Live yeast imaging identifies new role for mating blocksResearch Stories

Introduction

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers a signaling cascade, which represses mating and initiates meiosis.

In this research, Prof. Sophie Martin and the team from the University of Lausanne establish a system to demonstrate that mating blocks not only safeguard zygote ploidy but also prevent cell death caused by aberrant fusion attempts. This was done using long-term imaging and flow cytometry, and we identified previously unrecognized and independent roles for Mei3 and Mei2 in yeast zygotes.

Figure 1: Mei3 promotes G1 exit in stable diploid cells. (A) H1Δ17 diploid cells expressing mCherry and sfGFP from P- and M-cell-specific promoters pmap3 and pmam1 24 hours after removal of nitrogen. Arrows point to shmoo-like projections in mei3Δ and mei2Δ mutants.

Imaging Solution

These long-term fluorescence widefield imaging experiments were performed using a Prime BSI sCMOS camera in combination with a DeltaVision imaging platform and softWoRx software.

By pairing the balanced 6.5 um pixel of the Prime BSI with a 60x oil objective, high spatial resolution and great image quality were achieved. This was maintained over the long-term time-lapse experiments thanks to the reliability of the Prime BSI.

The Prime BSI is highly sensitive thanks to a combination of near-perfect quantum efficiency and low noise levels, and can image over a large field of view at a high speed.

Nematostella OPM Light SheetCustomer Stories

Dr. Rory Power

Advanced Light Microscopy, EMBL Heidelberg, Germany

Background

Dr. Rory Power is a staff scientist and engineer at the advanced imaging center of the EMBL headquarters in Heidelberg. Involved in a variety of projects, Dr. Power also oversees Ph.D. students involved in building custom light sheet imaging systems.

Dr. Power described their light sheet imaging system, “It’s an oblique plane microscope (OPM) that uses a single objective, which allows us to do light sheet microscopy in a more traditional inverted, epifluorescence setup with less restrictions on sample geometry and using a water dipping objective.”

“This system is for imaging of a little tentacle monster Nematostella, which are interesting from a morphological and behavioral point of view, how their muscle hydraulics and neural dynamics influence their development and motion. We are using our light sheet system to image contractile waves of motion within these animals.”

Figure 1: Animation (top) and image montage (bottom) of a Nematostella in motion, taken with the Kinetix sCMOS. Images are taken at two second intervals, scale bar is shown. A contractile wave of motion is seen from image to image. The figure is taken from referenced paper, Singh et al. 2022.

Challenge

Dr. Power told us about the imaging challenges he faces in his work, “The Nematostella cannot be constrained, these are fully grown animals that need to move around and undergo normal behaviors. These samples are moving freely in a droplet, so we only image them when they move into the camera’s field of view. We also want to go fast so we can capture dynamic motion while retaining a large FOV.”

In addition to this, Nematostella is a light-sensitive organism, meaning a low light regime is needed, reducing signal levels and requiring a highly sensitive camera. Due to their size (~1.5 mm in length, ~200 μm in width), the Nematostella can only be imaged with a light sheet when the body axis is in a certain orientation, further requiring a large FOV to increase the number of good imaging events.

Finally, in order to further increase the FOV, a low magnification objective is used which inherently has a lower numerical aperture, which can be challenging when using an OPM imaging regime.

The Kinetix has a larger sensor and much higher quantum efficiency, in both the visible and the UV, compared to our previous camera systems, we are happy with the results.

Solution

The Kinetix is an ideal solution for this imaging application, featuring both a very large 29 mm FOV combined with a very high speed of 500 fps across this entire sensor. The Kinetix is widely used for light sheet and means researchers no longer have to compromise.

Dr. Power described his experience with the Kinetix “The speed and the large chip of the Kinetix were the things that made it ideal for our application. Right now, the full speed of the Kinetix hasn’t even been leveraged in recent experiments, we can easily go a factor of 5x faster.”

“Software setup was absolutely fine for capturing images when triggered, everything worked so no problems. Introducing the camera to the system hardware was easy.”

