in order to detect the alternans patterns with FFI.”

By obtaining high-quality structural and functional information from the cardiac tissue models, Dr. Hörning works to improve the model and increase the biological relevance to the in vivo situation.

We can see high signal-to-noise voltage from hundreds of neurons at once, the Kinetix is the first camera that was able to do this. This will definitely be a game changer in the voltage imaging field.

Solution

The Kinetix sCMOS is a powerful solution for both large format and high-speed imaging. The large field of view (29 mm diagonal) and small pixel (6.5 μm) allow for high-resolution imaging across a large sample, while the high acquisition speeds (500 fps across the full-frame) allow for easy capture of fast, dynamic events such as calcium waves and other functional cellular activity.

Dr. Hörning told us about his experience with the Kinetix, “We got support from Teledyne Photometrics the whole time, it all worked well and I didn’t have any trouble. I’m happy it works so smoothly and I was able to get results.”

“We use Sensitivity mode but would also be interested in optimizing the Speed mode as this 8-bit mode decreases the data file size. Overall, the Kinetix is a very impressive camera and meets the needs of my research.”

Sub-cellular Oblique Plane MicroscopyCustomer Stories

Dr. James Manton

MRC Laboratory of Molecular Biology, University of Cambridge, UK

Background

Dr. James Manton develops new microscopy techniques in the MRC Laboratory of Molecular Biology, Cambridge. A recent development project involves an oblique plane microscope (OPM) with a Mr. Snouty solid-immersion objective, which combines the speed and efficient illumination of light-sheet with the ease of use of a traditional inverted microscope. Dr. Manton aims to do multicolor imaging at higher speeds than existing light-sheet systems allow while maintaining the standard sample presentation of an inverted microscope.

This light sheet imaging system has been designed for a wide variety of samples, including highly photosensitive Dictyostelium slime molds, T-cells, mouse fibroblasts, and other samples that require the gentle illumination of light-sheet.

Dr. Manton told us about their new light-sheet imaging system, “Because we are using a galvo mirror rather than moving the stage through the 3D acquisition, we can go five, ten times faster. This is particularly nice because a lot of the processes we want to look at, e.g., Dictyostelium are extremely fast. On our traditional light-sheet microscope we can acquire one volume a second, but here we are aiming for up to ten.”

Figure 1: Sub-cellular resolution OPM imaging using the Prime BSI Express sCMOS. The image shows a cyan stain for mitochondria within a single cell.

Challenge

As the OPM will involve imaging volumes at high speed, a suitably high-speed camera is needed to capture all the light coming from the microscope in real-time. This kind of high-speed imaging requires low exposure times, combined with the low illumination level of light-sheet and the highly photosensitive samples, a highly sensitive camera is also needed to maximize the use of the photon budget.

In addition, this imaging system has a fixed magnification (55.7x), meaning a specific pixel size is required in order for optimal Nyquist sampling and high-resolution imaging.

Dr. Manton also mentioned some issues with previous sCMOS cameras, “we had issues with gain variation on previous sCMOS solutions — when we used an ROI to look at a single cell the non-uniformity in the gain became really clear at these low signal levels. This also made deconvolution trickier.”

A new sCMOS imaging solution would need to have both low noise levels and no patterns or artifacts on the sensor, in order to have high sensitivity and reliable post-processing.

We can see high signal-to-noise voltage from hundreds of neurons at once, the Kinetix is the first camera that was able to do this. This will definitely be a game changer in the voltage imaging field.

Solution

The Prime BSI Express is a flexible, reliable and powerful sCMOS camera, featuring high speed combined with high sensitivity.

Dr. Manton described his experience with the Prime BSI Express “We knew we wanted an sCMOS-style camera because of their speed, pixel size, and sensitivity… The gain variation appears to be a lot flatter on the Prime BSI Express compared to typical sCMOS, resulting in superior raw image quality.”

“We have two [Prime BSI Express] on separate light paths split by a dichroic mirror. The system is run with MicroManager and the Photometrics device adaptor works just as expected, with nice, flat images at low light levels.”

