Page Type: Customer Story

iSIM Live Cell ImagingCustomer Stories

Dr. Daniel Dickinson

Department of Molecular Biosciences, University Of Texas, Austin

Background

The Dickinson lab aims to understand the generation of polarity in cells, namely when two ends of a living cell become molecularly distinct from each other. Polarity is vital for the proper function of cells and can become disrupted in diseases such as cancer.

Dr. Dickinson and his team employ a multi-disciplinary approach including fluorescence microscopy of live samples and quantitative image analysis. Working mainly with the embryos of the genetically manipulable model system, the nematode worm Caenorhabditis elegans, the Dickinson Lab uses imaging and single-molecule interrogation techniques, to determine the biochemical interactions that lead to the determination of the direction of the polarity axis and the timing of polarity establishment.

Tracking single molecules expressed at native levels in the C. elegans embryo allows them to measure diffusion constants, and using single-molecule pull-down techniques, establish protein-protein interactions during the biogenesis of cell polarity.

Figure 1: Images of dividing cells taken with the Kinetix22 on a Nikon Ti2 with a Visitech iSIM super-resolution confocal head. A) A C. elegans zygote, microtubules (green), and cell polarity marker PAR-2 (magneta). B) A dividing mouse embryonic cell embedded in 3D Matrigel, microtubules (cyan), cell tracking dye (magenta), and apical polarity marker Podacalyxin (green). C) An 8-cell C. elegans embryo, microtubule TPXL-1 protein (magenta), and cell polarity determinant PAR-3 (cyan).

Challenge

Tracking single molecules in time and space in living embryos requires a high spatial and temporal resolution from a detector. The embryo is sensitive to total illumination power, and the proteins of interest move in all dimensions of position (X, Y, Z) and time. Capturing the position and temporal information allows the lab to draw conclusions about molecular interactions contributing to the formation of cell polarity.

As Dr. Dickinson pointed out, “Low read noise is really critical”. The total amount of light collected is so low in any one frame. Without low read noise, high quantum efficiency, and a uniform camera background, they often couldn’t collect enough photons for precise measurements.

The Kinetix22 has a uniform background, high sensitivity and low read noise, allowing us to collect enough photons to make precise measurements.

Solution

The Kinetix22 sCMOS provides a unique solution, with both high speed and very low read noise options. Fast particle tracking in Speed mode provides robust data for the calculation of molecular diffusion constants, while Sub-Electron mode allows for precise molecular localization with limited signal thanks to the ultra-low read noise of less than 0.7 e-.

The Kinetix22 combines multiple modes for flexible yet powerful imaging, with Speed, Sensitivity, Sub-Electron, and Dynamic Range modes. Imaging with select modes across a large sensor, more than twice the size of typical sCMOS, allows for high-throughput and efficient imaging.

OPM Snouty Light-SheetCustomer Stories

Mr. Jacob Lamb, Dr. Edward Ward, Prof. Clemens Kaminski

Laser Analytics Group, Department of Chemical Engineering & Biotechnology, University of Cambridge, UK

Background

Dr. Edward Ward and Mr. Jacob Lamb are both researchers in the Laser Analytics Group, led by Prof. Clemens Kaminski. This group aims to develop imaging techniques to apply to biological systems, requiring the latest in optical and camera technologies.

Dr. Ward spoke about one of their latest developments, “We use light sheet and confocal microscopy, and we needed a solution with high-speed imaging, easier sample prep, easier mounting as well as high resolution. Using single objective OPM [Oblique Plane Microscopy] Snouty allows for faster imaging at a high resolution.”

By using a custom AMS-AGY ‘Snouty’ lens, the Kaminski Lab aim to image at a higher temporal and spatial resolution than traditional light sheet methods, obtaining volumetric high-contrast images at high speed, all with less sample prep and easier mounting due to the single-objective format. This light sheet system needs to be paired with a suitable camera in order to avoid any bottlenecks and maximize imaging throughput and efficiency.