The Kinetix sCMOS replaced a traditional sCMOS camera used for alignment/testing due to the better performance and allows for high-resolution imaging across a large FOV while maintaining a high speed.

Reference

Singh R., Subramanian K., Power R.M., Paix A., Ikmi A., Prevedel R. (2022) An oblique plane microscope for mesoscopic imaging of freely moving organisms with cellular resolution, bioRxiv 2022.07.15.500249; doi: https://doi.org/10.1101/2022.07.15.500249

PLIF Combustion ImagingCustomer Stories

Prof. Dirk Geyer, Martin Richter (M.Sc.), Adrian Breicher (M.Eng.)

Laboratory for Optical Diagnostics and Renewable Energy (ODEE), Department of Mechanical and Plastics Engineering, Darmstadt University of Applied Sciences, Darmstadt, Germany

Background

The group of Prof. Dirk Geyer, including Ph.D. students Martin Richter and Adrian Breicher, work towards the decarbonization of energy conversion. They told us about their research, “Our main research field is the combustion of promising new fuels for the future such as hydrogen and ammonia, which, unlike methane, contain no carbon in their molecular structure and therefore produce no CO2 emissions in the combustion process.”

“In our recent work, we are looking at the co-firing of methane (CH4), the main component of natural gas, and hydrogen (H2). We have many combustion systems operating with natural gas, and we could substitute some of the CH4 for H2, but this has effects on combustion. To understand the fundamentals of these effects, we investigated laminar flames with reduced complexity, such as Bunsen flames: if we burn pure CH4 we observe a smooth flame cone, if we add certain amounts of H2 cellular structures start to appear, so we are looking into the structure of these with planar laser-induced fluorescence (PLIF).”

“PLIF can analyze molecules/species that occur during the combustion process, such as the OH radical, which tell us where reactions are happening, and we can then map other measurements onto this.”

Figure 1: PLIF data and images of OH radicals within Bunsen flames at different methane:hydrogen ratios, acquired by the Kinetix sCMOS. The top row shows the raw OH radical signal, the middle row shows the same data normalized and averaged to reduce noise due to thermal effects, and the bottom row shows the Bunsen flame shape from the side, the dashed white line indicating the cross-section for the above data.

Challenge

Using PLIF to study combustion systems is a challenging application, Mr. Breicher told us more, “We use a specific wavelength to excite these OH radicals, which emit at a distinctive wavelength in the UV at ~315 nm, so we use an intensifier to increase the low signal and shift it towards visible light, which we image with a camera.

“The intensifier introduces a lot of noise and limits our spatial resolution, and is also 25 mm in diameter, ideally we would capture this in its entirety.” In addition, PLIF for combustion systems involves imaging a dim fluorescence signal against the bright background of a flame, requiring a highly sensitive camera with a high dynamic range.

The Kinetix has a larger sensor and much higher quantum efficiency, in both the visible and the UV, compared to our previous camera systems, we are happy with the results.

Solution

The Kinetix features a large sensor, high resolution, and high sensitivity, thanks to the combination of near-perfect 95% peak quantum efficiency (QE) and ultra-low noise contributions.

The group of Prof. Geyer told us more about their experience with the Kinetix, “The main reasons we got a Kinetix is the large sensor and much higher quantum efficiency in both the visible light and the UV compared to our previous camera systems. With the UV sensitivity, we can try to image native emissions without the intensifier.”

“The Kinetix is also a general improvement for the camera systems in our lab and will also be used in the future for other techniques like chemiluminescence imaging of flames due to the sensitivity over such a broad wavelength range, as well as the low noise.”

“We are happy with the results and look forward to using the Kinetix in future experiments, such as with more complex flames or other techniques.”

