Page Type: Customer Story

PaleoluminescenceCustomer Stories

Dr. Pierre Gueriau, SNSF postdoctoral researcher

Anom Lab, Institute of Earth Sciences, University of Lausanne

Background

Paleontologists such as Dr. Pierre Gueriau rely on the accurate observation and interpretation of fossil anatomy in order to elucidate the origin and evolutionary history of life. For this process, imaging techniques are absolutely essential, whether it is simply illustrating findings using optical photography/microscopy, or extracting information hidden to the human eye using advanced methods. The advent of x-ray computed microtomography (µCT) has revolutionized the field for 3D preserved fossils. In turn, photography under UV light or blue/green wavelengths has revealed a wide range of details completely invisible under visible light in flat fossils.

Figure 1: Images of a ~150-million-year-old shrimp fossil from Solnhofen Plattenkalk, Germany. The top image shows an optical photograph, while the bottom shows a false-color composite image composed of UV luminescence and reflection, revealing many parts faked with paint (black). Image was taken with a Prime 95B 25mm coupled to a multispectral macro lens.

Challenge

Luminescent signals can be extremely faint, requiring long (up to several minutes) acquisition times. Rather than bioluminescence, Dr. Gueriau’s application is closer to paleoluminescence as it focuses on fossils. As Dr. Gueriau mentions, “There are other visible and near-infrared wavelengths that could provide interesting additional contrasts. Therefore, paleontologists need a camera that is extremely sensitive from the UV to the near IR in order to exploit all possible sources of contrast.”

The Prime 95B offers a unique combination of extreme sensitivity, high speed and low noise performance from the UV to the near-infrared.

Solution

Dr. Gueriau is now using a Prime 95B 25 mm sCMOS camera. Dr. Gueriau spoke about his experience with the camera: “The Prime 95B 25mm offers a unique combination of extreme sensitivity (up to 95% quantum efficiency), high speed, and low noise performance from the UV-A to the near IR.” The Prime 95B 25 mm also provides a large 25 mm field of view, essential for imaging a variety of fossil sizes at high spatial dynamics, revealing particular anatomical details of these fossils.

High Flux Plasma SpectroscopyCustomer Stories

Dr. Hennie van der Meiden

Gijs Akkermans

Dutch Institute for Fundamental Energy Research (DIFFER), Eindhoven, Netherlands

Background

With the transition to sustainable energy and new energy technologies, research into controlled nuclear fusion is being carried out. In a worldwide collaboration, the tokamak ITER is being developed and built in Cadarache, France. This is one of the first steps for realizing power plants that can generate clean and reliable energy for the future.

The principal design concept of ITER is a doughnut-shaped vessel where the fusion fuel, a hot plasma of hydrogen isotopes, is contained by high magnetic fields and fusion power can be produced at a plasma temperature of ~100 million °C. Fusion products like helium and impurities have to be exhausted from the core plasma which results in high power loads of 10 MW/m2 (continuous), peaking at over 1 GW/m2 on plasma-facing components (PFCs) inside the exhaust systems. To address this challenge, a linear plasma generator Magnum-PSI was built to study the plasma-wall interactions (Fig.1).

Figure 1: A side cutaway view of the linear plasma generator Magnum-PSI. The plasma (magenta-colored beam) exhausted by the plasma source (right side) is confined by the max 2.5 Tesla magnetic field (superconductive magnet (dark blue)) and deposited on a sample (power flux <40 MW/m2 continuously and transients >1GW/m2). Different diagnostics are used for measuring the plasma conditions.

PhD student Gijs Akkermans and Dr. Hennie van der Meiden are involved in the research with the Magnum- PSI in order to test different materials under different plasma conditions similar to those in ITER and beyond. As they told us, “The power load in some areas of the tokamak is too high and can damage the plasma-facing components”.

In their experiments, a continuous or pulsed beam of plasma is focused onto a sample. Different diagnostics are used to monitor plasma conditions such as temperature, density, and calorimetry. Among these, a spectrometer system is used for measuring the population of states of atoms and molecules by collecting the photons originating from these particles. Inside the spectrometer, these photons are separated by wavelength and imaged using a scientific camera.

