Page Type: Customer Story

Super-Resolution Expansion Light SheetCustomer Stories

Prof. Ulrich Kubitscheck, Dr. Martin Schwarz, Mr Juan Eduardo Rodriguez-Gatica

Institute of Physical and Theoretical Chemistry, University of Bonn, Germany

Background

The lab of Prof. Ulrich Kubitscheck works to analyze living as well as cleared biological cell systems, developing novel quantitative light microscopy techniques. A combination of tissue clearing with light-sheet fluorescence microscopy (LSFM) is especially well suited to the fast analysis of complex arrangements of large cleared cell clusters and thus allows fast light microscopic access into the complex 3D architecture of neuronal tissue.

The limitations of LSFM and potential solutions to these were outlined by Prof. Kubitscheck: “LSFM cannot reveal the very fine details of neuronal networks since these structures are well below the optical diffraction limit and are of extreme complexity. A solution to this issue is realized by the combination of light-sheet fluorescence and expansion microscopy (LSFEM) allowing for the analysis of extended neural circuits in super-resolution… LSFEM is compatible with multicolor fluorescence imaging, thus enabling molecular contrast for diverse neuronal populations and nanoscale resolution within a single, large tissue preparation. LSFEM is optimally suited for the analysis of connectivity in large neuronal tissue regions.”

“Together with Dr. Martin Schwarz from the University of Bonn Medical School, we developed a novel tissue expansion pipeline and used LSFM and LSFEM to image very extended regions of mouse brain tissue in 3D. We can zoom in and out from meso- to nanoscale resolution, which means effective super-resolution, depicting the finest details of neuronal network parameters within a larger context.”

In this experiment, projections from the horizontal diagonal band of Broca (HDB) to the olfactory bulb (OB) in the mouse brain are visualized using LSFEM and a novel clearing protocol.

Figure 1: Optical section of an olfactory bulb from a cleared sagittal slice of a ChAT Cre-mouse, expressing endogenous EGFP (green) and nuclear staining with Hoechst (red). Acquired with a custom-built LSFM, Nikon 10x/0.3 N.A W objective. A) Image acquired with a typical CMOS, 12 separate acquisition tiles needed. B) Image acquired using Teledyne Photometrics Kinetix CMOS (on a similar sample), only 2 tiles were needed to cover the same region as A. C) Single plane of the area marked in B at 208 μm depth. D) Single plane of a separate area at 200 μm depth and higher magnification. Images courtesy of Juan Eduardo Rodriguez-Gatica.

Challenge

LSFEM involves huge specimen regions and consequently, a large number of image tiles acquisitions are needed to cover the complete field. These images often need to be overlapping image fields to allow stitching, which adds to both the time taken for an image to be produced and the computational effort to perform stitching of a large number of image tiles. By using a camera with a very large field of view, a smaller number of acquisitions would be needed, reducing both the time for acquisition and processing.

The current imaging experiments can often have an exceedingly long duration, during which time the sample is exposed to light and risks photobleaching. A highly sensitive camera with a low noise level is needed in order to reduce the exposure required to image the sample and avoid photodamage.

Finally, a small pixel size is also required in order to achieve diffraction-limited resolution, this technique combines the needs of light sheet and super-resolution imaging, meaning the camera also needs to operate at a high resolution at certain magnifications.

The large sensor of the Kinetix greatly reduces the number of required image tiles to cover the field of view… the signal to noise ratio is excellent.

Solution

The groundbreaking Kinetix CMOS features a large field of view, a 10 megapixel sensor with a small 6.5 μm pixel size, and a high sensitivity thanks to a combination of back-illumination and low-noise imaging modes. This makes the Kinetix an ideal solution for this demanding imaging application.

Prof. Kubitscheck told us about his experience with the Kinetix CMOS, “As demonstrated, the large sensor of the Kinetix greatly reduces the number of required image tiles to cover the interesting field of view ranging from the HDB to the olfactory bulb area of a 1.5x expanded and cleared mouse brain section. The signal to noise ratio is excellent.”

“Using the new Kinetix camera in combination with novel tissue clearing protocols and LSFM we can show in great detail and overextended regions that HDB neurons innervate different cell layers of the OB. Most notably, we can, due to the large field of view, the exquisite resolution and the possibility to image ~2mm thick sections, follow projections from individual HDB neurons up to their target region within the OB.”

