Delayed Fluorescence ImagingCustomer Stories

Dr. Anthony Hall

Anthony Hall Group, Earlham Institute, University of Norwich, UK

Background

The Anthony Hall group is a world-leading lab with a focus on understanding wheat genomics. Their work seeks to bridge the gap between traditionally used model plant organisms and crop species with real-world applications.

The biological circadian clock is the internal time-keeping mechanism within a living organism. It is entrained by external day-night cycles and is responsible for controlling a wide array of processes such as photosynthetic activity. A robust circadian rhythm is vital to overall plant fitness and controls many other factors such as flowering time and resistance to pathogen attack.

A valuable tool available to researchers who are investigating photosynthesis is to observe bioluminescence. The Anthony Hall group is interested in a form of bioluminescence called delayed fluorescence (DF), whereby photons of light absorbed by the plant are later re-emitted at a level proportional to the photosynthetic efficiency of the sample at that precise time. This measurement cycles with a circadian rhythm and can be used on any biological sample containing photosynthetic pigments. An advantage of DF over conventional luciferase experiments is that material can be used straight from the plant with no genetic modifications necessary.

Figure 1: Delayed fluorescence image of wheat leaves taken with the Retiga LUMO. A) Delayed fluorescence in 3 week old wheat leaves (cv.Cadenza) with a 60 second exposure time. B) Images 6h, 12h, 18h, and 24h after perceived dawn show the signal variation with the circadian rhythm. C) Normalized data for DF intensity over 3 days.

Challenge

DF has a very weak intensity compared to other forms of bioluminescence and requires long exposure times to capture all available signal. Image acquisition is triggered after a critical pause following lights-off to eliminate contamination from residual light and other forms of bioluminescence.

The primary challenge is to obtain a bright enough signal over several days for meaningful quantitative measurements of the oscillations to be made. Having a sufficient field of view for high throughput sample analysis is also important. Accuracy of camera shutter speed is essential for capturing all available DF signal without any noise from the light source turning off.

The previous technology used for delayed fluorescence measurements was based on expensive back-illuminated CCD cameras, which required cooling down to ~70°C to reduce dark current. The large pixels of these sensors also reduced resolution. These cameras significantly increase the cost of putting together and running a delayed fluorescence experiment.

Using the Retiga LUMO camera, we’re able to get high quality images as with a back-illuminated CCD for a fraction of the price, and without the inconvenience of cooling to minus 70 degrees.

Solution

The Retiga LUMO CCD camera from Teledyne Photometrics is precisely engineered to have low dark current at long exposure times, low noise readout, and to account for hot pixels. As a result, the Retiga LUMO provides equal or better performance than a back-illuminated CCD for a fraction of the cost. What’s more, the larger sensor with smaller pixels improves both field of view and resolution over the previous technology.

“With the Retiga LUMO, we get consistent image quality, no extra cooling is needed and we no longer need to wait an hour for the cameras to cool down,” says Dr. Anthony Hall, head of plant genomics. “We were able to use the same protocols in the Micro-Manager software, making it easy to get the cameras up and running.”

Gene Expression via Bioluminescent ReportersCustomer Stories

Dr. James Locke, Mr Mark Greenwood

The Locke Group Laboratory, University of Cambridge, UK

Background

The research performed by the Locke Group at the University of Cambridge focuses on developing a quantitative understanding of gene circuit dynamics. One of the gene circuits of particular interest is the circadian clock, the biological timekeeper. In plants the clock is highly important; the clock controls anticipation of day/night as well as responses to faster environmental changes.

The research team has found it is critical to observe the circadian clock at both the tissue level and single-cell level as traditional approaches that take an average from a population can obscure heterogeneous responses and novel dynamics.

Figure 1: Expression of GI:LUC, a transcriptional reporter for the circadian clock, in single Arabidopsis seedlings. The image was taken using the Retiga LUMO with a 20 minute exposure and 4×4 binning.

Challenge

Previously the team used luminescent reporter genes and EMCCD or back-thinned CCD cameras to monitor gene expression of the clock tissue specifically over several days. Although long exposure imaging with these cameras provides good signal sensitivity, often much of the spatial dynamic was lost to noise. The cost and physical size of the cameras also limited their throughput.

Using the Retiga LUMO camera, we’re able to get high quality images as with a back-illuminated CCD for a fraction of the price, and without the inconvenience of cooling to minus 70 degrees.

Solution

In an effort to better support their imaging needs, the team
purchased four Retiga LUMO™ CCD cameras from Teledyne Photometrics. Mr. Greenwood (Ph.D. student) shares, “We chose the Retiga LUMO camera because of its impressive sensitivity and low dark current which means we can achieve a high signal while minimizing noise, which is the key to long exposure imaging.”

Greenwood continues, “The low cost and physical size of the Retiga LUMO in comparison to other cameras designed for long exposure imaging, as well as the simple API, meant that we could increase our throughput considerably.”

