Page Type: Customer Story

Spinning Disk ConfocalCustomer Stories

Dr. Peter March, Senior Experimental Officer

University of Manchester, Bioimaging Facility

Background

The research being performed at the University of Manchester has a real-world impact beyond the lab. The team is at the forefront of the search for solutions to some of the most pressing issues in biology, medicine and health. The Bioimaging Facility delivers a broad range of state-of-the-art imaging solutions to the University, Faculty of Biology, and Medicine and Health. A key technology used in biological imaging of live cells is Spinning Disk Confocal Microscopy. Spinning Disk allows for long-term, high-speed, three-dimensional imaging of live samples with multiple channels of illumination.

Mitochondria (green, 488nm) and Actin filaments (red, 566nm).
Sample prepared by Vicki Allen, imaged using a Yokogawa CSU-X1 with a 63x oil, 1.4NA objective.

Challenge

One of the primary reasons for using Spinning Disk Microscopy is to generate confocal images without photobleaching or damaging live samples. “Bright cells are not necessarily healthy cells,” warns Peter March, senior experimental officer at the university. “Using less GFP in cells matches their natural behavior more closely,” he adds.

Correspondingly, sensitivity is among the most important features of a camera. Until recently, and due to their ability to achieve >90% quantum efficiency, EMCCD cameras were the preferred imaging device for Spinning Disk Microscopy. March and his team typically use a 60x objective, resulting in the need to address the large pixels of an EMCCD, which require extensive optical adjustments to reach acceptable sampling levels. This severely limits field of view, making samples harder to find and capture. Additionally, EMCCD cameras cause excessive, visible noise across the sample, even at high exposures.

The Prime 95B [Scientific CMOS camera] is the perfect camera for Spinning Disk – the image quality is a big improvement over our EMCCDs, and the field of view makes samples much easier to find.

Solution

The Prime 95B back-illuminated Scientific CMOS is the perfect match for Spinning Disk Microscopy because it delivers much greater image quality at a higher resolution than possible with a EMCCD camera. In addition, its sensitivity is much higher than other CMOS cameras currently on the market. March is most impressed with the increase of field of view as he shares “With EMCCD cameras, finding the sample was often an issue. Field of view is all-important, and the Prime 95B is a big improvement here.”

Without the excess noise factor of EMCCDs or the pattern noise seen in 2×2 binned front-illuminated sCMOS, March says, “The difference in image quality is huge”. The Prime 95B provides the ability to capture more of a sample at equal exposure times compared to EMCCD cameras, and it produces more impressive images as a result.

Structured Illumination Microscopy (SIM)Customer Stories

Dr. Guy Hagen, Research Associate,

University of Colorado, Colorado Springs

Background

Dr. Guy Hagen, Research Associate from the University of Colorado, Colorado Springs creates high performance image reconstruction methods and open-source software to process super-resolution microscopy data. In 2014 Dr. Hagen released ThunderSTORM, an ImageJ plug-in for automated processing of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) data.1 In 2016, he introduced SIMToolbox, a MATLAB toolbox for processing SIM data. It has the flexibility to process 2D and 3D images, including both optical sectioning and super-resolution applications, and can be used on data acquired from commercial systems.2 SIMToolbox also includes maximum a posteriori probability estimation (MAP-SIM), a super-resolution restoration method that suppresses out of focus light, improves spatial resolution, and reduces reconstruction artifacts.3 Dr. Hagen is currently developing live cell imaging using SIM, and is continuing to develop data analysis methods for super-resolution microscopy.

Cross section of a rabbit seminiferous tubule acquired using Dr. Hagen’s structured illumination microscopy system and a Prime 95B camera with a 100X/1.47NA objective (top row) or 40x/1.3NA objective (bottom row). Images were reconstructed using SIMToolbox [2]. Conventional widefield fluorescence images (left) display out of focus light, raw SIM images (center) display the illumination pattern, and the 3D SIM reconstruction (maximum intensity projection, right) shows high quality, optically sectioned images.