Figure 1: Two maximum intensity projection images taken with a single-objective light-sheet imaging system including a Kinetix22 sCMOS, OptoSplit III, and OPM Snouty lens. In blue is a 200 μm x 200 μm FOV image of microtubules in Cos 7 cells using the fast galvo scanning method on the OPM. In orange is a 1000 μm x 220 μm image of microtubules in live retinal ganglion cell axons growing out of a Xenopus laevis eye explant, using stage scanning on the OPM.

Challenge

Mr. Lamb outlined the challenges faced with their imaging system, “We want to image as fast as possible, a few volumes a second would be nice… but how fast we can image is entirely limited by the camera: by how short we can make the exposure time and still see a nice image, and by the readout speed of the camera.”

“For the camera, we also need high photosensitivity, with the OPM-Snouty system there are so many optical components and light can be lost between them.”

As well as needing a camera with high sensitivity and high readout speed, Dr. Ward also mentioned wanting to perform simultaneous multichannel imaging with three different channels. This is achievable by pairing a camera with a three-way splitter (such as the OptoSplit III from Cairn Research) but would reduce the available field of view to a third, meaning a large field of view camera is vital.

The Kinetix22 has a uniform background, high sensitivity and low read noise, allowing us to collect enough photons to make precise measurements.

Solution

The Kinetix22 sCMOS represents the latest in modern camera technologies, featuring high speed, high sensitivity, high spatial resolution with a 6.5 μm pixel, and a large 22 mm field of view. Fast imaging speeds can only be achieved by highly sensitive cameras, due to the reduction in exposure time needed to reach high speeds. With the combination of a large field of view and high readout rates, the Kinetix22 is well suited for high-throughput light-sheet applications, able to measure large volumes with high resolution.

Dr. Ward told us about his experience with the Kinetix22, “When we looked at other cameras, the Kinetix22 was by far the most sensitive… The pixel size is just right for Nyquist with Snouty, which is what we want.”

Mr. Lamb also mentioned using the Kinetix22, “We use the whole field of view of the Kinetix22 with an OptoSplit III, the sensor size is just right so we can split it and have the maximum field of view that our system allows… the software support is also great, the camera runs in MicroManager and is triggered through hardware in LabView.”

NIR/VIS Brain ImagingCustomer Stories

Friederike Seifert, Dr. David Fleck, Prof. Marc Spehr

Department of Chemosensation, Insitute for Biology, RWTH Aachen University, Germany

Background

The lab of Professor Marc Spehr is working on the olfactory system of mammals and the underlying mechanisms of pheromone detection. Pheromone detection is key for social communication in the animal world.

The main methods of choice utilized by Dr. David Fleck and Ph.D. student Friederike Seifert are electrophysiological techniques, in combination with imaging to visually observe the signal transmission in the vomeronasal organ (VNO) upon very sophisticated stimulation with pheromones. The imaging techniques range from simple backfilling of neurons, identifying subsets of neurons expressing certain types of receptors, to measuring activity by detecting signals from genetically encoded (GECI) or dye-based Ca2+ indicators.

Figure 1: Images comparing a typical CCD camera and the Moment CMOS. A) 150 μm thick coronal VNO slice, FPR-rs3 vomeronasal receptors fluorescently labeled with Venus protein. The CCD FOV is displayed on the Moment FOV as a white square. B) Magnified view of two neurons from the slice seen in A, displaying the differences in resolution and sensitivity.

Challenge

For a typical experiment, a slice of the VNO is mounted under a microscope with the aim to record an individual neuron using electrophysiology, with a patch pipette. Typically, near-infrared (NIR) illumination is utilized to focus on the tissue and the pipette in order to achieve high contrast. For this purpose, special coatings had to be used on older CCD cameras in order to get a sufficiently high sensitivity – this compromised sensitivity in the visible spectrum relevant for the imaging of fluorescent reporters and dyes.

Additionally, existing detection solutions had very small sensor areas resulting in being able to just observe a small fraction of the entire VNO sample. This also made it very difficult to make conclusions about the overall context and anatomy of individual cells.