Figure 2: The left figure shows a spectrum at middle fiber low-resolution setting. The right figure shows light from the plasma beam imaged on a fiber array and the spectra imaged with the Prime 95B. Each fiber corresponds to a certain plasma position of the beam, i.e. a radial profile of the beam is measured. The highest peak corresponds to a Balmer beta line (488 nm, n=4 > n=2).

Challenge

Cameras used for the spectroscopy need to be highly UV sensitive as the wavelength of interest is 340- 500 nm, with good signal-to-noise ratio for UV wavelengths.

For experiments with continuous plasma, different exposure times are needed depending on the intensity and wavelength of the signal, making the dynamic range important. The plasma pulses have a duration of 0.5 ms, and need to be sampled with time scales shorter than in 50 μs, making very fast shutter times desirable. In addition, both types of experiments need to capture the entire field of view without significant magnification, meaning that cameras with a large field of view are necessary.

The Prime 95B offers a unique combination of extreme sensitivity, high speed and low noise performance from the UV to the near-infrared.

Solution

The Prime 95B 25mm delivers on each of the challenges presented by spectroscopy and imaging of the radiated plasma photons, offering great quantum efficiency and low noise at UV wavelengths. As mentioned by Dr. Hennie van der Meiden “with the Prime 95B 25mm I can get a 10 μs exposure time with the fast mode, letting us image the induced plasma pulses and plasma fluctuations.”

The large sensor and wide field of view offered by the Prime 95B 25mm ensures that a large area can be imaged without having to compromise high speed and high resolution, all with very low levels of readout noise and dark current.

eSPIM Light SheetCustomer Stories

Prof. Bo Huang

Departments of Pharmaceutical Chemistry, Biochemistry and Biophysics; University of California, San Francisco (UCSF)

Background

Prof. Huang’s lab at UCSF aspires to generate a map of the entire proteome of endogenous proteins in human cells. Towards that goal they use and develop novel imaging modalities including eSPIM, a high numerical aperture epi-illumination form of selective plane illumination microscopy (SPIM). Using a galvanometer mirror to drive the position of an oblique light sheet, and a unique combination of re-imaging objectives angled to match the angle of the illumination sheet, they can image 34 slices per volume of 35x35x7 µm and collect 14 volumes per second. This allows for high-throughput volumetric imaging.

Figure 1: Live U2OS cells imaged by eSPIM. The cell nuclei are stained with Hoechst (blue), and two nuclear proteins are fluorescently labeled: FBL (green) and NPM1 (red). The image shows a maximum intensity projection of a small section of the field of view.

Challenge

Imaging with short exposure times is necessary to collect more than 450 frames per second and accurately combine them into 14 volumes, this demands the most out of the camera. Live-cell imaging at these rates will not allow increases of excitation laser power, as phototoxicity becomes an issue. Cameras with a limited field of view decrease the number of cells measured, requiring repeating experiments to generate sufficiently robust measurements. Prof. Huang told us they needed the camera to be most efficient at gathering signal, as well as have a high frame rate and appropriate pixel size for sampling the high resolution available in their optical design.

The Prime 95B offers a unique combination of extreme sensitivity, high speed and low noise performance from the UV to the near-infrared.

Solution

Prof. Huang told us they found the 95% quantum efficiency, low pattern/read noise, and 6.5 µm pixel of the Prime BSI gave them the light-gathering efficiency and sampling size to meet their needs. The angled light-sheet only fills half the sensor area allowing up to 125 frames per second. Prof Huang told us they found that the Prime BSI was substantially more robust than another, previously tested, camera from a different vendor that used the same sensor.

Super-Resolution Single Molecule LocalizationCustomer Stories

Assis. Prof. Matthew Lew

Department of Electrical & Systems Engineering, Washington University, Saint Louis

Background

The Lew lab is creating technology to image multidimensional trajectories of individual molecules in time and space, with one focus on understanding the structural dynamics of amyloid aggregation in diseases like Alzheimer’s.