“The new setup comprising the Kinetix camera will significantly improve our imaging capabilities and will likely lead to novel insight into the HDB to OB synaptome.”

DNA PAINT Super ResolutionCustomer Stories

Dr. John Danial, Prof. David Klenerman

Klenerman Lab, Yusuf Hamied Department of Chemistry, University of Cambridge, UK

Background

Dr. John Danial works as part of the Klenerman Lab to develop biophysical approaches to complex neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. In these diseases certain proteins can accumulate in the brain and form aggregates, these aggregates can be investigated using extremely sensitive super-resolution microscopy, to uncover the mechanisms of brain diseases.

The approach used by Dr. Danial involves using a super-resolution microscopy technique known as DNA-based Point Accumulation in Nanoscale Topography (DNA-PAINT), this allows for imaging with resolutions smaller than 20 nm, allowing Dr. Danial’s custom imaging system to study nanoscopic aggregates.

Figure 1: Image of nanorulers taken with the Prime BSI Express. Each of these nanorulers is made of DNA origami, with the distance between each bright spot measuring 40 nm. Successful imaging of these is used to establish the performance of an imaging system.

Challenge

DNA-PAINT involves taking many thousands of images and processing into a super-resolution image, this means any interference during the process can limit the overall image quality.

Dr. Danial spoke about a previous EMCCD solution, used due to the high sensitivity, but otherwise limited in terms of the small FOV limiting throughput, and the high-speed fan causing vibrations that perturbed measurements.

Typical CMOS solutions were also considered but were lacking in terms of noise characteristics and post-processing capabilities, both necessary for DNA-PAINT. A higher-quality CMOS solution was needed, with a large FOV, small pixel, and excellent noise characteristics.

The large sensor of the Kinetix greatly reduces the number of required image tiles to cover the field of view… the signal to noise ratio is excellent.

Solution

The Prime BSI Express presents an ideal solution for this application, featuring a large 18.8 mm diagonal FOV, a small 6.5 μm pixel (65 nm when paired with 100x magnification, ideal for oversampling), and extremely high sensitivity, thanks to the combination of near-perfect 95% quantum efficiency for maximizing signal collection, and CMS mode minimizing read noise to near 1 e-.

Dr. Danial told us about his experience “I am very happy with the Prime BSI Express, it is the most robust part of my imaging system and I appreciate the small form factor… I had no problems with the setup and integrating the camera into our custom LabView software using the SDK.”

Mechanosensitive dSTORM ImagingCustomer Stories

Dr. Alexandre Fürstenberg

School of Chemistry, University of Geneva

Background

The lab of Dr. Alexandre Fürstenberg deals in the development and application of optical spectroscopic and microscopic tools, with a focus on single-molecule imaging using smart fluorescence probes.

Dr. Fürstenberg told us of his lab’s recent research, “We are interested in developing fluorescent probes… we use probes that are sensitive to the lateral pressure in biological membranes, they can tell you about the composition of the membrane, the packing of the lipids, these are mechanosensitive fluorophores that are called flippers… there is a new version of these flippers that blink, and that’s exactly what we need for super-resolution microscopy.”

“Using these fluorescent flippers we want to look at the membranes of giant unilamellar vesicles (GUVs) and demonstrate super-resolution imaging with dSTORM.”

Figure 1: Combined images showing the membrane of a GUV captured with the Kinetix CMOS, shown both in super-resolution (orange) and diffraction-limited (white, reconstructed from a projection of all 6000 movie frames) for comparison purposes. The high resolution and low noise capabilities of the Kinetix allow for high image quality with super-resolution applications.

Challenge

Imaging GUV membranes at super resolutions levels is a demanding application for a scientific camera, due to the individual GUV cross-sections (with membranes filled with flipper probes) having low signal, and requiring many images over time to build up a dSTORM image. In addition, a large FOV is needed to contain larger GUVs so that tiling and stitching can be avoided, and high sensitivities are needed to detect fluorophores within the membrane.

Dr Fürstenberg discussed a previous camera solution, “I had an EMCCD camera already but it was 10 years old and too slow… we were really limited by the field of view, we wouldn’t have been able to look at the really large GUVs… we also need the sensitivity to look at individual probes at the GUV membranes which have very weak signals and which we could not really resolve before in certain lipid environments.”