The Kinetix22 has a uniform background, high sensitivity and low read noise, allowing us to collect enough photons to make precise measurements.

Solution

The Moment CMOS is an ideal solution for this demanding application, overcoming the problems a classically used patch-clamp camera had faced. The general sensitivity of the Moment between 200 1100 nm allows the camera to have excellent sensitivity in both the visible range and the NIR, without compromise. Sensitivity is much improved compared to the previous CCD solution, resulting in better signal-to-noise ratios, whether for brightfield visualization of the patch pipette or fluorescence imaging of individual neurons.

The Moment´s higher maximum speed of 50 fps gives more instant feedback while positioning the pipette, but also enables a higher recording speed of fluorescent and/or dye-based dynamics opening new insights into physiologically relevant processes.

With the much larger diagonal field of view of 17 mm, the members of the Spehr Lab can finally detect the VNO in a single field of view, resulting in a much higher throughput and more efficient sample handling, allowing for more experiments. This results in a system that is both simpler and more powerful.

Lastly, the ultra-compact size of the Moment (40 x 42 x 50 mm) and the extremely low weight (96 g) allow for easy integration into electrophysiological imaging systems, and the global shutter triggering plus passive cooling makes it perfectly suited for upright microscopes typically used for electrophysiological experiments.

Light-Sheet MicroscopyCustomer Stories

Dr. Rui Ma, Prof. Jochen Guck

Max Planck Institute for the Science of Light (MPL), Germany

Background

Dr. Rui Ma is working on the development of optical microscopy systems at MPL, and is currently involved
with building a light sheet microscope in order to image zebrafish, a biological model system used by
colleagues at MPL to study spinal cord injury.

This imaging is done with genetically modified zebrafish that express a fluorescent protein, such as GFP.
These fish are imaged with fluorescent light-sheet microscopy at 10x in order to image the entire fish, but
Dr. Ma has plans to image at higher magnifications with higher NA in order to image the spinal cord in more
detail.

Using a custom-built OpenSPIM-based light-sheet microscope, image stacks of zebrafish are taken at
different angles at different time points, and the samples are monitored for several days.

Figure 1: Zebrafish sample imaged with a Kinetix sCMOS on a custom light sheet system, built by Dr. Rui Ma. The image shows a zebrafish modified to express GFP, over 2 mm in length. Zebrafish sample provided by Dr. Stephanie Moellmert and Dr. Daniel Wehner.

Challenge

Live zebrafish make for large and dynamic microscopy samples, meaning an imaging system would need a sufficient field of view to capture an entire sample.

As well as a large field of view that matches up with a light sheet system that works with large samples, sensitivity is needed in order to image a range of different fluorophores at different wavelengths, requiring a camera with a high peak QE as well as a broad sensitivity across the visible spectrum.

The Kinetix22 has a uniform background, high sensitivity and low read noise, allowing us to collect enough photons to make precise measurements.

Solution

The Kinetix sCMOS is the ultimate solution for light-sheet microscopy, featuring a huge 29.4 mm field of view, combined with high speeds, high resolution, and sensitivity from 200-1000 nm range.

Dr. Ma uses the Kinetix with her custom-built light sheet system, and described her experience, “This system was built with the Kinetix in mind, we wanted to use the larger field of view, higher QE and faster framerate, compared with other cameras.”

“The Kinetix matches well to our light-sheet application, the large field of view is a big benefit for us, especially if we want to image our large samples without stitching.”

“Overall the Kinetix is a good camera. The zebrafish is in the center of our field of view and we can crop the sensor to capture it, this reduces the data size. We also tried other fluorescent proteins and the sensitivity is good for these too.”