Challenge

SIM is a widefield fluorescence technique that uses illumination patterns with high spatial frequency to illuminate samples. Algorithms applied to a combination of images taken with different phases and orientation of the illumination pattern are used to reconstruct a high-resolution image. Reconstruction of images routinely requires many images per focal plane. Fluorescent signals need to withstand photobleaching and the acquisition rate must be fast enough to observe live cell dynamics.

The Prime 95B [Scientific CMOS camera] is the perfect camera for Spinning Disk – the image quality is a big improvement over our EMCCDs, and the field of view makes samples much easier to find.

Solution

Because multiple images are required for the reconstruction of SIM data, a sensitive camera will allow for shorter exposure times and faster acquisitions, reducing phototoxic and photobleaching effects on samples.

The back-illuminated Prime 95B Scientific CMOS camera has a near perfect 95% quantum efficiency and large 11 µm pixels, making it extremely sensitive. The Prime 95B allowed Dr. Hagen to reduce exposure times to acquire a set of SIM images in about half the time required with typical sCMOS cameras.

Pixel-to-pixel and column-to-column gain variations which are present in typical sCMOS cameras can degrade SIM images during the reconstruction process. The Prime 95B reduces image artifacts by implementing several pixel noise filters to detect and correct dynamic fluctuations, and the static variation in gain and offset is calibrated for every pixel. Because of this, raw SIM images have a reduction in noisy pixels and visible readout lines, resulting in higher quality processed SIM images.

References

  1. Ovesný, M., Krížek, P., Borkovec, J., Švindrych, Z., and Hagen, G. M. ThunderSTORM: a comprehensive ImageJ plugin for PALM and STORM data analysis and super-resolution imaging. Bioinformatics (2014), doi: 10.1093/bioinformatics/btu202
  2. Krížek P, LukeŠ T, Ovesný M, Fliegel K, Hagen GM. (2016). SIMToolbox: a MATLAB toolbox for structured illumination fluorescence microscopy. Bioinformatics (2016), doi: 10.1093/bioinformatics/btv576
  3. LukeŠ T, Krížek P, Švindrych Z, Benda J, Ovesný M, Fliegel K, Klíma M, Hagen GM. Three-dimensional super resolution structured illumination microscopy with maximum a posteriori probability image estimation. Opt Express (2014), doi: 10.1364/OE.22.029805

Single Molecule TIRFCustomer Stories

Dr. Aleks Ponjavic

Klenerman Group, University of Cambridge

Background

The Klenerman group at the University of Cambridge investigates intracellular signalling in T-cells, a vital component of the human adaptive immune response. They are particularly interested in the kinetic-segregation model of T-cell signalling which proposes that signalling is only possible when CD45 molecules on the T-cell surface are sterically excluded from the T-cell receptor site.

The group observes these cell-surface molecules using single molecule TIRF microscopy to add further structural support for the kinetic-segregation theory.

Jurkat T-cells bound to fibronectin. CD45 molecules on the cell surface labelled with Alexa 488.
A. Left: Maximum intensity projection of 500 frame 33 ms exposures taken with the Evolve Delta EMCCD camera using 250x EM gain. Right: Line profile taken through the marked area of the image (yellow line).
B. Left: Maximum intensity projection of 500 frame 33 ms exposures taken with the Prime 95B. Right: Line profile taken through the marked area of the image (yellow line).
C. Left: Maximum intensity projection of 500 frame 33 ms exposures taken with the Prime 95B binned 2×2. Right: Line profile taken through the marked area of the image (yellow line).
Cross section of a rabbit seminiferous tubule acquired using Dr. Hagen’s structured illumination microscopy system and a Prime 95B camera with a 100X/1.47NA objective (top row) or 40x/1.3NA objective (bottom row). Images were reconstructed using SIMToolbox [2]. Conventional widefield fluorescence images (left) display out of focus light, raw SIM images (center) display the illumination pattern, and the 3D SIM reconstruction (maximum intensity projection, right) shows high quality, optically sectioned images.