The large sensor of the Kinetix greatly reduces the number of required image tiles to cover the field of view… the signal to noise ratio is excellent.

Solution

The Kinetix CMOS meets all the needs for this application, with a large FOV, high sensitivity thanks to 95% peak QE and minimal read noise, and high-resolution thanks to a large 10 megapixel sensor with a balanced 6.5 μm pixel size.

Dr. Fürstenberg outlined his experience with the Kinetix, “I needed a new camera so I went for the best one, I can see many advantages such as the really large field of view. We can do a factor of 10 better in terms of speed than the previous camera, this will help us for planning future experiments.

“It was pretty easy to set up the Kinetix thanks to the help from Photometrics staff and the camera also runs very smoothly with MicroManager, this was all a big selling point… It’s also great not to worry about having to center the region of interest as well, before we had to position our samples exactly at the center of the field, this problem is gone now with the Kinetix.”

Reference

José García-Calvo, Jimmy Maillard, Ina Fureraj, Karolina Strakova, Adai Colom, Vincent Mercier, Aurelien Roux, Eric Vauthey, Naomi Sakai, Alexandre Fürstenberg, and Stefan Matile, (2020), Fluorescent Membrane Tension Probes for Super-Resolution Microscopy: Combining Mechanosensitive Cascade Switching with Dynamic-Covalent Ketone Chemistry, Journal of the American Chemical Society 2020 142 (28), 12034-12038, DOI: 10.1021/jacs.0c04942

Single-Molecule ImagingCustomer Stories

Prof. Madhavi Krishnan, Dr. Timothy Bennett

Krishnan Group, Physical and Theoretical Chemistry, Merton College, University of Oxford

Background

The Krishnan Lab, headed by Prof. Madhavi Krishnan, is involved in the study of soft condensed matter at the nanometre scale, at the University of Oxford. This involves applied physics and photonics, physical chemistry of charged interfaces, and single-molecule imaging. This interdisciplinary group works with advanced nanofabrication and optics, requiring powerful and sensitive detectors.

Current research areas involve work with single molecules, including spatio-temporal control, trapping, high precision measurements, modeling electrostatics on the nanoscale, and soft matter interactions. By measuring changes in macromolecule electric charge with high precision, the Krishnan Lab aims to read out 3D conformation and small differences of charge in single macromolecules in real-time.

Figure 1: An image taken in the Krishnan Lab with the Prime BSI Express. The image shows single molecules (bright spots) within fluorescent features on a surface fabricated with nanoscale channels. The single-molecule signals are clearly visible above the background.

Challenge

Single-molecule imaging presents numerous challenges, as outlined by Prof. Krishnan, “Imaging single molecules labeled with a single fluorophore can be challenging as single fluorophores generally emit relatively weak signals. We also need to work at high speeds to visualize the motion of the molecules.”

The low fluorophore density, combined with the weak signal and the need for low exposure in order to achieve high speeds, means that the level of signal is extremely low and any detector used with this system would need to maximize signal collection. In addition, at these low signal levels, noise can have a significant impact and a suitable detector would also need a low read noise, in order to minimize the effects of noise and maintain a high signal-to-noise ratio.

In addition, a large field of view (FOV) would be beneficial as it would allow the capture of more molecules and events with every acquisition. In order to achieve both high speeds and large FOVs, the detector would need a high base speed. Previous EMCCD solutions for this single-molecule work could only reach the required speeds at small FOVs, limiting throughput.

The large sensor of the Kinetix greatly reduces the number of required image tiles to cover the field of view… the signal to noise ratio is excellent.

Solution

The requirement for high sensitivity and high speed across a large FOV makes the Prime BSI Express an ideal solution. The combination of near-perfect 95% QE for maximizing signal collection, and the sub electron read noise in CMS mode for minimizing noise levels, make the Prime BSI Express highly sensitive for single-molecule applications.

