Page Type: Customer Story

Neural OptogeneticsCustomer Stories

Dr. Scott Cruikshank

Research Associate Professor of Neuroscience
Brown University, Rhode Island, USA

Background

Dr. Cruikshank and colleagues are interested in information processing in the brain, focusing on circuits linking the brain’s neocortex and thalamus. These circuits are critical to sensation, perception and learning. Using optogenetic probes, fluorescent and bioluminescent proteins and electrical recording, they can activate and or inhibit particular pathways and measure changes in downstream processing.

Figure 1 (A) Dual fluorescence image of a live 300 μm thick coronal brain slice taken from an NTSR1-Cre mouse using a 4x air immersion objective. Red signal indicates retrograde labeled neurons in the cortex and thalamus. Green signal shows the localization of the channelrhodopsin-EYFP signal in axons of layer 6 neurons projecting the LP thalamus.
(B) Higher magnification image of the corticothalamic ChR2-EYFP terminal labeling at the approximate position of the boxed region in panel A (imaged with a 40x water immersion objective). The high sensitivity of the Prime BSI allowed identification of the corticothalamic terminal zone using relatively low light intensity, preventing unnecessary activation or bleaching of the opsin and fluorescent protein.

Challenge

The group often image axon terminals that contain channelrhodopsin and other opsins tagged with fluorescent proteins. These terminals are fine structures and are sometimes only weakly fluorescent, causing problems with signal to noise in the resulting images.

Dr. Cruikshank told us, “We have typically used a fast sCMOS camera that can acquire images at 100 Hz (full frame) but the quantum efficiency of that camera is just 60%, and the signal to noise ratio is generally low at fast frame rates.”

Common solutions to weak signals can include illuminating with higher intensities or for a longer duration, but this can excessively activate the channelrhodopsins used to control the neuronal excitability. Uncontrolled activation of the channelrhodopsins can lead to prolonged changes in neuronal behavior, confounding the results of the experiments. On the other hand, lowering the illumination intensities and/or exposure times enough to minimize channelrhodopsin activation can degrade the ability to detect and image labeled nerve terminals.

Using the Prime BSI to image weakly illuminated and sparsely labeled fluorescent neurons, we are able to avoid uncontrolled activation of channelrhodopsin while imaging the signal in axon terminals using short exposure times

Solution

The group is now using the Teledyne Photometrics Prime BSI back-illuminated sCMOS camera.

Dr. Cruikshank told us, “The Prime BSI, with 95% quantum efficiency and 6.5 μm pixels, was a great solution to our issues of light detection and required resolution. We were also very happy to see that the fixed pattern noise was lower than that of our previous sCMOS camera.”

He continued, “Using the Prime BSI to image weakly illuminated and sparsely labeled fluorescent neurons, we are able to avoid uncontrolled activation of channelrhodopsins while imaging the signals in axonal terminals using short exposure times”.

Dr. Cruikshank concluded, “We are happy with the Prime BSI and also with Teledyne Photometrics as a company. The service and support from Teledyne Photometrics has been very good. Their people are helpful, patient, experienced, and seem to have excellent technical knowledge.”

TIRF MicroscopyCustomer Stories

Dr. Kristina Ganzinger

Group Leader

Christian Niederauer

PhD Student

Marko Kamp

Facility Technician

Physics of Cellular Interactions Group at AMOLF

Background

The group of Dr. Ganzinger is interested in the basic physical principles of immunological signaling. The group uses synthetic biology to reconstitute signaling pathways, and single molecule imaging to enable them to understand how receptors and ligands work together to transmit signals through the cell.

In particular, the group is interested in how the length of time the ligand is associated with the receptor can provide a means for a receptor to differentiate between multiple ligands. By labeling the receptor subunits their individual diffusion can be visualized prior to ligand binding events. Once the ligand is added, colocalization of the subunits can be observed and receptor interactions inferred, as well as the kinetics of different ligand binding.

Figure 1 Snippet of a movie taken with one of the receptor-proteins on a membrane. The single molecules are localized and fit with a Gaussian to find their position with sub diffraction-limited precision.

Challenge

The group uses site-specific organic fluorophores to label the different subunits of the receptor of interest to ensure they have a large enough photon budget to track the molecules with enough signal. As they are monitoring multiple receptors and ligands, the experiments involve three color imaging, potentially including the far-red, and thus a camera with a high quantum efficiency over a broad spectral range is essential for their research.

The subunits can also diffuse quickly, meaning a camera with a high frame rate is needed to permit accurate tracking. Likewise, in order to track the molecules simultaneously, the sensor must be split into three. As such, a camera with a large field of view is also essential.

Using the Prime BSI to image weakly illuminated and sparsely labeled fluorescent neurons, we are able to avoid uncontrolled activation of channelrhodopsin while imaging the signal in axon terminals using short exposure times

Solution

The group is now using the Teledyne Photometrics Prime BSI on their custom home built TIRF system to simultaneously image in up to three colors at high frame rates.

Christian told us, “We picked the Teledyne Photometrics Prime BSI camera partly due to the large field of view, which is essential as we split the sensor to permit imaging with two/three colors simultaneously, but also for the broad quantum efficiency suitable for imaging at multiple wavelengths. We also needed a camera that could offer high frame rates as the receptor subunits diffuse quite quickly.” He went on to say that, “The Prime BSI also offers the ideal pixel size to match Nyquist with our 60X objective offering us the best resolution possible”.