Challenge

The group needs to ensure reliable detection of single molecules so high signal to noise is of great importance. To achieve this, they have been using the Photometrics Evolve® 512 EMCCD camera but made the decision to purchase a Prime 95B because of its high sensitivity combined with the multiple benefits of CMOS architecture. Aleks Ponjavic, postdoctoral researcher with the Klenerman group, told us, “I was very interested to compare the high sensitivity of the Prime 95B to an EMCCD for single molecule imaging.”

The Prime 95B [Scientific CMOS camera] is the perfect camera for Spinning Disk – the image quality is a big improvement over our EMCCDs, and the field of view makes samples much easier to find.

Solution

When asked how the Prime 95B compared to an EMCCD camera, Aleks told us, “I find the performance of the Prime 95B to be comparable to state-of-the art EMCCDs but at lower cost and higher speed.” He went on to say, “I would definitely choose the Prime 95B over an EMCCD for any high sensitivity application that would benefit from the high speed offered by a CMOS camera.”

Microfluidic Live Yeast ImagingCustomer Stories

Dr. Marc Fouet, Postdoctoral Researcher

University of California, Berkeley

Background

The Rine lab at the University of California, Berkeley is working towards understanding mechanisms underlying establishment, maintenance, and epigenetic inheritance of gene silencing in yeast. The lab has developed a genetic strategy to capture transient losses of gene silencing of heterochromatin in S. cerevisiae, and translating these dynamic processes as a permanent modification of fluorescence expression. By using the CRASH (cre-reported altered states of heterochromatin) reporter, red fluorescent yeast cells become green once gene silencing by Sir proteins has been lost.1 Monitoring fluorescence of yeast colonies can be used to describe dynamic epigenetic phenomena quantitatively. Recently, the Rine lab began using high-throughput yeast aging analysis (HYAA) chips2 with high-resolution time-lapse microscopy to monitor the relative life span of yeast and understand better the relationship between gene silencing and aging.3 Dr. Marc Fouet, a postdoctoral researcher in the Rine lab, is working on solutions to detect events leading to a lower signal of fluorescence and on the microfluidic process intensification to image an increasing number of single cells.

This microfluidic HYAA chip2 contains single-cell traps and are used with time-lapse microscopy in aging assays of S. cerevisiae.
Figure 1 is an image from a chamber with cells carrying the CRASH (cre-reported altered states of heterochromatin) reporter that induces a permananet and hertibale switch from expressing red fluorescent protein (RFP) to expressing green fluorescent protein (GFP) when loss of silencing of the auxiliary mating-type locus HMLα2 occurs.1
Figure 2 is a close up view of a single trap with single cells that are budding. They can be expressing either RFP (fig. 2A), GFP (fig. 2B), or expressing both colors when they are switching (fig. 2C), scale bar represents 10µm.

Challenge

Rare transcription events within heterochromatin occur in approximately 1/1000 cell divisions and cells divide every 90 minutes. In order to capture these rare events, 40 images are taken of 5,000 yeast cell traps for each sample. Images are captured every 5 to 10 minutes to measure cell division and fluorescent protein levels. Phototoxicity and photobleaching should be minimized, and resolution is increasingly important when machine learning algorithms are used to segment fluorescence data.

The Prime 95B [Scientific CMOS camera] is the perfect camera for Spinning Disk – the image quality is a big improvement over our EMCCDs, and the field of view makes samples much easier to find.

Solution

The Prime 95B Scientific CMOS (sCMOS) camera has a 95% quantum efficicency (QE) and can detect low levels of fluorescent proteins in yeast. The high sensitivity of the Prime 95B camera allowed Marc to lower the exposure time to reduce phototoxicity and photobleaching.

The sensitivity and large field of view reduces acquisition time of the microfluidic chip. In addition to faster exposure times, the large field of view (18 mm diagonal) reduces the amount of images needed per chip and decreases acquisition times.