Quantum Simulation via Atomic Optical LatticesCustomer Stories

Alexander Impertro, Julian Wienand, Prof. Monika Aidelsburger

Quantum Optics Group, Ludwig-Maximillian University of Munich, Germany

Background

Alexander Impertro and Julian Wienand are both PhD students in the group of Prof. Monika Aidelsburger and Prof. Immanuel Bloch at the LMU Munich, working on experiments with ultra-cold atoms for analog quantum simulation.

They took us through the concepts behind their work, “The idea is that by taking very cold atoms and trapping them in lattices generated by interfering laser beams, one can simulate the behavior of electrons in a real solid. The atoms hereby play the role of the electrons and the potential landscape generated by the laser beams mimics the ion crystal. This effectively simulates quantum phenomena in real solids with the benefit that the atoms can be controlled in a targeted way, as typical length scales in the analog quantum simulator are thousands of times larger than in real solids.”

“The quantum simulator is highly tunable. By changing the depth of the lattice and the value of an external magnetic field, the dimensionality of the lattice, the strength of the atom tunneling, or the magnitude of the interactions can all be adjusted, in order to simulate tailored systems and phenomena.”

The Quantum Optics Group requires an imaging setup in order to determine which site of their lattice is occupied with atoms and which site is not. In order to do this, the lattice intensity can be increased to ‘freeze’ the atoms in place, then near-resonant lasers can be used to excite the atoms, these atoms then fluoresce and the scattered photons can be detected with a camera.

Figure 1: Fluorescence image of ultra-cold cesium atoms pinned in an optical lattice, taken
with the Kinetix22 sCMOS.

Challenge

Mr. Impertro and Mr. Wienand described the challenges they face, “Exciting the atoms with our laser heats them up and will cause them to move, so the atoms only fluoresce for around 100 milliseconds to 1 second before they tunnel to another site of the lattice. During that time, we need to acquire an image where we see the single atoms in their lattice sites. In order to get high signal-to-noise ratios, we require high quantum efficiencies and low electronic noise combined with a high frame rate.”

“The better the signal to noise ratio, the shorter we have to image in order to get the same image quality, so what’s important is that the camera has low noise. In order to carefully analyze the quality of the imaging technique, it is also important to take many images of the same atom cloud. Typically, we want to take 10 or more images at exposures of 100 ms each and a small delay between them.”

Another challenge is that the detected wavelength is in the near-infrared (NIR) region of the spectrum, at 852 nm. This means a camera with a broad QE range and good NIR sensitivity is required in order to achieve good signal-to-noise ratios. For high-quality and high-resolution images it is further beneficial to work with small pixel sizes in order to reduce the complexity of the optical setup. Moreover, having a large sensor available significantly simplifies finding a first signal from the atomic cloud, thereby simplifying experimental alignment procedures.

Using the Retiga LUMO camera, we’re able to get high quality images as with a back-illuminated CCD for a fraction of the price, and without the inconvenience of cooling to minus 70 degrees.

Solution

The Kinetix22 sCMOS is a flexible and powerful camera with numerous physical science applications, particularly for quantum-based projects. The large 22 mm sensor, fast readout speeds, small pixels, and sub-electron read noise result in an ideal solution for this application.

Mr. Impertro and Mr. Wienand told us about their experience with the Kinetix22, “Previously, we used a camera with much longer readout times, around 800 ms. Compared to this the Kinetix22 is a huge improvement for us as we can take several measurements of the same cloud one after another.”

“We found that the Kinetix22 Sub-Electron mode gave us a great signal-to-noise ratio, so we plan to use this mode in the future for our experiments. What we also like about the Kinetix22 is that the pixel size is small, so we don’t need to work with large magnifications. A previous camera with 13 μm pixel size meant we had to use a magnification of at least 100x, now thanks to the 6.5 μm pixel size of the Kinetix22 we can use 40-60x, which makes our imaging path much shorter.”

“The 22 mm chip size is also much larger than our previous camera and this makes it much easier to find the atoms in the system, which is very useful. We used the USB connector with the Kinetix22, this was really easy plug and play, this was something we liked a lot.”

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.

Using the Retiga LUMO camera, we’re able to get high quality images as with a back-illuminated CCD for a fraction of the price, and without the inconvenience of cooling to minus 70 degrees.

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.

Using the Retiga LUMO camera, we’re able to get high quality images as with a back-illuminated CCD for a fraction of the price, and without the inconvenience of cooling to minus 70 degrees.

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.

Using the Retiga LUMO camera, we’re able to get high quality images as with a back-illuminated CCD for a fraction of the price, and without the inconvenience of cooling to minus 70 degrees.

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.

Using the Retiga LUMO camera, we’re able to get high quality images as with a back-illuminated CCD for a fraction of the price, and without the inconvenience of cooling to minus 70 degrees.

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.

Using the Retiga LUMO camera, we’re able to get high quality images as with a back-illuminated CCD for a fraction of the price, and without the inconvenience of cooling to minus 70 degrees.
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.

Using the Retiga LUMO camera, we’re able to get high quality images as with a back-illuminated CCD for a fraction of the price, and without the inconvenience of cooling to minus 70 degrees.

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.

Using the Retiga LUMO camera, we’re able to get high quality images as with a back-illuminated CCD for a fraction of the price, and without the inconvenience of cooling to minus 70 degrees.

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.”