In collaboration with Jan Bieschke, UCL, the lab has developed a single-molecule localization microscopy (SMLM) technique called TAB, transient amyloid binding, to image the structure of amyloid protein aggregates over periods of hours to days. Using a combination of engineered point spread functions (PSFs) such as Tri-spot and a new robust statistical estimation algorithm, they unmix molecular position and orientation within raw SMLM data, allowing them to follow translation and rotation (azimuthal and polar angles and wobble) of molecules over time.

Figure 1: Imaging amyloid bodies with SMLM using the Prime BSI. a) A super-resolution image of an image of an amyloid beta (Aβ1-42) fibril network reconstructed using SMLM with the Prime BSI. Colorbar: localization count. Scale: 1 μm. b) Photons detected per localization from the fibril in (a) using the Prime BSI and a control camera. The Prime BSI detects more localizations (165192) with a higher median brightness (948 photons) compared to a control camera (143414 localizations, median brightness of 799 photons) using the same exposure time. (c) A stabilized white light source with low brightness (approximately 60 photons per pixel per frame) tests whether the photons detected by each camera matches a statistical Poisson process. Black solid line marks the default cutoff p-value of 0.05; pixels with values greater than 0.05 are considered to match a Poisson distribution, whereas pixels with smaller values are significantly different from a Poisson distribution. The Prime BSI matches the Poisson process better compared to the control camera.

Challenge

Localizing individual molecules in super-resolution fluorescence microscopy is photon limited, with the precision improving by the inverse of the square root of the number of photons collected. Beyond standard SMLM, orientation-sensing SMLM (using the Tri-spot PSF), requires the fixed photon budget of a single molecule to be spread across a larger area of the detector.

With less than 1000 photons detected within each localization in TAB imaging, previous sCMOS cameras struggled to produce robust orientation resolved images due to suboptimal quantum efficiency and sensitivity. Further, pixel to pixel variability in electronic gain adds undesired noise to photon counts in each pixel, thereby polluting sensitive measurements of molecular orientation.

The Prime 95B offers a unique combination of extreme sensitivity, high speed and low noise performance from the UV to the near-infrared.

Solution

The Prime BSI’s high quantum efficiency, low read noise, and very low fixed pattern noise improves the precision of measuring molecular orientation using a variety of PSFs. The higher quantum efficiency enables more molecules to be detected and increases the photons collected from each localization. Reading out photon counts from the camera that match closely the Poisson statistical distribution ensures that the imaging achieves the best-possible shot noise limit. Having more localizations, each with better precision, creates super-resolved images with higher resolution and resolve heterogeneities in molecular orientations that could not be observed before.

Imaging Neurotransmitter ReleaseCustomer Stories

Prof. Kirill Volynski

Institute of Neurology, University College London

Background

The Volynski group are primarily interested in understanding cellular regulation of synaptic release of neurotransmitters which forms the basis of communication among neurons in the brain.

The Volynski group uses fluorescent probes, such as vesicular release sensor synaptophysin-pHluorin (sypHy). They visualize synaptic transmission at the level of single synapses in order to understand the basic mechanisms of transmitter release and also the cellular mechanisms of synaptopathies – paroxysmal neurological disorders caused by mutations in synaptic proteins. These disorders include some forms of epilepsy, migraine and ataxias.

The sypHy probe consists of a pH-sensitive GFP variant fused with the vesicular membrane protein synaptophysin. sypHy is widely used to image synaptic vesicle exo- and endocytosis. At rest, the sypHy fluorescence inside of a synaptic vesicle is quenched by the acidic vesicular pH. Upon synaptic vesicle exocytosis, caused by neuronal spiking, the sypHy probe becomes exposed to the extracellular medium with neutral pH, which leads to an increase in its fluorescence. Quantification of the fluorescence signal provides a means to monitor the response of individual synaptic boutons to different stimuli, thus providing a readout for the effects of disease-linked mutation effects on synaptic function.

Figure 1. Representative image of mouse cortical neurons expressing the sypHy reporter. A small region of interest (right panels corresponding to the white box on the original image) containing several synaptic boutons is shown before and after stimulation with 40 action potentials at 40Hz. Action-potential-evoked fluorescence responses in the labeled boutons are shown on the graph bottom right.