The Prime BSI Express has a clean, pattern-free bias and low noise CMS mode. Combining this with the near-perfect 95% quantum efficiency results in a highly sensitive camera that can run at 95 fps across the full sensor, allowing for high-speed imaging across large volumes.

Multifocus and Snouty Light Sheet MicroscopyCustomer Stories

Dr. Florian Ströhl

Department of Physics and Technology, The Arctic University of Norway

Background

Dr. Florian Ströhl leads a group of physicists to develop advanced microscopy systems, including a new light-sheet imaging system. This custom light-sheet system involves a single-objective oblique plane microscopy (OPM) approach using the Snouty lens, as well as additional capabilities for 3D imaging.

Dr. Ströhl explained what his imaging system can do, “Snouty scans through the sample and produces opticallys ectioned images. There is a technique called multifocus microscopy that uses multiple focal planes at the same time, and we can optically section all of these planes as well. This allows us to record a full volume in a single camera frame.”

This dynamic imaging system allows for 3D imaging at high speed and with a high resolution, once paired with a suitable camera. While intended for use on a range of different samples, Dr. Ströhl described an example of the kind of sample his group intended to image, “We are using this system to image human cardiomyocytes (heart cells) that are grown on flexible posts. The cells attach to these posts and beat, this beating becomes directional and they align, resulting in heart muscle that is in more of an adult state.”

Figure 1: Fixed BPAE cells imaged with the Prime BSI Express on the multi-focus system. The image shows actin labeled with phalloidin.

Challenge

These cardiomyocyte samples are highly challenging to image, as Dr. Ströhl mentioned, “The problem is that we are trying to image this large lump of tissue which is beating very fast, so we need to do 3D imaging at a high speed. The whole tissue is constantly moving.”

This requires an imaging system that is fast enough to image multiple 3D volumes a second while retaining high resolution. This system is also working with a low signal level, which means a suitable camera must have low noise levels while also retaining a high speed.

We can see high signal-to-noise voltage from hundreds of neurons at once, the Kinetix is the first camera that was able to do this. This will definitely be a game changer in the voltage imaging field.

Solution

The Prime BSI Express is a highly flexible camera that features high imaging speeds, high sensitivity and high resolution across a large sCMOS field of view.

Dr. Ströhl uses two Prime BSI Express cameras for his advanced imaging system, due to the ability to easily swap cameras between systems depending on the sample need, such as having one camera on a 3D Snouty system and another doing high-speed imaging, or having both cameras on one system for simultaneous multichannel imaging.

When asked about the performance of the Prime BSI Express cameras, Dr. Ströhl said, “Now we have a lot more pixels, which translates to a lot more voxels… The [Prime] BSI Express is the whole package, with flexibility, high speed, high sensitivity, and many pixels.”

“The USB 3.1 Gen2 is actually really nice to have as well. The camera setup was smooth… I have tested the cameras and they worked as intended, the speed, FOV, and sensitivity were exactly to spec.”

3D Axially Swept Light-Sheet MicroscopyCustomer Stories

Dr. Stephan Daetwyler, Prof. Reto Fiolka

Fiolka Lab, UT Southwestern Medical Center, Dallas, TX

Background

The lab of Prof. Reto Fiolka develops new, transformative technologies to image across scales: from sub-cellular imaging to imaging of whole organs. In the Fiolka Lab, Dr. Stephan Daetwyler is a postdoctoral researcher who builds, programs and applies advanced light-sheet microscopy systems to image dynamic processes in live biological organisms.

Dr. Daetwyler told us more of his work, “Amongst other innovations, the Fiolka lab has been a pioneer in a technique known as axially swept light-sheet microscopy or ASLM. ASLM excels in high-resolution 3D imaging of subcellular structures and signaling over extended volumes. I apply ASLM to study the dynamic behavior of single cells in developing zebrafish embryos. Interestingly, the development of vasculature is a highly dynamic process, and many thin, fine sprouts are formed. To reveal the behavior of these subcellular structures in vivo, ASLM is ideal.”