Light Sheet Optical ThermometryCustomer Stories

Dr Brian Patton, Mr Petros Hadjichristodoulou
Department of Physics, University of Strathclyde, Glasgow

Background

Dr. Brian Patton and Ph.D. candidate Petros Hadjichristodoulou are involved in microscopy development and incorporating technologies into imaging. One such technology is the use of nanodiamonds (NDs), these contain natural defects known as nitrogen-vacancy centers that can be modulated by illuminating the NDs, which will emit fluorescence. NDs are also non-toxic and photostable, and as such are used by the lab of Dr. Patton as biological probes for thermometry, measuring the temperature at a cellular level, even measuring temperature gradients across a whole cell population.

Dr. Patton explains his research, “Our first ND application will be thermometry, cells generate heat as a by-product of chemical processes, and so cellular activity can be measured via thermometry. This is a huge step towards more challenging measurements, such as measuring magnetic fields generated by biological activity, so once thermometry measurements are feasible you can look at sensitivity, and get interesting answers to biological questions.”

One current project for Dr. Patton is the design of a novel technique that combines several factors, namely using ND thermometry to study entire nematode worms (C. elegans) at the nanoscale using light-sheet microscopy with adaptive optics.

Figure 1: Optical bench setup with Prime BSI sCMOS (purple).

Challenge

While C. elegans have been studied with ND thermometry in widefield, or imaged using light-sheet microscopy, and light-sheet systems have been developed that use adaptive optics, the combination of light-sheet imaging, adaptive optics, and optical thermometry has not been done before. This results in a highly complex and demanding technique that presents unique and potentially unforeseen challenges.

When using a light-sheet, typical challenges involve the need for high resolution across a large field of view, while maintaining a low noise level for high sensitivity. Alongside this, when imaging large volumes it is important to have a high-speed detector in order to capture a sufficient number of volumes per second, especially with whole-organism light sheet.

The Kinetix22 has a uniform background, high sensitivity and low read noise, allowing us to collect enough photons to make precise measurements.

Solution

An ideal camera solution is the Prime BSI sCMOS, which features high resolution (2k x 2k sensor with 6.5 μm pixel), a large FOV, and fast imaging speeds. The Prime BSI is a high sensitivity sCMOS, featuring both high quantum efficiency (QE) for maximizing signal collection across a wide range of wavelengths, and low-noise CMS mode for minimizing noise levels.

Dr. Patton shared his thoughts on the Prime BSI, “The final version of our system on the table was designed for the Photometrics [Prime BSI]… the speed and QE we thought were really good, as well as the fact that it extended into the emission wavelength range for the ND.”

“Our volumetric imaging requires speed, so a CMOS is a natural choice. The Prime BSI matches particularly well with our required performance. In particular, the dedicated PCIe board allows us to stream the data from large datasets directly to storage, and this allows us to do the post-processing. This is helpful when developing and debugging new imaging approaches and when imaging moving animals. For future development, the range of acquisition modes will allow us to try algorithmic improvements, and the sensitivity increases the chances of success when in low signal-to-noise regimes.”

Mr. Hadjichristodoulou also told us about software, “We are able to run the [Prime BSI] using Python, and downloaded the Python wrapper from the Photometrics website. The PVCAMtest software is also great as you can access all the functionality of the camera as well before implementing it in software.”

Fluorescence and ElectrophysiologyCustomer Stories

Prof. Naoki Yamawaki

Department of Biomedicine, Aarhus University, Denmark

Background

Prof. Naoki Yamawaki is a neuroscientist at Aarhus University, who told us about his research, “We are interested in understanding how stress affects the neuronal connections and communication in the brain, I am responsible for running the lab, doing experiments, mentoring, teaching students, and more.”

Prof. Yamawaki’s main research approaches include microscopy for imaging the brain and the neuronal circuits within, often using viral tracing and optogenetics combined with electrophysiology. These experiments are done on both healthy brains and brains in various disease states, in order to examine how certain neural networks act in these states.

Figure 1: A 4x brightfield image showing a recording pipette and the cytoarchitectonic structure of the cortex, taken with the Moment CMOS.