The Prime 95B camera is a very versatile camera and is useful in multiple applications in the Rine group. In addition to time lapse imaging, the Prime 95B camera can acquire full frame images at 82 fps in the 12-bit mode. This is fast enough to capture rapid events that occur frequently in microfluidic chips, for example, loading of a cell trap.

References

  1. Dodson AE, Rine J. Heritable capture of heterochromatin dynamics in Saccharomyces cerevisiae. Elife (2015), doi: 10.7554/eLife.05007.
  2. Jo MC, Liu W, Gu L, Dang W, Qin L. High-throughput analysis of yeast replicative aging using a microfluidic system. Proc Natl Acad Sci U S A (2015), doi: 10.1073/pnas.1510328112.
  3. Schlissel G, Krzyzanowski MK, Caudron F, Barral Y, Rine J. Aggregation of the Whi3 protein, not loss of heterochromatin, causes sterility in old yeast cells. Science (2017), doi: 10.1126/science.aaj2103.

Single Molecule ImagingCustomer Stories

Prof. Madhavi Krishnan

Krishnan Group, University of Zürich

Background

The electrostatic properties of macromolecules—specifically, their electrical charge and interior dielectric characteristics— are a vital component of their function as they contribute to the physical basis of mechanisms that range from molecular recognition, signalling and enzymatic catalysis to protein folding and aggregation, and are of fundamental relevance in experiment and theory.

The Krishnan group at the University of Zurich are pioneering the use of the “electrostatic fluidic trap” to perform novel experiments in the spatial control, manipulation, and measurement of nanoscale matter in solution. Their primary focus is on biological molecules such as proteins and nucleic acids but some experiments also involve inorganic entities displaying interesting photonic properties.

The unique “field-free” trap offers high-precision measurement of the effective electrical charge of a single molecule in solution. They are able to measure a macromolecule›s electric charge with the precision of a single charge and below (<1e-). One of their goals is to use this approach to read out three-dimensional conformational changes or fluctuations in single macromolecules in real time.1, 2

Figure 1: Atto 532 labelled DNA fragments in electrostatic fluidic traps (indicated by green circles) arranged in rectangular lattices.
Image acquired with the Prime 95B under 40x magnification with an 8 ms exposure time using the full frame (1200×1200 pixel) field of view.

Challenge

Professor Krishnan shared, “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 in the electrostatic fluidic trap.”

This means that the group is using very low exposure times so they are constantly working in a low signal to noise environment.

The group had previously been using an EMCCD camera for this work but to reach the required speed they could only use a small field of view which only allowed them to visualize a few molecules at a time.

The Prime 95B [Scientific CMOS camera] is the perfect camera for Spinning Disk – the image quality is a big improvement over our EMCCDs, and the field of view makes samples much easier to find.

Solution

The near-perfect 95% quantum efficiency of the Prime 95B Scientific CMOS camera provides sensitivity that is equivalent to an EMCCD but with the high speed and large field of view expected of a CMOS device.

Having the ability to go 82 frames per second with high sensitivity on the large 1200×1200 sensor with an 18.66 mm diagonal increased the number of single molecules that could be detected in a single frame by a large margin. Professor Krishnan told us, “The high speed and large field of view of the Prime 95B are a massive advantage for our work.”

References

  1. Ruggeri, F., Zosel, F., Mutter, N., Różycka, M., Wojtas, M., Ożyhar, A., Schuler, B. & Krishnan, M. (2017) Single-molecule electrometry. Nature Nanotechnology May;12(5):488-495. doi: 10.1038/nnano.2017.26. Epub 2017 Mar 13.
  2. Mojarad, N. & Krishnan, M. (2012) Measuring the size and charge of single nanoscale objects in solution using an electrostatic fluidic trap. Nature Nanotechnology 7, 448–452. doi:10.1038/nnano.2012.99