Figure 1: In axially swept light-sheet microscopy (ASLM), a tightly focused Gaussian light-sheet is swept through the sample in its propagation direction. Thereby, the acquisition of the signal by the camera chip is synchronized with this sweep by controlling the rolling shutter of the sCMOS camera using Programmable Scan Mode: only pixel rows on the chip are active that correspond to the thin beam waist. Therefore, only the thin beam waist of the light-sheet contributes to the final image, resulting in high axial resolution and optical sectioning.

Challenge

ASLM requires a fast and flexible scientific camera to best synchronize the camera readout with the sweep of the light sheet. Therefore, light-sheet techniques such as ASLM require advanced camera modes that can control the camera readout speed and direction at a high timing accuracy.

Dr. Daetwyler discussed this need, “In ASLM we need to sweep this narrowly-focused beam across the camera chip and co-ordinate acquisition with the sweep of the light sheet.”

In addition, due to sweeping light sheet illumination, parts of the sample are only illuminated for a short time compared to conventional light-sheet acquisition. This results in comparably low levels of signal, meaning a sensitive camera with a high quantum efficiency is required.

We can see high signal-to-noise voltage from hundreds of neurons at once, the Kinetix is the first camera that was able to do this. This will definitely be a game changer in the voltage imaging field.
Figure 2: Maximum intensity projection of the endothelial cells forming the intersegmental vessels (ISV) in zebrafish at 1.5 dpf, labeled with the vascular marker Tg(kdrl:Hsa.HRAS-mCherry). The color encodes the depth in the z-direction of the 3D data, deconvolved with double-blind deconvolution. Data acquired on a Prime BSI Express sCMOS camera.

Solution

The Prime BSI Express is a powerful, flexible solution for 3D ASLM imaging. It allows for sub-cellular resolutions across a large sample. Furthermore, the presence of a 16-bit HDR mode with a low-noise CMS mode allows for high sensitivity imaging at a high dynamic range.

Importantly, the Prime BSI Express also features Programmable Scan Mode (PSM), where the direction and speed of camera readout can be controlled. This is ideal for techniques such as ASLM where the beam sweep had to be synchronized with camera readout.

Dr. Daetwyler told us of his experience with the Prime BSI Express, “I developed imaging software for the Prime BSI Express and Programmable Scan Mode using Python and PyVCAM. The setup was as easy as it can get for scientific cameras, the Prime BSI Express is a reliable camera that does what I want it to do!”

Neuronal Single-Molecule TIRFCustomer Stories

Mr. Marco Schnieder, Prof. Jürgen Klingauf

Institute of Medical Physics and Biophysics, University of Münster, Germany

Background

Marco Schnieder is a PhD student in the group of Prof. Klingauf, whose research focuses on neuroscience, mainly the physiology of synaptic transmission, and the mechanisms of synaptic vesicle recycling, in particular endocytosis.

The cellular and protein machinery involved in synaptic transmission is investigated in the Klingauf Lab using extremely sensitive high- and super-resolution imaging techniques on cultures of living neurons. Fluorescence microscopy is combined with electrophysiology, where all neurons are excited by electrodes simultaneously in order to trigger action potentials, at which point synaptic transmission of vesicles between neurons can be observed.

Mr. Schnieder uses pH-sensitive probes such as pHluorin to track synaptic vesicles at both the pre- and post-synaptic neurons, as well as observing protein machinery involved in synaptic vesicle fusion, such as the SNARE protein complexes. This is all done with live neuronal cultures and imaged with TIRF.

Figure 1: Neuronal cells imaged using the Prime 95B sCMOS camera. Image is part of a stack where stimulation is applied, two regions of interest are shown as part of the full stack, where endocytosis and synaptic vesicles can be observed in motion through the cell.

Challenge

The first challenge was the sample size, as described by Mr. Schnieder, “The vesicles and endocytosis machinery are so small, far beyond the classical diffraction limit at ~40 nm. Nevertheless, we would like to observe them in living cells. While electron microscopy is suitable for high resolution, it is not suitable for imaging living cells, this is why we use fluorescence microscopy.”