Combined with this sensitivity, the Prime BSI Express operates at high speeds, 95 fps across the full sensor. This allows for imaging of fast, dynamic single-molecule events while still maintaining a large FOV with a high resolution, thanks to the balanced 6.5 μm pixel size

Single Microalgae PhototaxisCustomer Stories

Dr. Jorge Arrieta, Dr. Marco Polin

Mediterranean Institute for Advanced Studies (IMEDEA), University of the Balearic Islands

Background

Dr. Jorge Arrieta is a postdoctoral researcher at IMEDEA in the lab of Drs. Marco Polin and Idan Tuval, whose research area involves analysing the motility and behavioural in response to external stimuli of the microalgae Chlamydomonas reinhardtii. These microalgae are a popular model system for study of eukaryotic flagella, photosynthesis, and the diurnal cycle, and can detect light and then reorient themselves towards or away from it, a response known as phototaxis. This is linked to the photosynthetic activity of the cell in a way that is not well understood. Clarifying the link between photosynthesis and phototaxis will allow researchers to uncover new strategies for light management in microalgae, as well as manipulate these organisms using light for potential use in bioreactors.

The link between phototaxis and microalgae metabolism is a research focus for Dr. Arrieta, “In particular we are aiming to look at the link between light-induced changes in motility and photosynthetic metabolism. The latter can be probed at the single-cell level through chlorophyll autofluorescence.”

Figure 1: Changes in autofluorescence of a single C. reinhardtii chloroplast, taken with the Kinetix CMOS. High sensitivity is needed to detect minute fluctuations in fluorescent signal.

Challenge

These experiments involve studying phototaxis in individual cells held on micropipettes and subject simultaneously to independent light-stimulation to the eyespot and the chloroplast. As Dr. Arrieta outlines, “We will look at metabolism by monitoring minute changes in chlorophyll autofluorescence. For this, we will need a high sensitivity and low-noise camera.”

The cells respond differently to high and low light stimuli, moving towards light in the former case and away from it in the latter. As Dr. Arrieta mentioned, “We aim to focus on the range that results in positive phototaxis. This translates into low levels of single-cell chloroplast autofluorescence. Therefore, a sensitive camera was needed to detect minute fluorescent signals and, in particular, their changes in time.”

The large sensor of the Kinetix greatly reduces the number of required image tiles to cover the field of view… the signal to noise ratio is excellent.

Solution

The Kinetix CMOS is a highly sensitive imaging solution for low light applications that require fine levels of signal quantification for delicate samples. Imaging of small, sensitive samples is well suited to the Kinetix in Sensitivity Mode, which features near-perfect 95% QE to maximize signal collection, and low read noise CMS technology to minimize the effects of noise.

With the new Sub-Electron Mode, the Kinetix reaches read noise levels of 0.7 electrons, allowing for highly sensitive imaging of very small signals, without the impact of read noise.

Dr. Arrieta told us about his experience with the Kinetix, “The camera works well, we can measure fluorescence and in the range of parameters we are aiming to work in the camera is responding very well, a very nice device… the high resolution of the Kinetix works really well and the setup was straightforward.”

Super ResolutionCustomer Stories

Prof. Suliana Manley, Dr. Luc Reymond, Dr. Willi Stepp, Mr Chen Zhang

Laboratory of Experimental Biophysics, EPFL, Lausanne, Switzerland

Background

The Laboratory of Experimental Biophysics (LEB) at EPFL, headed by Prof. Suliana Manley, develops and uses fluorescence imaging techniques combined with live-cell imaging and single-molecule tracking to determine how the dynamics of protein assembly are coordinated.

Dr. Stepp and Mr. Zhang are researchers in the LEB, with Mr. Zhang studying the cell cycle dynamics of bacteria using super-resolution imaging techniques such as STORM and SIM, while Dr. Stepp operates as a microscope and optics specialist, enhancing imaging systems and instrumentation. Dr. Stepp spoke about the LEB: “We try to get the right microscope technique for the problem at hand… people come to us with imaging problems and we try to solve it with any technique that is out there.”

Figure 1: Mycobacterium smegmatis genetically modified to express the cell wall synthesis protein wag31, tagged with GFP in orange. The cell outline in blue was labeled with a novel fluorophore attached to an amino acid compound that incorporates into the peptidoglycan component of the cell wall.

Challenge

The LEB uses a range of different imaging systems and techniques, including custom-built microscopes providing specialized capabilities. Dr. Stepp outlined some of the detector needs at the LEB, “for our current project we can have issues expressing enough fluorophores in the bacteria, so there we need high sensitivity, especially as the experiments are performed on our iSIM, which is quite a photon hungry setup… for other projects on mitochondrial dynamics, we also need speed and field of view… if we have one camera that could do it all that would be the best.”