Challenge

Prof. Volynski was initially capturing images with a 512×512 EMCCD camera, however, imaging routines often consisted of several seconds which led to problems with drift in the temperature of the EMCCD sensor accompanied by a drift in the camera gain and ultimately the signal. Although this can be corrected posthoc it is non-trivial and complicates the analysis.

In addition to this, Prof. Volynski also wanted to image at higher speeds to capture the fluorescence associated with neurotransmitter release as well as synchronizing the camera with electrophysiological traces. However, when using the EMCCD, speed was limited to 30 fps and often frames were dropped randomly, which led to difficulties in the acquisitions and syncing.

The Prime 95B offers a unique combination of extreme sensitivity, high speed and low noise performance from the UV to the near-infrared.

Solution

The Volynski group is now using the Teledyne Photometrics Prime 95B to image synaptic transmission. Professor Volynski told us that “Stability and speed were the key issues here, and once we started using the Prime 95B the problem of instability totally disappeared. We switched to the Teledyne Photometrics Prime 95B and now have very stable responses, the camera works faster, and the SNR is very comparable to EMCCDs” He shared that, “We can acquire the same region of interest faster with the Prime 95B compared to the EMCCD.”

Professor Volynski went on to say that, “Now, we have a very robust protocol for detecting synaptic responses both at the level of single boutons and populations of synapses which provided us with a reliable and fast method for testing the synaptic effects of the disease-linked mutations”.

Spinning Disk ConfocalCustomer Stories

Dr. Emma Sigfridsson

Centre for Clinical Brain Sciences, University of Edinburgh, UK

Background

The group of Professor Seth Grant are interested in whole-brain synaptome mapping with an aim to understand more about how synaptic proteins are distributed and the role the molecular heterogeneity of synapses plays in health and brain disease.

There are more than 1000 genes encoding proteins in the excitatory postsynaptic density alone, and the group aims to catalog as many synaptic proteins and protein subtypes in both excitatory and inhibitory pre and post-synaptic densities.

The group mainly works with fixed mouse brain sections that contain genetically-encoded fluorescent synaptic proteins. So far, the group has categorized two proteins: PSD95 and SAP102, both found in excitatory postsynaptic densities. They have monitored the changes in the distribution of these proteins throughout different stages of development and aging in mice.

The group is also working to map the synaptome of models of disease as there is a link between mutations in the genes encoding proteins found in the postsynaptic density of excitatory synapses and neuropsychiatric and neurodevelopmental disorders in humans.

In addition, the group looks at human post-mortem tissue using immunohistochemistry to investigate synaptic proteins in the human brain and in the context of human neurodegenerative diseases such as Alzheimer’s.

Figure 1: Fixed neural sample from a mouse with enhanced green fluorescent protein (eGFP) tagged to postsynaptic density protein 95 (PSD-95), taken with the Prime BSI.

Challenge

The Grant group was previously using an Andor XD Revolution Spinning Disk system with an EMCCD camera. The main issue they faced was speed and throughput, partly due to the small imaging FOV and long exposure times needed for certain fluorophores.

The group found it necessary to image using a 100X objective in order to obtain maximum resolution. However, the brain slices are very large, around 16 mm x 8 mm in area, thus imaging at high magnification with limited FOV was very time-consuming. The FOV of the EMCCD is ~11 mm diagonal, however, the FOV is further restricted by using an additional 2x magnification changer to achieve Nyquist imaging with the large pixels of the EMCCD. With this small FOV, imaging whole coronal or sagittal sections of mouse brains can take more than 10 hours to image just two channels.

The group also found signal levels of certain fluorophores challenging. For example, they use the Venus YFP fluorophore to visualize an activity dependant protein, Arc. The combination of a weak fluorophore and Arcs low-level baseline expression posed an imaging challenge.

The Prime 95B offers a unique combination of extreme sensitivity, high speed and low noise performance from the UV to the near-infrared.

Solution

The group is now using dual Prime BSI cameras on a Nikon Ti2 and Spinning Disk system. Dr. Sigfridsson told us “The speed is much improved in the new system. Depending on the settings and the fluorophores, we are now running at around 2-3 hours for a sample compared to the previous 10 hours. When we use the second camera, we can do it in half the time again.”