Challenge

Patching tissue samples and using recording pipettes is a highly technical technique that brings challenges, both in order to patch a select area of the tissue, to ensure a good signal quality can be maintained during recording, and to avoid damaging brain tissue samples.

Prof. Yamawaki mentioned he had previously used a CCD camera; these devices often have limited speeds (less than 10 fps) and high levels of read noise (more than 10 e-), making it a challenge to image dynamic live samples or to detect low signals and image in low-light environments in order to avoid photobleaching.

A new imaging solution would need to feature both high speeds and high sensitivity, as well as high resolution and a large field of view in order to optimally image brain tissue slices and detect electrophysiological signals or fluorescently-labeled neurons. For this application, it was important to choose a flexible yet powerful camera.

The Kinetix22 has a uniform background, high sensitivity and low read noise, allowing us to collect enough photons to make precise measurements.

Solution

The Moment CMOS is the right tool when the goal is fast, high resolution imaging in a simple and highly compact package. The Moment achieves 50 fps across a 7 megapixel sensor, all with low read noise and a 17.5 mm diagonal field of view.

Prof. Yamawaki told us about his experience with the Moment, “We use the Moment for two things: ex vivo electrophysiology and fluorescence microscopy, in both cases we care about the sensitivity and resolution (bit depth and pixels) for imaging… In addition to sensitivity and resolution (chip size), I also considered the potential frame rate and controllability, especially its compatibility with NI data acquisition board (DAQ) and
open-source software.”

“The Moment has been working great so far, it is flexible, has low-noise, and was easy to install… [Teledyne] Photometrics staff were very helpful in every aspect such as determining the camera, purchasing, and troubleshooting.”

Organoid Light Sheet ImagingCustomer Stories

Dr. Franziska Decker

Control Theory and Systems Biology Lab, ETH Zürich, Switzerland

Background

Dr. Franziska Decker aims to follow the differentiation of tissues and the development of organoids in 3D using a custom-built light sheet microscope. This work is mainly done with kidney organoids and gastruloids.

Experiments involve the light sheet imaging system in order to scan through layers of cells in an organoid. By taking z-stacks through the organoid at regular intervals over the long term, growth can be monitored. Due to the large size of the samples, more penetrative wavelengths are preferred, leading Dr. Decker to use near-infrared fluorescent proteins such as miRFP.

Figure 1: Image of a gastruloid taken with the Iris 15 on a light sheet microscopy system. The organoid is labeled with a DNA marker miRFP-H2B, which emits in the far red.

Challenge

Dr. Decker outlined some of the challenges she faces when imaging, “Ideally we want to use light sheet microscopy to watch organoids grow up to a millimeter or more in size, for that we need a large field of view with enough resolution to be able to see the nuclei of the cells for tracking.”

“We sometimes have 300-400 z-stack planes to image in order to get through the entire organoid, if we do that with three different channels it’s also nice to have speed so imaging doesn’t take a full hour.”

A suitable solution for this light sheet application would require a large field of view, a small pixel size for high resolution and low magnification (16x), as well as speed in order to increase the throughput and perform more efficient multidimensional imaging of large samples over the long term.

The Kinetix22 has a uniform background, high sensitivity and low read noise, allowing us to collect enough photons to make precise measurements.

Solution

The Iris 15 is designed for large field of view, low magnification light sheet imaging, making it the ideal solution for this application. With a small 4.25 um pixel and a massive 25 mm field of view thanks to the 5056 x 2960 pixel array, the Iris 15 is a high-throughput sCMOS with very high resolution, all operating at 30 fps.

Dr. Decker told us about her experience with the Iris 15, “we talked to various companies and in the end, I decided on the Iris [15], mainly because of the small pixel size and huge field of view, it is ideal for the imaging we want to do… so far I really like it and I’m happy with the images and the integration with Micro-Manager.”