The camera needs for each imaging system and each type of sample are varied in terms of resolution, sensitivity, field of view, and speed, meaning the ideal solution would be a highly flexible detector that can move between different imaging systems, yet is powerful enough to deliver sufficient acquisitions across these different systems.

The large sensor of the Kinetix greatly reduces the number of required image tiles to cover the field of view… the signal to noise ratio is excellent.

Solution

The Kinetix CMOS is a highly powerful camera that can be easily adapted to a number of different imaging systems, featuring a large 29 mm field of view with a balanced 6.5 um pixel size, a very high speed of over 500 fps full frame, all with low read noise and high quantum efficiency.

Dr. Stepp discussed his experience with the Kinetix, “We have a number of imaging systems that would be suitable for the Kinetix… the speed was interesting paired with the big field of view as our samples have short time ranges.”

“The build quality is great, the mounting feels solid, and the initialization is faster than our other cameras so that makes imaging faster on startup in the morning… We had [the Kinetix] set up on our system pretty quickly and our first images were great, very impressive.”

Whole Heart Light SheetCustomer Stories

Dr. Leonardo Sacconi

Cardiac Imaging Group, National Institute of Optics, National Research Council, Italy

Background

Dr. Leonardo Sacconi is a senior researcher at the National Institute of optics and group leader of the Cardiac Imaging Group, which aims to develop novel imaging modalities for research into cardiovascular physiology and pathology. Using techniques such as calcium imaging, multiphoton excitation, and light sheet, Dr. Sacconi is able to investigate both the structure and function of cardiac tissue.

Dr. Sacconi further explained one experimental focus, “We use advances in tissue transformation, staining and processing to reconstruct the entire anatomy of cardiac tissue, especially mouse hearts, in order to have a mesoscopic reconstruction of the heart with subcellular resolution. This is to establish a correlative framework where we can apply our functional data to the structure of the model.”

This work is done on entire mouse hearts in 3D, with hearts being optically cleared using CLARITY or SHIELD protocols. Using a newly-developed light sheet system based on the mesoSPIM initiative, the large heart samples (~1 cm in diameter) can be imaged with an axially scanned light sheet to the resolution of 5 µm.

Figure 1: Preliminary image of a clarified (CLARITY based) mouse heart stained with an Alexa Fluor conjugated WGA using the mesoSPIM imaging system in combination with the Kinetix CMOS. The white square represents the full field of view of the camera, capturing the entire heart in one acquisition. The magnified red area represents the resolution at 2x magnification.

Challenge

The sample size is in the order of centimeters, as Dr. Sacconi is imaging entire cleared mouse hearts. This means a very large FOV is beneficial, otherwise, experiments would involve long acquisition sessions with many images stitched together, also requiring large computational commitment. Dr. Sacconi ran into these issues with a previous CMOS camera solution, “The previous CMOS camera we used had a small sensor and wasn’t able to acquire the entire heart, so we decided to switch.”

This large FOV also needs to match a low magnification, standard low mag (2-4x) objectives typically have a low numerical aperture (limiting resolution) and a small FOV. Dr. Sacconi saw this limitation and planned to use a custom lens, “The FOV of normal low mag objectives is normally very limited, so we needed to design a new telecentric lens, 2x magnification and 0.1 NA, with a large field of view and flatness of field.”

With a large FOV, more data can be captured with every acquisition, increasing throughput and simplifying experiments. In addition, high speed is beneficial to match with the axially scanned light sheet, which requires a camera with a fast-rolling shutter, by synchronizing these mechanisms light sheet images can optimally be acquired across large samples.

On top of this, a high resolution and high sensitivity are also beneficial, capable of imaging such a large sample with suitable image quality.

The large sensor of the Kinetix greatly reduces the number of required image tiles to cover the field of view… the signal to noise ratio is excellent.

Solution

The Kinetix CMOS meets the needs of this application, featuring a huge 29.4 mm FOV with a 10 megapixel sensor (3200 x 3200) and a balanced 6.5 µm pixel. This allows for imaging across a large area while at a high resolution, ideal for the large sample size.

The combination of the Kinetix and a 2x custom telecentric lens results in a FOV in the order of 1-2 cm, allowing the entire clear mouse heart to be captured in just one acquisition. As Dr. Sacconi says, “The different in cameras was amazing… the flatness of the field is most impressive, and the high frame rate is also important, this will be an interesting feature for future work.”