Dr. Sigfridsson went on to say, “One of the selling points for the BSI was that we didn’t need to use the 2x magnification to reach Nyquist. The Prime BSI has the ideal pixel size to achieve Nyquist using the 100X objective, whilst also maximizing imaging area.”

Dr. Sigfridsson also stated that, “The biggest differences we have seen are speed and the increase in signal to noise. The EMCCD looks messier because you have additional noise from the multiplication process, which we no longer have with the Prime BSI.”

Super-Resolution Spinning Disk ConfocalCustomer Stories

Dr. Urs Ziegler
Dr. Joana Delgado Martins

Center for Microscopy and Image Analysis, University of Zurich, Switzerland

Background

Dr. Joana Delgado Martins is a research associate at the Center for Microscopy and Image Analysis, a facility at the University of Zurich, which is run by Dr. Urs Ziegler. The center has recently acquired a new Olympus SpinSR SoRa system along with two Prime BSI cameras.

Dr. Martins works closely alongside researchers to improve imaging in a variety of different applications. Dr. Martins explained, “We have a wide variety of users within the facility, so the systems are used for investigation of a host of different applications and sample types, ranging from whole organism research to live cell and tissue imaging.”

The facility required a system and camera combination that would provide the best imaging for a variety of users and applications. One group uses the facility to perform time-lapse imaging of zebrafish embryos. Through the expression of mCherry in the nuclei and a neuronal marker, it is possible to see the structure very nicely with spinning disk systems.

Figure 1: Imaging of zebrafish embryos. Left) Image of three zebrafish embryos taken with a large field of view over a software grid. Right) A single embryo labeled with nuclear (red) and neuronal (green) markers, scale bar 40 μm. Sample courtesy of Greta Ebnicher, laboratory of Prof. Darren Gilmour, University of Zurich.

Challenge

Dr. Martins told us that, prior to the purchase of the SpinSR and BSI system, the systems available were limited in their ability to permit live-cell imaging at high resolution. Often, users suffered low sensitivity and photodamage issues. Dr. Martins told us “We were not able to perform some live-cell experiments because we didn’t have enough signal and would have to expose cells to high light doses which frequently lead to photodamage.”

Dr. Martins went on to mention that “We wanted to find a system that could offer sensitivity and the best performance to a majority of users. Due to the broad range of applications, the biggest challenge was finding a system that suits most of the users most of the time for their specific applications.”

The Prime 95B offers a unique combination of extreme sensitivity, high speed and low noise performance from the UV to the near-infrared.

Solution

The Center for Microscopy and Image Analysis at the University of Zurich is now using the combination of the Olympus SpinSR super-resolution spinning disk system with dual Prime BSI cameras.

Dr. Martins told us, “For high-resolution investigations, the cameras and the SpinSR together have been really a great advantage in terms of what we can image. The system combines the high speed of spinning disk with the field of view and sensitivity of the Photometrics cameras. Together it allows us to do live imaging with weak samples, giving great performance… For all our experiments the cameras have been really nice in terms of field of view and sensitivity. We have a big field of view for fixed tissue or live-cell imaging expressing fluorescent probes.”

Dr. Martins added, “We have also seen a great improvement in photobleaching and photodamage. Partly because of the sensitivity of the camera but also because of the Real-Time Controller (RTC) implementation by Olympus which works well with the Prime BSI, allowing reductions in the exposure during live imaging which really helps.”

The facility is now also trying out other super-resolution approaches such as SRRF (Super-resolution radial fluctuations) where multiple images (typically 50-100) are required to derive a single super-resolved output image. The Prime BSI cameras are ideal to acquire such data sets with relatively low exposures and fast sampling allowing fast acquisition of super-resolved multicolor images. Dr. Martins said “By combining the two Prime BSI cameras with super-resolution spinning disk confocal microscopy we are able to acquire super-resolved multicolor images… Overall, we are very happy with the performance of the cameras and spinning disk combined.”