Plant BioluminescenceCustomer Stories

Dr. Matt Jones

Institute of Molecular Cell & Systems Biology, University of Glasgow

Background

Dr. Matt Jones leads a group of circadian biologists working at the University of Glasgow, who research internal circadian rhythms in plants and how they respond to light at different times of day, with an aim to manipulate these responses and improve plant growth and crop yields. Plants typically use their circadian rhythm to measure day length and enable appropriate responses to environmental cues. Dr. Jones is studying this system using a light source to illuminate plant samples, modifying the colour, duration, intensity, and timing of the illumination and determining any changes in response.

Dr. Jones uses transgenic plants that excess firefly luciferase, a light-emitting enzyme, which enables these plant samples to emit endogenous light. This light is measured using bioluminescence, collecting the emitted light over a long exposure (>10 minutes). Changes in emitted light are measured over several days, determining the circadian rhythms of these samples.

Figure 1: Image shows high throughput imaging of bioluminescence signals from hundreds of plants (each bright spot is a single plant sample) across long exposures, taken with the Retiga LUMO CCD.

Challenge

The lab of Dr. Jones uses bioluminescence imaging to study plant responses to light, which typically involves very long exposures (20 minutes and more) in order to collect luminescence from plant subjects over time. CMOS cameras face issues with dark current build-up when operating at such long exposures, meaning CCD technologies are required.

Hundreds of plant samples need to be imaged at once, within a dark box (to avoid autofluorescence). This means a suitable CCD would need a large field of view (FOV) and a low noise, in order to maximize throughput and collect bioluminescence from a large number of plants over the long term, in ultra low light conditions.

Dr. Jones commented on the need for throughput, “It has to be high throughput as we have to capture every plant in every image, automating this with a camera makes this a lot easier… the camera is running non-stop for at least seven days.”

The Kinetix22 has a uniform background, high sensitivity and low read noise, allowing us to collect enough photons to make precise measurements.

Solution

The Retiga LUMO CCD is the Teledyne Photometrics answer to ultra-low light luminescence imaging, specifically designed to capture these signals using long exposures up to 60 minutes. Combining this with deep sensor cooling, ultra-low noise modes, advanced correction technologies (Defective Pixel Correction and Dynamic Dark Frame Correction) and a 16 mm diagonal FOV, the LUMO is a powerful choice for bioluminescence imaging.

Dr. Jones spoke to us about his experience with the LUMO, “We went for the LUMO because we wanted to guarantee the bioluminescence signal… One of the benefits of using the LUMO is that we can control it using our open-source software… the noise reduction features have been awesome, it’s worked really well.”

“We have exclusively been using LUMO cameras in our Glasgow lab, it’s very difficult for competitors to match [Teledyne Photometrics] on price now that we have our own imaging system, the flexibility is very useful for me… and the support you provided was great when we were looking for extra camera settings.”

Simultaneous Multichannel SMLMCustomer Stories

Dr. Ian Dobbie, Dr. Jingyu Wang

Dynamic Optics and Photonics Group, University of Oxford

Background

Both Dr Ian Dobbie and Dr Jingyu Wang work with advanced optics and imaging systems at the University of Oxford. One of their projects aims to achieve the maximum possible 3D imaging resolution in multicolour single-molecule localisation microscopy (SMLM). This involves the use of a 4π-SMS microscope that projects 4 interference images onto the camera.

In order to visualise 2 or 3 dyes simultaneously, as well as collecting the light transmitted by the dichroic through the conventional fluorescence imaging path, this imaging system also collects the light reflected by the dichroic away from the fluorescence imaging path, the so-called ‘salvaged fluorescence’. The ratio of the conventional to salvaged fluorescence intensity can be used to distinguish between two or three dyes with closely spaced emission wavelengths, allowing simultaneous multichannel 3D SMLM with a single excitation laser.

As the multichannel data is collected simultaneously on a single camera, drift between channels is non-existent, and chromatic aberration is minimised as the emission wavelengths are very close. This means that spatial relationships between the molecular locations are reliable down to and below the 3D resolution, of about 20 nm in all directions.

Figure 1: A reconstructed image showing localisations of multiple dyes, imaged simultaneously. This setup involved
two sCMOS cameras (including the Prime BSI) to provide a ratio and form multicolor localisation images.