X-ray Beam CharacterisationCustomer Stories

Dr. Roelof van Silfhout

The Department of Chemistry, KU Leuven, Belgium

Background

Dr. Roelof van Silfout is a researcher at KU Leuven, working on a variety of projects mostly concerning x-rays. Dr van Silfout explained one of his experiments, “We are interested in defining or measuring the position of a hard x-ray beam. We have an indirect detection system where we use a very high-resolution scintillator material that is lens-coupled to a camera, so the camera is focused on the back of the scintillator while the x-rays enter from the front. The camera is then at a 45° angle from the light path to avoid any damage to the camera sensor. We measure back-reflected x-rays, these reflected x-rays have a very low intensity, 7 orders of magnitude less intense than the x-ray beam itself.

Dr van Silfout’s research interests involve the application of x-rays in structural studies of materials at the atomic scale, as well as optical characterization of these x-ray beams. With a role at both KU Leuven and the European Synchrotron Radiation Facility (ESRF, France), Dr van Silfout has a strong track record of innovation and experimentation with high-precision instruments for x-rays.

Figure 1: Horizontal x-ray beam focusing with the Kinetix22 sCMOS. The top row shows graphs of the row summation of each corresponding image below, the solid line indicating horizontal x-ray beam profile and dashed line indicating vertical (only the horizontal plane is being focused. The bottom row shows images of x-ray beam adjustment taken with the Kinetix22 sCMOS. The label r’ indicates the radius of curvature of the mirror above and below the optimum of 693 nm.

Challenge

X-rays detected by the camera are of an extremely low intensity, requiring a highly sensitive camera in order to have a sufficient signal-to-noise ratio to detect the x-ray beam scatter. Dr. van Silfout told us about the challenges he encounters, “We typically have to sum the detected signal and perform procedural background reduction in order to detect these x-rays… Because the x-rays have low signal levels, this pushed us towards using dedicated cameras with low noise, I’m always on the lookout for cameras which have good performance, high sensitivity, and fast readout.”

“I got very interested in sCMOS, I usually do photon transfer curves to measure camera performance. However, some sCMOS cameras have two different gain amplifiers which are merged by software, and somewhere in the middle there can be huge anomalies which are really awkward when collecting high dynamic range images, introducing artificial issues.”

The Kinetix has a larger sensor and much higher quantum efficiency, in both the visible and the UV, compared to our previous camera systems, we are happy with the results.

Solution

The Kinetix22 sCMOS is a powerful solution for indirect x-ray imaging, equipped with sub-electron read noise levels that ensure even the weakest x-ray signals can be reliably detected. Dr. van Silfout described his experiences with the Kinetix22, “For me, the reasons the Kinetix22 was a good choice was that it perfectly matches our aperture at 22 mm, it has the really low noise characteristics that we were looking for, it is very fast, and the range of readout options and software compatibility is really useful when you need to synchronize your imaging setup.”

“The 22 mm FOV was a very good selling point as it matches our C-mount microscopes, the small 6.5 um pixel size was also a very strong point, as it gave us a high resolution… I also like the ability to make multiple regions of interest around the sensor, you can do it very nicely in software control, it’s very fast if not simultaneous.”

“The photon transfer curves with your cameras did not have the anomalies we would usually see with other sCMOS… I looked at other sCMOS cameras, for me, it was a no-brainer to pick the Kinetix22. I’m very happy with it, it really does what it promises.”

“The support I got from Photometrics from my initial contact was excellent, looking at the whole picture in terms of software support, the features of the camera, and of course the price, for me it was definitely a no-brainer. I can’t think of a reason not to buy it!”

Single-Molecule FRETCustomer Stories

Prof. Keith Weninger

Department of Physics, North Carolina State University, US

Background

The lab of Prof. Keith Weninger develops single-molecule fluorescence methods to study biomolecular systems, with a particular focus on FRET to study proteins involved in DNA mismatch repair. Prof. Weninger further explained his research, “I do single-molecule FRET experiments on tethered DNA molecules with surface-immobilized TIRF microscopy.”

“Most of our lab is focused on DNA mismatch repair. After copying DNA, the DNA polymerase has an error rate, and a set of proteins follow behind and proofread, repairing any base-base mismatches. DNA polymerase makes a mistake one in every million bases, and the proofreaders improve this by a factor of a thousand, resulting in one in a billion errors from DNA copying in cells. We are interested in these proteins and how they work, and when they don’t work right it’s associated with various cancer phenomena.”