Optogenetics of Living BiofilmsCustomer Stories

Prof. Fan Jin
Dr. Shuai Yang

Shenzhen Institute of Advanced Technology (SIAT), Chinese Academy of Sciences, Shenzhen, China

Background

Bioprinting is an increasingly applied technique that facilitates the precise placement of biological substances – such as living cells, DNA, proteins, and growth factors – for the computer-aided fabrication of biologically
active materials with a prescribed organization.

Prof. Fan Jin’s group in SIAT is engaged in synthetic biology. One of their main research interests is the precise regulation of bacterial phenotype at the single-cell level and its application in synthetic biology. They have developed a new bioprinting strategy for dense bacterial communities with a prescribed organization on a substrate. Using a combination of optogenetic tools and microprojection, they could directly manipulate the cyclic dimeric guanosine monophosphate (c-di-GMP) levels in single Pseudomonas aeruginosa cells, which allows the living bacteria to form patterned biofilms following prescribed illumination.

Figure 1: Living P. aeruginosa printed onto biofilms in a prescribed pattern. Once these bacteria were activated through optogenetic manipulation, they spell out SIAT.

Challenge

The illumination intensity in bioprinting is low in order to reduce photobleaching, therefore it asks for a camera with high sensitivity to achieve a high signal-to-noise ratio. In the initial experiments of their previously published paper (Huang et al. 2018), they used an EMCCD camera for imaging. Due to the decay of EM gain, they found the signal-to-noise ratio decreased over time and they needed to calibrate their EMCCD regularly to ensure quantification accuracy. In addition, the small field of view and large pixel size of the EMCCD also presented problems.

The Prime 95B offers a unique combination of extreme sensitivity, high speed and low noise performance from the UV to the near-infrared.

Solution

Being attracted by high-quality images in the relevant literature, Prof Jin’s group decided to look into the Teledyne Photometrics Prime BSI back-illuminated sCMOS camera.

Dr. Shuai Yang, an assistant research associate from Prof. Jin’s lab, highly appreciates the extremely high quantum efficiency of Prime BSI. Without the influences of excess noise factor and gain decay, they could consistently get images with a high signal-to-noise ratio. Dr. Yang told us: “The most amazing thing is, although we have known about the high quantum efficiency of Prime BSI, we are still impressed by its perfect sensitivity in low light imaging”.

The Prime BSI has a 6.5 μm pixel, which results in a greater spatial resolution under 60x magnification compared with the previous EMCCD solution (13 μm pixel). At the same time, the larger field of view of the Prime BSI massively increased the number of cells in one acquisition, shortening the time of the entire experiment.

Additional Information

Yajia Huang, Aiguo Xia, Guang Yang and Fan Jin (2018) Bioprinting Living Biofilms through Optogenetic Manipulation, ACS Synth. Biol. 7, 1195−1200.

Lab Link: http://jin.isynbio.siat.ac.cn/wordpress/?p=464

FRET Voltage ImagingCustomer Stories

Prof. Jiulin Du

Dr. Jianan Liu
Dr. Rongwei Zhang

Institute of Neuroscience, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Chinese Academy of Sciences (CAS), Shanghai

Background

Prof. Jiulin Du’s group is engaged in understanding the mechanisms of how multiple sources of sensory information are integrated in our central nervous system in order to execute behaviors. They investigate neuronal activity through in vivo electrophysiological recording and optical imaging on the model organism zebrafish. However, such a recording strategy is invasive and difficult to scale up for high throughput recording, while other options such as calcium imaging ignore the subthreshold membrane potential fluctuations which also play an essential role in the development of neuronal computation. Because of this, they hope to develop a novel near-infrared (NIR)-excited voltage nanosensor that can directly monitor the changes in membrane potential.

Most optical voltage sensors need to be excited by high-intensity visible light, these wavelengths of light suffer from distortion and scattering in deep tissues, and prevent long-term imaging due to photobleaching. NIR excitation has deeper penetration and lower phototoxicity, permitting long-term recording of membrane potential fluctuations of neurons from deep brain areas.