Challenge

Maximizing signal collection is vital with single-molecule applications. In this system, the illumination laser wavelength is blocked with several stacked emission filters. This salvaged fluorescence signal is also a small fraction of the conventional fluorescence signal so sensitivity and high signal to noise are critical in this application. Additionally, with large 3D volumes requiring a high number of image frames to localize a large number of single molecules, high frame rates are important while preserving the sensitivity and signal to noise.

The Kinetix22 has a uniform background, high sensitivity and low read noise, allowing us to collect enough photons to make precise measurements.

Solution

The high speeds, low read noise, and high quantum efficiency make the Prime BSI a great solution for this application. Dr Dobbie was pleased with the Prime BSI, “In this extremely challenging imaging application we find that the Prime BSI is an excellent camera. We have had excellent support from Photometrics assisting us in utilizing this state-of-the-art camera in a cutting-edge super-resolution optical microscope.”

Single Molecule TIRFCustomer Stories

Roman Barth

Cees Dekker Lab, Department of Bionanoscience, Delft University of Technology, Netherlands

Background

The Cees Dekker lab at TU Delft works with single-molecule imaging techniques in order to explore life at the nanoscale. Roman Barth is a PhD candidate in the Cees Dekker Lab, running experiments using the two TIRF single-molecule imaging systems in the lab.

These imaging experiments are focused on proteins that interact and bind to DNA, particularly structural maintenance of chromosome (SMC) proteins. Mr. Barth explains further, “A strand of DNA is tethered to a surface with two biotin handles, in between these handles the DNA is loose and moves around… we flow fluorescently labeled proteins over the DNA, and image protein-DNA and protein-protein interactions with multiple colors.”

The overall project looks at SMC proteins and how they extrude a loop of DNA, with an aim to understand DNA looping and interactions of loop extruders with other DNA-interacting proteins on chromosomes within cells.

Figure 1: Individual DNA strands tethered to a surface and stained with SYTOX orange, imaged with the Prime BSI Express CMOS in low-noise CMS mode (inferno-mpl LUT applied in software).

Challenge

When looking at many DNA strands on a surface, high-throughput imaging is a must. Mr. Barth told us of a previous EMCCD solution that limited throughput, “The field of view was small, just 512×512 pixels, so we just didn’t see much on it. We wanted to enlarge the FOV with a larger camera and more pixels, as well as change our magnification from 100x to 60x as we were over magnifying.”

“The more DNA I can image, the better the statistics per experiment, which is helpful for experiments with a low probability of the event we want to observe. SMC protein loop extrusion usually only happens for a few minutes in vitro, the more events we capture within this critical frame right after protein flush in, the better the statistics and the less often we need to repeat something.”

As the signals from the DNA are bright and sufficient speed can be achieved with 100 ms exposure, the best solution would be a larger field of view (FOV) camera with a small pixel that can maximize throughput while matching to 60x magnification.

The Kinetix22 has a uniform background, high sensitivity and low read noise, allowing us to collect enough photons to make precise measurements.

Solution

The Prime BSI Express is an ideal solution for this application, featuring a 2048×2048 array of small 6.5 μm pixels across a 19 mm diagonal FOV, increasing the available imaging space. Mr Barth spoke about his experiences with the Prime BSI Express CMOS, “60x magnification works well with the [6.5 μm] pixel size of the Prime BSI Express, we are at a pixel size of 100 nm at the sample, just what it should be.”

“We spoke to you immediately and it was quite clear we were going to go with [Teledyne] Photometrics, we didn’t even talk to another company… Setting up [the Prime BSI Express] and letting it talk to the computer was as simple as plugging in a USB, there was almost nothing you can do wrong… totally user-friendly and we see everything we want to see.”

“I was really surprised how small it is, I have a lot of space suddenly! It was also nice that you provided the USB and triggering cables with software, it’s everything you need and works well through MicroManager.”