Light Sheet and Single Molecule TrackingCustomer Stories

Dr. Martin Lenz, Senior Research Associate,

Cambridge Advanced Imaging Centre (CAIC),
University of Cambridge

Background

The Cambridge Advanced Imaging Centre (CAIC) at the University of Cambridge develops modern imaging techniques to answer some of the most pressing and challenging biological questions. Keeping in mind the needs and demands of biologists, one of the current developments is a localization based 3D super-resolution microscope. One of its applications include investigation of Notch pathway transcription factor dynamics in Drosphila salivary gland cells and mapping out the arrangement of chromatin inside Drosphila spermatocytes. Working in close collaboration with biologists requires CAIC to adapt and apply technological advancements in biomedical imaging to answer some of the most challenging questions in the field.

Figure 1: A) Raw image of spermatocytes using double-helix point spread function and B) 3D reconstruction from the raw data. Localizations in different colors represent their axial position.

Challenge

The research team uses single molecule tracking (SMT) to explore protein dynamics in living tissues of Drosphila and Zebrafish. A complete picture of different diffusing populations require images with high signal to noise ratio (SNR) at low excitation laser powers and short exposure times. One of the key points for achieving this is an efficient collection and detection of emitted photons.

To investigate the architecture of chromatin in Drosphila spermatocytes the team uses single molecule detection in combination with double-helix point spread function (DHPSF). This can give high-resolution in all three spatial dimensions. Losses in generating DHPSF and splitting the number of photons into two lobes of the DHPSF, requires highly efficient collection of single molecule emissions for this technique to be successful.

The Prime 95B [Scientific CMOS camera] is the perfect camera for Spinning Disk – the image quality is a big improvement over our EMCCDs, and the field of view makes samples much easier to find.

Solution

Dr. Lenz, Senior Research Associate at CAIC shares, ”The Photometrics Prime 95B Scientific CMOS (sCMOS) camera will help us to progress in both SMT as well as chromatin mapping projects. The high QE of the 95B compared to other sCMOS cameras currently used by us, will allow us to improve our investigation of protein dynamics and extend SMT to more challenging samples.”

Single molecule light sheet microscopy is one of the key applications for future work that will benefit the most from the increased sensitivity. For this application, the larger than usual pixel size of 11µm together high quantum efficiency will be highly advantageous.

Mizar TILT Light SheetCustomer Stories

Dr. Paul Maddox, Assistant Professor,

The University of North Carolina at Chapel Hill, Biology Department

Background

Conventional light sheet fluorescence microscopy (LSFM) is performed with two objectives oriented orthogonally to each other so that one objective introduces the light sheet and the other detects the fluorescence signal. However, this orientation requires the detection objective to be placed slightly away from the sample to prevent the two objectives colliding in space. Therefore, a long working distance detection objective is necessary which means that high NA, oil-immersion objectives are incompatible with the conventional LSFM design.

This presents a problem for the detection of cellular or subcellular structures which require a high NA detection objective and coverslip-based mounted samples for the superior resolution and light collection efficiency.

The Mizar TILT overcomes this problem by removing the illumination objective and introducing a tilted light sheet through a photomask and cylindrical lens which can be made to converge at the working distance of high NA objectives. In this way, high magnification and high NA (60x, 1.49), oil-immersion objectives can be used to image coverslip-based mounted samples.

Maximum intensity projection of fission yeast expressing LifeAct-mCherry
Image acquired on the Mizar TILT under 150x magnification with a 1.49NA TIRF objective, 0.2 µm step size. 100 ms exposure using the Prime 95B scientific CMOS camera, cropped to 550×750 pixels.
Sample kindly provided by Dr. Dan Mulvihill, University of Kent https://www.kent.ac.uk/bio/profiles/staff/mulvihill.html

Challenge

Like all light sheet systems, the Mizar TILT is designed to minimize photodamage and photobleaching to live samples by reducing the light source intensity and reducing exposure times. This allows for longer acquisitions to be made to monitor live processes over longer timescales.