As well as requiring high spatial resolution, this application also benefits from high temporal resolution, as the events occurred on a second/sub-second scale. Other techniques such as STORM were available but were not used due to the speed limitations.

Sensitivity is also vital for quantitative research; one aim is to determine the number of pHluorin molecules within vesicles by the fluorescence intensity. The signal flux is very small, meaning a highly sensitive camera with high signal collection but low read noise is necessary.

We can see high signal-to-noise voltage from hundreds of neurons at once, the Kinetix is the first camera that was able to do this. This will definitely be a game changer in the voltage imaging field.

Solution

The Prime 95B sCMOS is an ideal solution for this application, as outlined by Mr. Schnieder, “The Prime 95B is really advantageous because it’s a CMOS, so it doesn’t enhance the noise of the measurement as EMCCDs do. Furthermore, the 95% quantum efficiency is really good for these dense culture measurements as our change in fluorescence intensity is low… there is a positive difference in the quality of the data compared to our other CMOS.”

The 95% QE, large pixel, and low read noise of the Prie 95B result in EMCCD-like levels of sensitivity, while also maintaining CMOS advantages of a larger field of view and higher speed. With the low noise, intensity changes can be identified in order to perform quantitative analysis while maintaining high image quality.

Regarding the pHluorin imaging, the lab further increased its confidence in being able to resolve numbers of present and signal-contributing pHluorin molecules during exo- and endocytotic events. This enables a more reliable and more quantitative analysis.

High-Speed Single Molecule Light SheetCustomer Stories

Dr Aleks Ponjavic

School of Physics & Astronomy/Food Science and Nutrition, University of Leeds, UK

Background

The lab of Dr. Aleks Ponjavic develops fluorescence microscopy techniques in order to study live-cell samples, including particularly mechanically delicate and photosensitive samples such as T cells. These live samples undergo complex processes and require high-speed imaging, sensitivity, and nanoscale resolution in order to determine the behavior of individual proteins within the cells.

To this end, Dr. Ponjavic uses a single-objective oblique-plane (OPM) light sheet imaging system that is tuned for high-speed single-molecule imaging of live T cells at the nanoscale using two different experimental methods, as explained, “There are two methods for this project, one is to just image single molecules as quickly as possible with the goal of approaching live localization microscopy, and the second is to do high-sensitivity flow cytometry, flowing cells through a light sheet and then quantifying receptors on these cells, also as quickly as possible.”

This results in a high-throughput imaging system that images T cells, intracellular proteins, and cell-cell calcium signaling at the nanoscale, making use of super-resolution probes such as the spontaneously photo-switching fluorophores.

Figure 1: Image of the Kinetix sCMOS set up on the OPM light-sheet imaging system, optimized for high-speed single-molecule imaging of live cells.

Challenge

The highly sensitive nature of the sample and the complex design of the imaging system bring a number of challenges.

Firstly, T cells are very sensitive and undergo unpredictable changes when adhered to a surface, requiring the preparation of gels in order to embed the cells in suspension. This in turn cuts down on movement so that samples can be more easily located and imaged. These cells are also very sensitive to light, requiring the characteristic low light dose of light-sheet and a highly sensitive detector.

Secondly, the system needs to operate at very high speeds while retaining sensitivity. Dr. Ponjavic discussed a previous camera solution for this system, “We have an intensified high-speed camera but there are issues with intensifiers such as aberrations and low quantum yield, I wanted a more robust setup that was more sensitive at high speeds.” A camera that can operate at very high speeds with a high quantum efficiency (QE) and low noise levels is therefore required.

Lastly, this super-resolution single-molecule localization light sheet system requires a flexible camera solution, due to the galvo-based OPM descanning of the light sheet through the gel, the need for high-speed imaging, the large number of frames needed for the desired nanoscale resolution, the low signal levels, and more.

We can see high signal-to-noise voltage from hundreds of neurons at once, the Kinetix is the first camera that was able to do this. This will definitely be a game changer in the voltage imaging field.