“We do different things, such as build short DNA molecules with mismatches and tether them to a surface and flow the proteins over them. Or we can put a polystyrene bead onto tethered DNA to see the motion and range. For our FRET imaging, we can put FRET fluorophores on the DNA, protein or one on each.”

Figure 1: Top image shows two Prime 95B sCMOS cameras on a Cairn TwinCam for simultaneous multichannel imaging. The bottom images are FRET for the red laser excitation, showing the acceptor channel (left) and donor channel (right), each acquired by a separate Prime 95B.

Challenge

This format of single-molecule microscopy can be highly challenging, due to the combination of a very low signal level and the need for high-speed imaging in order to capture molecular dynamics. Prof. Weninger described his need for speed, “In every experiment we want to push temporal resolution, we basically always work at the limits of technology and so we want to image as fast as we can to capture conformational dynamics and interaction, using sub-millisecond exposures. There are also a variety of different timescales so we need to be flexible.”

“We are at a very low signal, photon-counting level as we are doing single molecule fluorescence. We can increase the intensity of the light source but this can result in bleaching, so we also need good triggering of experiments to minimize bleaching too.”

The Kinetix has a larger sensor and much higher quantum efficiency, in both the visible and the UV, compared to our previous camera systems, we are happy with the results.

Solution

The Prime 95B sCMOS is an ideal solution for high sensitivity single-molecule microscopy, featuring near-perfect 95% signal collection thanks to back-illumination, low noise levels, along with a large 11 μm ideal for high magnification molecular imaging work.

Prof. Weninger described his imaging experiences, “We set up two Prime 95Bs on a TwinCam and they produce comparable data to our previous EMCCDs. We were using them for slow imaging but now we can go fast. We can also use the TwinCam for polarization.”

The use of two cameras allows for high-speed, high-throughput FRET imaging, with specifications well matched for a highly sensitive TIRF imaging system.

Live-Cell Single-Molecule FluorescenceCustomer Stories

Prof. Christof Gebhardt, Mr. Devin Assenheimer

Institute of Biophysics, Ulm University, Germany

Background

Prof. Christof Gebhardt, along with PhD student Devin Assenheimer and the team from Ulm University told us about their research. “We perform single-molecule fluorescence microscopy both in vitro and in vivo within cells, with the aim to also image small organisms. We use a light sheet-like illumination scheme where the sheet thickness can be set using a pinhole. This, in combination with organic dye fluorescent labels, gives us sufficient signal to noise for single-molecule detection even at very high temporal resolutions. We can basically image almost anywhere in a cell, we often image a couple of microns above the glass surface.”

“With live cell single-molecule experiments, we can measure the kinetics of biological molecules. For example, we can look at molecular motors and investigate how they move and measure their velocities. Conditions used in vitro with purified proteins create an artificial environment, so velocities measured in vitro might be different than what is happening in vivo in the live cell.”

Figure 1: A single molecule within a live cell imaged using single-molecule fluorescence with the Prime BSI sCMOS. The molecule in question is a molecular motor, with the motion indicated by the pink tracking line.

Challenge

Traditional single-molecule fluorescence microscopy of fixed cells carries its own challenges, using live cells introduces additional complexity. Prof Gebhardt explained further, “Live cells cannot be permeabilized and cleared to minimize autofluorescence. Thus the background is higher compared to imaging in fixed cells.”

“To image single molecules, we need the spatial resolution high enough to distribute the fluorescent signal over a couple of pixels on the camera sensor. Therefore the magnification is such that we only have one cell in the field of view.”

“Another challenge is that live cells do not tolerate high laser power. To measure molecular kinetics, we typically want to go to a high temporal resolution of 100 Hz, so 10 ms exposure. Since we cannot increase the laser power above a critical value, this means the signal of a single molecule is only a few hundred photons. Thus, we are interested in a low read noise in order to get a good signal-to-noise ratio.