One way to reduce exposure times is to use a more sensitive camera. CMOS devices are typically used in LSFM for the combination of a large field of view and fast speed but the sensitivity isn’t that high. EMCCD cameras are more sensitive than CMOS devices but suffer from small fields of view and slow speeds which makes them unappealing for LSFM applications.

The Prime 95B [Scientific CMOS camera] is the perfect camera for Spinning Disk – the image quality is a big improvement over our EMCCDs, and the field of view makes samples much easier to find.

Solution

The back-illuminated Prime 95B Scientific CMOS camera with an almost perfect 95% quantum efficiency and large 11µm pixels is the perfect fit for the Mizar TILT.

The sensitivity of the Prime 95B is equivalent to an EMCCD but it retains the field of view and speed advantages of a CMOS camera. Furthermore, the larger pixels of the Prime 95B allow for high-resolution imaging with higher magnification objectives which the Mizar TILT was designed to use. This allows exposure times to be reduced and cells to be imaged for much longer with high detail.

Dr. Paul Maddox, assistant professor at the University of North Carolina at Chapel Hill is the creator of the Mizar TILT and founder of Mizar Imaging, shares with us, “The Prime 95B Scientific CMOS camera is, right now, the best solution we have found for TILT imaging. The outstanding quantum efficiency and pixel size allow imaging of a wide diversity of samples of varying brightness whilst enabling Nyquist sampling in space and time for even the most challenging samples. Coupling the Prime 95B to the TILT generates an extremely powerful imaging system!”

SRRF and Super-Resolution MicroscopyCustomer Stories

Dr. Ricardo Henriques, LMCB Group Leader, UCL Senior Lecturer, Experimental Optics Leader,

Medical Research Council Laboratory for Molecular Cell Biology (LMCB), University College London (UCL)

Background

The Henriques group use various super resolution microscopy techniques to investigate cell signalling and host-pathogen interactions as well as creating and developing technology for cell biology research.

A big challenge in super-resolution microscopy is the requirement for intense illumination but this is usually phototoxic and incompatible with live-cell imaging. To tackle this problem the group developed a new approach – Super Resolution Radial Fluctuations (SRRF) – which enables super-resolution imaging using any fluorophore with far lower illumination intensities than conventional super resolution techniques.

LifeAct-GFP labelled fission yeast
Left: Widefield image
Middle: Image after applying the conventional NanoJ-SRRF algorithm
Right: Image after applying the in-development novel version NanoJ-SRRF algorithm optimized for CMOS devices (to be named SRRF4CMOS).
All images acquired with the Prime 95B scientific CMOS camera with a 10 ms exposure time (yielding 1 super-resolution frame per second).

Challenge

The Henriques group recently started using the Prime 95B Scientific CMOS (sCMOS) camera for some of their work. Dr. Henriques told us, “We’ve been actively using the Prime 95B as one of our main cameras for low-signal and super-resolution imaging at UCL. The Prime 95B Scientific CMOS is an outstanding camera, particularly due to its low-noise, high-sensitivity and large field-of-view.”

The group is also using the Prime 95B as they adapt the SRRF algorithm for use with CMOS cameras. Dr Henriques explains, “Super-resolution microscopy using our SRRF method was designed for EMCCD cameras but we are currently updating the SRRF algorithm for improved quality and performance when using data from modern CMOS devices to take advantage of the large field of view and higher speeds available.”

The accuracy of SRRF is directly related to the speed of acquisition so a faster camera would be advantageous.

The Prime 95B [Scientific CMOS camera] is the perfect camera for Spinning Disk – the image quality is a big improvement over our EMCCDs, and the field of view makes samples much easier to find.

Solution

The updated algorithm is expected to be released soon. Below are images obtained with the Prime 95B camera to show how SRRF is evolving to work with Scientific CMOS sensors.