Solution

The Kinetix sCMOS is an ideal solution for this imaging system, with a unique combination of 95% peak QE, sub-electron read noise levels, and extremely high speeds. With both speed and sensitivity available, there is no longer a need to compromise.


This combination of factors allows the Kinetix to be highly flexible, able to image at high speeds with low noise levels over a large field of view, well-suited to dynamic live samples, and rapid techniques such as flow cytometry. Dr. Ponjavic told us about his experience with the Kinetix, “All I wanted was high speed without making a compromise on sensitivity, and this was it.”

iSCAT and TIRFCustomer Stories

Dr. Francesco Reina, Prof. Christian Eggeling

Institute for Applied Optics and Biophysics, Faculty of Physics and Astronomy, University of Jena, Germany

Background

Dr. Francesco Reina is a postdoc in the lab of Prof. Christian Eggeling, taking part in quantitative imaging research. The lab of Prof. Eggeling is known for the application of fluorescence correlation spectroscopy (FCS) and STED-FCS to life sciences research, and Dr. Reina is building an imaging system that combines imaging techniques involving single-molecule tracking, namely interferometric scattering (iSCAT) and total internal reflection fluorescence (TIRF) microscopy.

Dr. Reina further explained his research, “These techniques are able to give you information on the single-molecule level at very fast frame rates, especially in the case of iSCAT. We can obtain information complementary to the FCS measurements, looking at the diffusion of molecules on surfaces such as cell lipid membranes and dynamic live cells. By using iSCAT and TIRF simultaneously we hope to achieve high levels of fluorescence specificity at a high sampling rate.”

The combination of iSCAT and TIRF allows for detailed, quantitative interrogation of living cells at the single-molecule level, and this demanding technique requires a flexible, powerful camera.

Figure 1: iSCAT images taken using the Prime BSI Express sCMOS. The larger image shows gold nanoparticle-tagged lipids diffusing on a supported lipid bilayer across the full FOV of the Prime BSI Express. The smaller magnified insert shows a 40 nm gold nanoparticle on a glass surface.

Challenge

Due to this system combining both TIRF and iSCAT, the imaging needs of both techniques need to be met by a single camera. TIRF requires high sensitivity, high spatial resolution, and high speeds (typically at high magnifications), and iSCAT requires even higher speeds still, in order to obtain adequate statistics of fast-moving molecules.

Dr. Reina told us about the other challenges of his work, “We focus on single cells or portions of single cells, we really want to go down to the single-molecule level. Ideally, we want to run at over 100 fps across the full camera sensor, and eventually split the camera sensor in half in order to perform TIRF and iSCAT simultaneously on a single camera sensor.”

“A big issue with some components of this imaging system is integration into a single hardware control software, previous camera solutions did not integrate well into our preferred software, ImSwitch.”

We can see high signal-to-noise voltage from hundreds of neurons at once, the Kinetix is the first camera that was able to do this. This will definitely be a game changer in the voltage imaging field.

Solution

The Prime BSI Express is a highly flexible and powerful back-illuminated sCMOS camera that delivers high sensitivity at a high speed, across the entire sensor.
Dr Reina described his experience with the Prime BSI Express, “The [Prime BSI Express] gives us flexibility, we can go to a very small homogeneous field of view and acquire as fast as possible, or increase the region to find new samples, without difficulty, and we can repeat this for the whole experiment.”

“With [Teledyne] Photometrics we have been able to integrate all our components into our external hardware control software ImSwitch that we are very fond of using, we use the PyVCAM wrapper and it all works very well, we are very happy about that.”

“Setting up the [Prime BSI Express] was very straightforward, it works great out of the box. [Teledyne] Photometrics’ customer support was very available to us and very quick.”

Functional Light Sheet Calcium ImagingCustomer Stories

Dr. Asaph Zylbertal, Dr. Issac Bianco

Department of Neuroscience, Physiology & Pharmacology, University College London, UK

Background

The Bianco Lab at UCL aims to understand the logic of brain circuits that control behavior. Using two‑photon and light-sheet microscopy with small, optically transparent larval zebrafish expressing genetically encoded calcium indicators (GECIs), researchers such as Dr. Asaph Zylbertal record activity at single-cell resolution throughout the brain.