”This application requires a robust and flexible imaging device that can image with high sensitivity without losing spatial or temporal resolution, all at a high speed and across a large field of view.

The Kinetix has a larger sensor and much higher quantum efficiency, in both the visible and the UV, compared to our previous camera systems, we are happy with the results.

Solution

The Prime BSI sCMOS is the ideal solution for this application, with low overall noise and a large camera sensor with a small pixel size. Prof. Gebhardt described his experiment with the Prime BSI, “Before, we were working with EMCCD cameras due to the high signal to noise, but now we feel that the sCMOS takes over in that respect. We can also benefit from higher temporal resolution even in a low-light application. We can make the regions of interest small and go for a very high speed.”

“The big advantage of these sCMOS cameras is the high framerate possible with low signals. One of the reasons we got the Prime BSI was because it is fast with a large field of view, along with the low noise levels.”

Mr. Assenheimer spoke in terms of software and ease of use, “We are using the Prime BSI with MicroManager, the experience with the camera is good, really easy to implement. I’ve worked with it for a while now and not experienced problems.”

The Prime BSI sCMOS also allows for future improvements of experiments, with the large field of view and advanced hardware triggering systems allowing for simultaneous multifocal or multichannel imaging when paired with a splitter.

Single Molecule Localisation MicroscopyCustomer Stories

Prof. Rainer Heintzmann and Alexander Jügler

Biological Nanoimaging, Leibniz Institute of Photonic Technology (Leibniz-IPHT)

Background

Senior Ph.D. student Alexander Jügler works in the Heintzmann Lab, which studies super-resolution imaging applications such as single-molecule localization microscopy (SMLM), and works on improving the phototoxicity and resolution of such techniques while making them easier to work with.

As Mr. Jügler mentioned, “In my imaging, I create local minima and shift them by a few nanometers using a spatial light modulator. We are able to detect the nanometer shift but to evaluate the local minima quality we need to find stable and bright fluorescent particles a few nanometer in size. The 110 nm microbeads we are using right now are way too big.

“Later we will use all kinds of biological samples with the intent to develop a system that is able to analyze toxic fungi, which create pores a few nanometres in size.”

Figure 1: An image of the Prime BSI CMOS on an optical bench, set up for single molecule localisation microscopy in the lab of Prof. Rainer Heintzmann.

Challenge

When performing super-resolution imaging, especially for SMLM, it is vital to have a high signal-to-noise ratio in order to best detect these particles. This requires both maximizing the signal collection with a high quantum efficiency and balanced pixel size, as well as minimizing the noise level with low read noise.

In addition, Mr. Jügler stated, “A big field of view to track the particles is also needed, a big advantage of our technique is that we have to track across a big FOV.” As the imaging system and technique can make use of a large FOV, it would be best paired with a camera that also features a large sensor size.

The Kinetix has a larger sensor and much higher quantum efficiency, in both the visible and the UV, compared to our previous camera systems, we are happy with the results.

Solution

The Prime BSI has a remarkable signal-to-noise ratio due to the near-perfect 95% quantum efficiency and the extremely low read noise, all while running at a high speed with a large sCMOS field of view. This makes the Prime BSI a great solution to SMLM imaging.

As outlined by Mr. Jügler, “I am working in this super-resolution field and trying to implement these techniques, which is why I need very good cameras with high quantum efficiency, and are very fast and do a good job at high speed. In our lab, we have a number of cameras available and I have decided on the Prime BSI because it has these characteristics.”

“My overall statement is that the [Prime BSI] is very good for my experiments. For me the experience of the camera and the quantum efficiency was great. Many students want to have a camera and I was lucky to get it! It’s one of the best cameras in our lab right now.”

The Prime BSI is running in MATLAB in the Heintzmann lab and Mr. Jügler mentioned how easy it was to implement the Prime BSI in the system with this software.

Spectral Optical Coherence TomographyCustomer Stories

Prof. Aart Verhoef

Department of Soil and Crop Sciences, College of Agriculture & Life Sciences, Texas A&M University, US

Background

The Verhoef Lab designs novel laser light sources to enhance existing and support novel imaging methods. Their systems might result in signals ranging from the detection of a few photons for super-resolution imaging, through to differentiating small differences amongst many tens of thousands of photons.