Figure 1: High fidelity stable optical responses of voltage nanosensors in HEK293 cells.
A: Pseudocolor images showing luminescence change of UCNP-labeled HEK293 cells before, during, and after 100 mV depolarization. B/C: Examples and summary of UCNP luminescence changes (red line) in response to repetitive depolarization (black line) of different frequencies (0.1, 1, 10,20, and 50 Hz).

Challenge

The nanosensor is constructed by engineering Förster resonance energy transfer (FRET) efficiency between the outer membrane-anchored upconversion nanoparticle (UCNP), and the membrane-embedded dipicrylamine (DPA). When the neuron is at resting state, FRET is occurring and the luminescence of UCNPs are absorbed by DPA. When the neuron is depolarized, FRET stops and the luminescence of UCNPs is increased. As a result, the emission intensity of the nanosensor can report changes in membrane potential.

The FRET signal is dim and shows a small response to voltage changes, so for this application, Prof. Du’s group needed a camera with high sensitivity to capture as many photons as possible. In addition, since neuronal spikes only last a few milliseconds, a high-speed camera with a fast scanning rate is necessary to detect the membrane potential changes.

The Prime 95B offers a unique combination of extreme sensitivity, high speed and low noise performance from the UV to the near-infrared.

Solution

After comparing different EMCCD and sCMOS cameras, Prof Du’s group chose the Teledyne Photometrics Prime 95B sCMOS camera to detect their FRET signals.

Dr. Rongwei Zhang, an associate researcher from the Du lab, told us: “Prime 95B has a very high quantum efficiency, which is really helpful for our voltage nanosensor imaging… We used Prime 95B to detect membrane potential-relevant fluorescent signals for the use of our voltage nanoparticle sensors in cultured cells and zebrafish larvae. Compared to other EMCCD and sCMOS cameras, [the] Prime 95B can detect very dim signals, achieving a high signal-to-noise ratio and fast imaging rate, giving good spatiotemporal resolution.”

T Cell Calcium ImagingCustomer Stories

Professor Johannes Huppa
Iago Doel-Perez, MSc

Institute of Hygiene and Applied Immunology, Medizinische Universitat Wien, Austria

Background

Prof. Huppa and team study T cells, cells that play an important role in the immune response. These T cells can recognize antigens and distinguish between friend (normal cells) and foe (viruses or infected/dangerous cells) in our bodies. By studying this recognition of antigens by T cells, Prof. Huppa and the team aim to discover more about the innate and adaptive immune system.

Ph.D. student Iago Doel-Perez outlined his research: “We work with both human and mouse T cells, studying the interaction of live T cells with a platform that allows us to control the activation of T cells.” When these T cells activate there is an influx of calcium ions (Ca2+), meaning that calcium imaging presents an easy and quick discrete marker to show activation and deactivation of T cells through fluorescent imaging.

Figure 1: A frame from a time-lapse of T cells stained with the Fura-2 calcium indicator.
Left) T cell activation. Blue T cells are inactivated (base calcium) and green T cells are activated (increased calcium as shown by the scale in AU).
Right) Same cells but with a localization tracker, showing the direction and distance the T cells moved, as well as the velocity (color corresponds to the scale below the image, units are μm per second).

Challenge

Iago explained: “We want to record the moment that the T cells activate in a controlled presentation manner, so we know what we are providing the cell with as information and then we can check the output of this cell. How fast does the activation happen and how many cells?”

Iago uses the calcium indicator Fura-2 to determine when the T cells activate and in order to analyze the number of active cells and their behavior. For this, Prof. Huppa and the team need a camera with a wide field of view in order to image as many cells as possible. Iago recalled: “With a previous EMCCD camera we could only record a quarter of the area so we imaged four times less cells so we could not test as many conditions.”

The Prime 95B offers a unique combination of extreme sensitivity, high speed and low noise performance from the UV to the near-infrared.

Solution

Iago told us his experience with the Prime 95B: “A big advantage is the larger field of view and also the smaller pixel size than our EMCCD, we can really capture cells on a very wide area, which allows us to record many cells and get an appropriate statistical description of the system… The large field of view combined with the higher resolution let us use a very low magnification, this is a really big advantage with the [Prime 95B] camera.”

Iago went on to mention: “The camera was very easy to work with, we are using μManager and everything worked out of the box.”