Dr. Zylbertal explained more about his work, “I am using our light-sheet microscope to look at tens of thousands of neurons expressing GECIs across the whole zebrafish brain, to see activity and estimate what each individual neuron in the fish brain is doing at any given time.”

“After acquiring the raw data, we use a segmentation algorithm to find neurons in each imaging plane, we then deconvolve the calcium signal from these neurons to estimate spikes, this gives us a representation of activity across the brain.”

While some light sheet experiments look at the anatomy of the zebrafish, the aim of Dr. Zylbertal is to observe functional activity within the zebrafish brain.

Figure 1: Video stack of the brain of a zebrafish larva, taken with the Kinetix sCMOS. Each image is of a different z-plane through the sample, which is expressing the GCaMP6f calcium indicator under the elavl3 promotor (elavl:GCaMP6f, Wolf et al. 2017). Dorsal view, anterior side is up, the view is approx. 420 x 710 x 150 μm3.

Challenge

Calcium imaging requires high speeds in order to capture the dynamic signaling between neurons. However, to monitor populations of cells throughout the brain, it is also necessary to capture large imaging volumes. Thus, the detector needs to be capable of both high speed and a large field of view (FOV) for this high‑throughput application.

Dr. Zylbertal told us about the experimental challenges, “When we are using fast indicators, we would like to be able to image as many volumes as possible over time, all with high resolution so we can integrate lots of pixels in order to get a higher signal to noise.”

This application needs a combination of speed and sensitivity, all across a large FOV.

We can see high signal-to-noise voltage from hundreds of neurons at once, the Kinetix is the first camera that was able to do this. This will definitely be a game changer in the voltage imaging field.

Solution

The Kinetix is an ideal solution for this application, featuring extremely high speeds (500 fps across the full frame), high sensitivity (<1 e- read noise and 95% QE signal collection), and a huge FOV (29 mm diagonal), all in one camera.

The Kinetix Speed Mode operates at 500 Hz across the full 10 megapixel sensor, enabling the Kinetix to capture rapid calcium signals from neurons in real-time. Combining this with the high sensitivity and large FOV allows for volumetric functional imaging across the whole zebrafish brain while retaining high resolution to resolve individual neurons.

Dr. Zylbertal outlined his experience with the Kinetix, “The advanced triggering is very important. We synchronize the camera with the light sheet and use pseudo-global shutter”

“With the Kinetix, we are imaging at a larger FOV and a higher speed than previously, which is what we need for this application. Experiments can last for hours and we want to react to the images as they come. With the Kinetix, we have the higher signal to noise ratio needed to extract activity from the neurons.”

Reference

Wolf, S., Dubreuil, A.M., Bertoni, T. et al. (2017) Sensorimotor computation underlying phototaxis in
zebrafish. Nature Communications 8, 651. https://doi.org/10.1038/s41467-017-00310-3

High-Throughput Spinning Disk ConfocalCustomer Stories

Dr. Tom Lummen

D-BSSE Single Cell Facility, Department of Biosystems, ETH Zürich, Switzerland

Background

Dr. Tom Lummen is a microscopy engineer at ETH Zürich University, and spoke to us about the imaging facility he works with, “I’m part of the microscopy team that operates the imaging core facility, we provide 20 high-end automated microscopes for the imaging needs of the users of our department… we are always scouting for new or expanded functionalities for these systems.”

“We have a very broad array of imaging applications, these range from small subcellular features in live cells that have to be visualized on short timescales, all the way to the development of very big 3D organoids or tissue cultures that need to be monitored for weeks. The name of the game is flexibility and modularity!”

“Spinning disk confocal microscopy is becoming more in demand, as data acquisition needs are moving towards getting single-cell parameters but with very good statistics and/or time resolution. Our inverted spinning disk systems are geared towards live-cell observations for dynamic samples, but we have also seen an increase in demand for high 3D spatial throughput.”