One lab focus is optical coherence tomography (OCT), which uses the ability of light to interfere with itself to map structures in tissues. Spectral OCT uses multiple colors of light to map a tissue in depth all at once.

Figure 1: Bessel beam OCT setup. The spectrometer uses a Prime 95B sCMOS camera to acquire spectra with a high spectral resolution and frame rate. Using a Bessel beam, the depth of focus of the setup is more than doubled compared to a Gaussian beam setup.

Challenge

Spectral OCT generates a map of the position in depth, but in one shot measurement. The changes in intensity due to interference of a broad-band input source when unmixed mathematically give the same results as when moving the reference arm.

Spectral OCT uses broad-band light sources centered around 1 μm for good tissue penetration. Strong focusing of the light in the sample arm results in a high lateral resolution in the focal plane of the focusing lens, but this resolution deteriorates fast away from the focus. In order to improve the lateral resolution away from the focal plane of the OCT scan lens, the Gaussian beam illumination can be replaced by Bessel beam illumination, at the expense of optical losses.

With less power returned from regions of the sample, a camera with a large dynamic range, high sensitivity, and a large chip size is needed in a custom-built spectrometer.

Typically, a camera with only a few lines of pixels is used in such spectrometers. However, when working with Bessel beams (which have a pi phase-shift between the central peak and the surrounding weak ring), such cameras do not allow to distinguish between a phase-shift across the vertical direction of the beam and the absence of fringes.

The Kinetix has a larger sensor and much higher quantum efficiency, in both the visible and the UV, compared to our previous camera systems, we are happy with the results.

Solution

The Prime 95B meets the requirements for a camera with a large dynamic range, large field of view, and high sensitivity.

Prof. Verhoef told us about the Prime 95B CMOS, “The Prime 95B offers a pixel size of 11 μm, that allowed us to construct a compact spectrometer matching the spatial resolution of the OCT imaging system without dramatically oversampling the resolution. The large chip size of the 95B almost exactly matched the spectral extent of our broad-band Bessel beam light source. The high frame rates that can be achieved with the 95B allow us to obtain high-resolution OCT images within a short time. The 95B allowed us to demonstrate a substantial improvement of focal depth provided by Bessel beam OCT.”

Novel Hyperspectral ImagingCustomer Stories

Prof. Silas Leavesley

Department of Chemical and Biomolecular Engineering, University of South Alabama, US

Background

The interdisciplinary laboratory led by Prof. Silas Leavesley and Dr. Thomas Rich is working to develop novel hyperspectral imaging systems for microscopy and endoscopy. Using rapidly controllable light sources with precise spectral selection they aspire to be able to increase the number of individual sensors and probes detected concurrently. This will allow measurements of spatial and temporal fluctuations in various signal transduction cascades in cells, tissues, and organisms.

Figure 1: Spectral excitation-scanning images of a 6-label slide (Abberior with autofluorescence). Excitation wavelengths were sampled over a range of 360-540 nm, at a 5 nm increment, using a Xe arc lamp and Sutter VF 5 filter assembly equipped with Semrock VersaChrome filters, while image data were acquired using a 555 nm long-pass emission filter and Prime 95B sCMOS camera. The raw fluorescence signal can be visualized as Total Fluorescence (the summed intensity across all wavelength bands) or RGB false-colored (a visualization of 3 selected and false-colored wavelength bands). A spectral library was constructed from single-label controls (plot at right). The spectral image cube was then unmixed using non-negatively constrained linear unmixing. The residual (unaccounted for) signal is displayed as both total signal, called root-mean-square (RMS) Error, and the relative error, as a percent of the RMS signal in each pixel, called RMS % Error. Linearly unmixed images were false colored for visualization and were also merged, shown by the False Colored Unmixed.