Overall the Kinetix is a great solution for large FOV light-sheet applications, able to image across a wide area with high resolution, high speed, and high sensitivity.

Imaging Photon Bose-Einstein CondensateCustomer Stories

Mr Chris Toebes, Prof. Pepijn Pinske

Adaptive Quantum Optics, Faculty of Science and Technology, University of Twente, The Netherlands

Background

The Adaptive Quantum Optics Lab in University of Twente performs advanced research on the physics of quantum light for applications in (quantum) information science and technology. PhD student Chris Toebes works in this lab with photon Bose Einstein condensates (BECs).

Mr Toebes explained his work, “We are working on computations based on light, using our photonic BEC system to simulate optimization problems and infer solutions to those problems by carefully monitoring the light that comes out of the system.”

A photon BEC is generated at room temperature using a system of curved, highly reflective mirrors placed within microns of each other, and filled with fluorescent dye. The BECs are generated in a microstructural patterning on the mirror, with the end goal is to have hundreds of BECs all communicating in a network.

Figure 1: Images from the Prime BSI Express, each showing two photon BECs (the bright spots on the far left and far right) and the interference in-between (dimmer spots in-between the bright spots). As the BECs are able to send photons between each other they generate standing wave patterns. These are two different configurations, where the top image is out of phase (4 dots) and the bottom image is in phase (5 dots). The phase of this system can be used as different states for computational purposes.

Challenge

Only a small portion of light can leak outside this system so a highly sensitive camera is necessary, as mentioned by Mr Toebes, “At the threshold where the BEC forms, intensity can be very low. This is where it is useful to have very sensitive cameras. When we measure the light that comes out we need a high signal-to noise ratio… In other operating regimes, the photonic BEC system might be unstable and we need statistics on a lot of data, so a high acquisition rate is required.”

Due to the exploratory nature of this work, the Adaptive Quantum Optics Lab requires a camera that is highly flexible, featuring high sensitivity, minimal noise, high speeds, a large field of view, that can be adapted to
custom software.

The large sensor of the Kinetix greatly reduces the number of required image tiles to cover the field of view… the signal to noise ratio is excellent.

Solution

The Prime BSI Express is a compact, powerful, flexible CMOS camera that is well suited to this application, featuring a number of different modes including high speed mode (95 Hz full frame, far more at small ROIs), CMS low noise mode, and high dynamic range mode. This kind of flexibility enables the camera to work within this system both now and into the future.

Mr Toebes outlined his experience with the Prime BSI Express, “The camera is really a crucial part of the operation… We considered other technologies but our main reason for going for this was the higher sensitivity and the ease of day to day operation, compared to the hassle of intensified cameras and lack of speed or field of view with EMCCDs.”

“We really need to write our own software for this system, a program that analyses images and extracts data. I wrote my own graphical interface to operate the camera and the Python support is very convenient, I didn’t have any issues at all.”

“The initial setup was as painless as could be, it worked immediately, and the large sensor will really come in handy when we go to large systems.”

Imaging Live Cell ExocytosisCustomer Stories

Mr. Benji Bateman, Dr. Lin Wang

Central Laser Facility, Science and Technology Facilities Council, Swindon, UK

Background

Mr Bateman is a Link Scientist working with Dr. Lin Wang in the Central Laser Facility (CLF), which contains everything from lasers the size of a room to compact benchtop lasers. Mr Bateman uses lasers with microscopes to do fluorescence imaging for the academic community, who bid for time at the CLF for their research. Mr Bateman and others at the CLF need to constantly keep up to date with the latest imaging technologies in order to best enable a wide range of imaging needs.

The CLF caters to many different applications, including super-resolution techniques such as Stochastic Optical Reconstruction Microscopy (STORM) for extremely high-resolution imaging, in very low imaging photon budgets, which need detectors capable of photon counting for STORM reconstruction, especially under cryogenic conditions.

Figure 1: Chinese Hamster Ovary (CHO) cells transfected with wild-type EGFR labelled with 5 nM EGF- Alexafluor488. TetraSpeck fiducial markers added and imaged at cryogenic temperatures.