Figure 1: Five color imaging on fixed Fucci4 HeLa cells. Images are sum intensity projections taken with a 100x, NA 1.45 oil immersion objective and a 25mm Kinetix FOV. Recorded on a Nikon Ti2 microscope equipped with a CrestOptics X-light v3 spinning disk confocal scanner, a Lumencor Celesta light source, and a Teledyne Photometrics Kinetix sCMOS camera. Fucci4 HeLa cells developed by Bajar et al. (2016). Image credit: Javier Casares, D-BSSE, ETH Zurich.

Challenge

Spinning disk microscopy allows for fast imaging of live samples, but this technique can involve several challenges. Dr. Lummen described some of these challenges he has encountered, “Within microscopy there is always a trade-off when setting up a system, you can’t have all the specs at the same time. What we try to go for are dedicated systems, but we try to keep them as tuneable as possible.”

“We identified the need for a dedicated inverted spinning disk system that is geared towards high throughput observation, whether spatial or temporal. This is when we started to explore what camera technologies were out there.”

A suitable camera for high-throughput live-cell imaging at high speeds (to pair up with the speed of a spinning disk) would need to be highly flexible, have a large field of view (FOV), small pixels for high sampling resolution, high readout speeds and high sensitivity to detect the dynamics of moving samples.

We can see high signal-to-noise voltage from hundreds of neurons at once, the Kinetix is the first camera that was able to do this. This will definitely be a game changer in the voltage imaging field.
Figure 2: Four-color dual camera imaging of a fixed mouse brain slice. Image shows a maximum intensity projection composite of 8×9 acquisition tiles taken with a 20x, NA 0.75 air objective with a 25mm Kinetix FOV and a 15% tile overlap. Recorded on a Nikon Ti2 microscope equipped with a CrestOptics X-light v3 spinning disk confocal scanner, dual Teledyne Photometrics Kinetix sCMOS cameras, and a Lumencor Celesta light source.

Solution

The Kinetix sCMOS was identified as a well-fitting solution for such dynamic live-cell spinning disk applications. Dr. Lummen shared his experiences with the Kinetix, “Imaging live-cell dynamics is one of the main reasons why the Kinetix is the right product for us, because the large field of view lets us image more cells in the same time span, which can proportionally increase the data throughput that we
can offer to our users.”

“We generally optimize our imaging setups on a per-experiment basis and tune for field-of-view, speed, resolution, sensitivity, or whichever trade-off thereof is most appropriate. The flexibility the Kinetix offers means that we’re not camera-restricted to one or two types of experiments; in principle, it can be configured for any of these optimized regimes.”

“The combination of a very large FOV, the small pixel size, and the high-speed specs of the Kinetix was really tempting to explore and we chose it for our new flexible high throughput system. It gives us a big boost to the FOV so we can get more cells per timepoint, or fewer tiles per composite large image. The combination with the large FOV of both the Nikon Ti2 and the CrestOptics X-Light v3 spinning disk confocal scanner let us configure a dual-camera large FOV setup. This enables us to further duplicate the data throughput or to perform true concurrent two-channel live-cell imaging in dynamic samples. In addition, with the Lumencor Celesta, this setup can do NIR imaging, adding an extra spectral channel we
can use.”

“For versatile systems, we often work with Nikon as the software is quite modular and open, and we can script more complex and conditional experiments…With the Kinetix directly coupled to the Nikon Ti2 we don’t see any hard vignetting at the corners of the chip and we can utilize the full 29 mm field of view, which is a big increase compared to our previous generation 19 mm sCMOS. The high sensitivity allows to run the disk at maximum speed, good for fluorescence observation of live cells.”

Reference

Bajar B., Lam A., Badiee R., Oh Y-H,, Chu J., Zhou X., Kim N., Kim B., Chung M., Yablonovitch A.,
Cruz B., Kulalert K., Tao J., Meyer T., Su X-D and Lin M (2016) Fluorescent indicators for
simultaneous reporting of all four cell cycle phases, Nat Methods 13, 993–996 https://
doi.org/10.1038/nmeth.4045