Challenge

Acquisition of hyperspectral image data on timescales relevant to biochemical and cellular processes requires rapid selection of precise excitation wavelengths and rapid, low-noise image acquisition of resulting excitation-scanning spectral image stacks.

Given the aim of acquiring 1-5 spectral image data sets of 30 wavelength bands each second, a fast, sensitive low-noise camera is an absolute requirement to generate data of sufficient signal-to-noise ratio for interrogating cellular processes.

Previous work using widefield imaging with EMCCD cameras was successful, but the field of view and slow readout speed of EMCCD detectors limited the spatial and temporal sampling of the desired measurements. Now, Prof. Leavesley’s lab is aiming to acquire similar spectral data using spinning disk confocal microscopy to allow 3D, timelapse, and spectral imaging capabilities.

The Kinetix has a larger sensor and much higher quantum efficiency, in both the visible and the UV, compared to our previous camera systems, we are happy with the results.

Solution

The Prime 95B is a highly sensitive CMOS camera with a large field of view, matching a near-perfect 95% quantum efficiency with low read noise for a high signal-to-noise ratio when imaging.

Prof. Leavesley told us about his experience with the Prime 95B CMOS, “The Prime 95B, with its high quantum efficiency, large FOV, high readout speed, and low readout noise allowed us to gather spectral cubes at N spectral volumes per second, image cubes at >1 Hz frequencies, and will be a key component in developing future 5-dimensional imaging approaches.”

OpenSPIM for Cleared Fish BrainCustomer Stories

Dr. Franziska Curdt, Ms Laura Ziegenbalg

Institute for Biology and Environmental Sciences, University of Oldenburg, Germany

Background

Dr. Franziska Curdt and PhD student Laura Ziegenbalg use fluorescent microscopy techniques to investigate the magnetic senses of fish, namely the magnetic imaging of putative magnetoreceptors. To this end, Dr. Curdt has built several imaging systems to image large tissue samples, including a magnetoscope and an OpenSPIM-based light-sheet microscope.

Dr. Curdt told us about her research, “We are imaging volumes of cleared tissue in order to find out more about the magnetic sense of certain species, as the origin of this sense is still a bit enigmatic”

Ms. Ziegenbalg explained the choice of sample, “We want to find out how fish can perceive the Earth’s magnetic field, this involves finding brain regions that correlate with magnetic stimuli and building a reference brain atlas. While this atlas exists for zebrafish, these fish are not migratory and it is not established if they can sense the Earth’s magnetic field, so we are building a 3D atlas for the migratory rainbow trout.”

Figure 1: 3D reconstruction of a cleared zebrafish brain from three different perspectives, imaged with the Prime BSI Express. The size of the brain is approximately 5 x 2 x 1.5 mm.

Challenge

While light-sheet microscopy with zebrafish is well established and often used as a model organism or to optimize imaging protocols, work with rainbow trout is less common. While the zebrafish brain is on the millimeter scale, the brain of the rainbow trout is many times larger, up to 2 cm. This large-format tissue imaging requires a large camera sensor in order to avoid excessive tiling and stitching.

In order to use the full aperture of the 4x objective, a small pixel is needed in order to achieve sub-cellular spatial resolution and optimize for Nyquist at low magnifications.

The Kinetix has a larger sensor and much higher quantum efficiency, in both the visible and the UV, compared to our previous camera systems, we are happy with the results.

Solution

The Prime BSI Express sCMOS features a small pixel and a large pixel array, allowing for large images to be obtained that feature high resolution at low magnifications. The high signal collection and low read noise of the Prime BSI Express maximize the signal-to-noise ratio and result in high-quality images, suitable for quantitative imaging and the construction of a 3D brain atlas.

Dr. Curdt described their experience with the Prime BSI Express, “The small pixel size of the Prime BSI Express combined with the relatively large sensor is a big advantage. We are also planning a future application involving calcium imaging. We got the Prime BSI Express with this in mind so we can do fast recording due to the high imaging speeds.”

“It was simple to get the Prime BSI Express set up in MicroManager, it worked immediately. No difficulties, no problems. I also really like the customer service of Teledyne Photometrics.”