Challenge

These CLF STORM imaging systems previously used EMCCD technologies, but these ran into issues with the dynamic range as Dr. Bateman outlined, “We’re looking at samples in liquid nitrogen vapour and they bounce and wobble around, in order to correct for that we use bright TetraSpeck beads as fiducial markers, these are a lot brighter than the samples we measure and the well-depth of EMCCDs weren’t sufficient to capture all of the fluorescent signals, we needed something with a larger dynamic range for the beads and the STORM information.”

Mr. Bateman also mentioned they would benefit from a larger pixel for greater sensitivity, “We did try using a traditional CMOS with 6.5 µm pixels and but the sampling rate was mismatched to our choice of the microscope objective and solid immersion lens combinations, leading to overall magnifications up to 450x. We require the best STORM measurement precision possible in order to answer the biological questions our user groups have under investigation. This is only achievable with correctly sampled fluorescence point spread functions and high detector quantum efficiency. The Prime 95B met both of these requirements and currently supports all of our cryogenic user groups with their experiments.”

The large sensor of the Kinetix greatly reduces the number of required image tiles to cover the field of view… the signal to noise ratio is excellent.

Solution

The Prime 95B CMOS meets the needs of super-resolution imaging systems while improving on previous EMCCD solutions in dynamic range, speed, field of view, and resolution while maintaining high sensitivity thanks to maximizing signal collection and minimizing noise.

Mr. Bateman spoke about the benefits of the Prime 95B, “We like the 95% quantum efficiency, the 11 µm pixel size, and the speed advantage are all benefits for us, there isn’t really a good reason for us to use EMCCD any more… Another bonus of [the Prime 95B] would be the enhanced field of view and the larger number of pixels, especially compared to an EMCCD which has a very small chip size… We’ve coupled two 95Bs into a Cairn TwinCam using the full chip size for each channel.

Multi-Patch Clamp ElectrophysiologyCustomer Stories

Dr Maurizio Pezzoli, Prof. Henry Markram

Neural Microcircuitry Laboratory, EPFL, Switzerland

Background

Dr. Maurizio Pezzoli works in the Neural Microcircuitry Laboratory, performing whole-cell patch clamping in acute slices in order to analyze local microcircuitry. The main approach is through multiple patch-clamp, as Dr. Pezzoli says “the lab has the first 12 patch system so we can put 12 pipettes in one slice and see how close neurons talk to each other”.

“The effort of the lab is to try and understand how different types of neurons are connected and how the bigger circuit is connected, my main focus would be around the somatosensory cortex and somatosensory thalamus, but we are also testing some other parts such as hippocampus.”

To this end, Dr. Pezzoli performs electrophysiology experiments on ~300 μm slices of brain tissue, using multiple patch pipettes to record neuronal function. However, generating a clear image of the tissue is vitally important to ensure the right neurons are patched with the micropipettes.

Figure 1: Image of the electrophysiological setup used in the Neural Microcircuitry Laboratory, showing the microscope, Prime 95B CMOS as a detector, and multiple patch pipettes positioned around a sample.

Challenge

Dr. Pezzoli outlined some of the challenges of his work: “We need a clear image in an environment that is mostly black and white, we are also working with infrared as this can go through the 300 μm tissue slice… our patching is all done live and you need good optical guidance when approaching with multiple pipettes when cells are in thick tissue, and to see the right time to do the patch, you really need to see things properly.”

“We push the contrast a lot and require a sensitive camera, secondly we need it to be very fast as we only have a small opportunity to patch the cell then it’s gone, we also need great resolution as you’re looking at very tiny surfaces and if you increase the optics too much you lose the working distance, so there are limits. We have a working distance of around 2 mm but as we put more pipettes we are limited in our XY directions.”

This challenging application requires a flexible, highly sensitive camera with a large dynamic range.

The large sensor of the Kinetix greatly reduces the number of required image tiles to cover the field of view… the signal to noise ratio is excellent.

Solution

The Prime 95B CMOS is an ideal solution for this application, with a near-perfect 95% QE for maximizing signal collection, and a high speed across a large field of view. The large dynamic range in 16 bit mode will allow for the identification of even the smallest neuron while pushing the brightness and enabling a good contrast in the image, assisting the patch clamping process.

Dr. Pezzoli has been using the Prime 95B for his patch clamping experiments: “It makes the patching easier, the main benefit for me is that it improved my patching as I can see better, particularly with the tiny interneurons…. the setup experience was quite straightforward, it is always nice to receive new equipment in the lab and it worked very easy.”