Super-Resolution MicroscopyCustomer Stories

Dr. Kyle Douglass, Post-Doctoral Researcher

The Laboratory of Experimental Biophysics EPFL
Suliana Manley Lab, Lausanne, Switzerland

Background

Dr. Kyle Douglass, a research scientist at the EPFL, has spent the past several years developing high-throughput and automation methods for super-resolution fluorescence microscopy. The Laboratory of Experimental Biophysics, which is led by Prof. Suliana Manley, uses these techniques to study the structural biology of multi-protein complexes such as chromatin foci, the bacterial division machinery, and the centrosome.

From the perspective of the technology, these structures share a common theme in that they require large datasets of high quality images to computationally combine into a structural model which can possibly consist of one or more disordered components. It is therefore imperative to acquire as much data as possible and to ensure that it meets the exacting standards required by the computational reconstruction pipelines.

Image shows a montage taken from a particle library showing images of centriolar protein Cep152. Each particle is from a single centriole. The raw library typically consists of over 1,000 particles.

Challenge

These multiprotein complexes are well-suited to super-resolution approaches like STORM and PAINT because they are too small to see with traditional light microscopy. Furthermore, their rich and often heterogeneous composition precludes a complete study with electron microscopy , but this problem is easily overcome with multi-color super-resolution. Unfortunately, not every available dye is bright and stable. The quality of the measurements depends critically on how many photons can be recorded from each dye molecule, which means that the protein maps that are reconstructed from weak dyes will suffer from a loss of resolution and quality.

The Prime 95B [Scientific CMOS camera] is the perfect camera for Spinning Disk – the image quality is a big improvement over our EMCCDs, and the field of view makes samples much easier to find.

Solution

Because every photon counts, Douglass and colleagues upgraded their cameras to the Photometrics Prime 95B. The high sensitivity and large field of view allows the researchers to simultaneously image numerous structures at the same time while capturing even more photons than before. The increased throughput and quality of the data is paying dividends.

Neuroscience and Calcium ImagingCustomer Stories

Prof. Geoffrey Murphy

Molecular & Behavioral Neuroscience Institute
University of Michigan

Background

Dr. Geoffrey Murphy, professor of physiology at the University of Michigan’s Molecular & Behavioral Neuroscience Institute studies the how the mammalian brain encodes, stores and retrieves information. Dr. Murphy explains, “We do a lot of mouse molecular genetics, in vitro neurophysiology, and mouse behavior. We are interested in the dendritic architecture, and the calcium signaling within the dendritic structure versus the somatic structure.”

Fluorescence imaging of the fluorescent reporter for vesicle cycling (VGLUT1-pHluorin, vGpH) in hippocampal neurons.
A: Images of fluorescent intensity before (Pre), during (Stim) and after electrical stimulation (100 pulses, 10 Hz) of neurons.
B: Enlarged view of an axonal segment (from outlined region in A).

Challenge

For many neuroscience researchers, speed, sensitivity, and resolution are all critical to visualizing small changes in calcium signals in different regions of the neuron. Dr. Murphy told us, “Being able to image at 100 Hz is very important to us. That allows us to get high resolution, rapid calcium signals.” Additionally, the samples to be imaged require a sensitive camera to visualize as the samples are often thick and can be troublesome to image through.

The Prime 95B [Scientific CMOS camera] is the perfect camera for Spinning Disk – the image quality is a big improvement over our EMCCDs, and the field of view makes samples much easier to find.

Solution

For Dr. Murphy, the 11µm pixel size and speed of the Prime 95B Scientific CMOS (sCMOS) provided a solution to the challenges of calcium imaging in neurons. Dr. Murphy told us, “Previously, we had been using cameras that had a lower frame rate and worse resolution, so using the Prime 95B allowed us to not only enhance the frame rate that we were using to acquire images, but get higher resolution too. For us, that meant being able to look at subcellular structures in real time so the speed is certainly something that we like. Also, it’s a really easy camera to use and it interfaces well with our preferred software application [for further analysis].”