Christian concluded, “It used to be that people would go for EMCCDs for their high quantum efficiency. However, back-illuminated sCMOS cameras now offer the same quantum efficiency but with the larger field of view which, for us, was essential for our multicolor acquisitions.”

Point Spread Function EngineeringCustomer Stories

Dr. Christy Landes

Professor of Chemistry, Electrical & Computer Engineering, and Chemical & Biomolecular Engineering
Rice University, Texas

Background

Christy Landes’ lab at Rice University uses point spread function (PSF) engineering by phase modulation to increase the information recoverable from a two-dimensional image. Phase modulation in three-dimensional (3D) super-resolution fluorescence microscopy is achieved by mixing the phase of fluorescent light in the Fourier plane of the microscope’s detection path with a specially designed phase mask. In an alternate implementation of this geometry, the Landes group has shown that rotation of the phase mask can be used to increase the time resolution of traditional wide-field imaging experiments. The Landes lab has also designed a novel stretching lobe phase mask that can encode both temporal and 3D information in separable observables in the PSF response.

Using these approaches, they study single-molecule structure-function dynamics in chromatographic separations and in live cells. Specifically, they aim to understand early endosomal trafficking, and protein dynamics at interfaces, with spatial and temporal precisions better than otherwise available from existing optics and detectors.

Figure 1 (A) A particle imaged using a standard fluorescence microscope appears as an airy disk PSF and the PSF response is identical when the particle is below or above the focus (left). The same PSF appears as a bi-lobed structure in PSF engineering using a DHPM, where the two lobes rotate in opposite directions depending on the position of the particle in z-axis. PSF rotation over time can give information in either three-dimensional depth (z) or sub-frame time (t). (B) Three-dimensional plot of a recovered vesicle trajectory using DHPM imaged using fast frame-rate (10 ms) of the Photometrics Prime 95B camera. Fast frame-rates allowed more information to be gained about the vesicle trajectory which would otherwise be lost in slow frame-rate cameras. Corresponding simulated spinning disk trajectory for the same vesicle in two-dimension at 500 ms time-step is plotted at the bottom.
Figure courtesy: Jorge Zepeda O., graduate student in the Landes lab.

Challenge

Chayan Dutta, Post doc in the Landes group, explained their imaging challenges, “Current sensor technology cannot achieve the sensitivity and speed with which we aspire to interrogate our samples. Phase modulation of the emitted light using rotating phase mask allowed us to achieve at least 20 times faster temporal resolution. However, hardware noise and lack of sensitivity diminished our ability to discriminate these “sub-frames” as precisely as we believed possible. Using EMCCD cameras, we could amplify small signal changes and sample quickly but at the expense of the excess noise factor inherent in electronic signal amplification. CCD sensors also have limited size, so capturing the full field of view (FOV) available from the microscope was challenging.”

Dr. Landes went on to say, “We tried older sCMOS technologies but their lower quantum yield and higher pixel noise meant that we were not able to improve our 3D or temporal localization precision”.

Using the Prime BSI to image weakly illuminated and sparsely labeled fluorescent neurons, we are able to avoid uncontrolled activation of channelrhodopsin while imaging the signal in axon terminals using short exposure times

Solution

Dr. Landes told us, “Using the Teledyne Photometrics Prime 95B back-illuminated sCMOS camera moved us closer to our theoretical maximal information recovery. The larger FOV of CMOS chips compared to CCD recovered more of the field of view possible from the microscope. The back-illuminated sensor increased the quantum efficiency of signal capture at a lower noise level than in our EMCCD cameras. This allows for a faster frame rate with shorter integration times or a lower illumination intensity to give the signal to noise level needed to recover the extra information encoded by the PSF engineering.”

Chayan Dutta added, “The larger size of the CMOS chip of the Prime 95B also allowed us to image molecules with different excitation/emission wavelengths simultaneously on our wide-field epi-fluorescence microscope, which could be particularly useful for other biological applications.”

Dr. Landes concluded, “The high quantum yield for photon detection in the Prime 95B means that now we can be confident that we can detect almost every precious photon in our low signal measurements”.

Imaging With A Quantum Light SourceCustomer Stories

Dr. Markus Gräfe
MSc. Marta Gilaberte-Basset

Optical Quantum Technologies
Fraunhofer Institute for Optics and Precision Engineering

Background

Dr. Gräfe and Ms Gilaberte-Basset’s research and development centres around quantum imaging light sources. The group is currently building a new light source that will make use of quantum imaging to permit excitation of samples in the UV range, whilst detecting in the visible range, through the manipulation of correlated photons.

The new light source offers promise for applications in cell biology due to the necessity for imaging at 400 nm in the UV coupled with the lower available QE in this range. Once developed, the new light source will permit imaging of a subject with UV light, whilst permitting detection in the 800 nm region where camera QE is much higher. The light source is based on a photon pair source, which permits the generation of correlated photon pairs. One of the photons is at 396 nm and the other is at 810 nm, one photon interacts with the object whilst the other is sent to the detector. The quantum nature of the correlated photons permits detection at the camera.

Once developed and optimized, the system will then be tested with optical setups to see how useful it may be for biological imaging in custom imaging setups.

Figure 1 A high-magnification Prime BSI image of the photon-pair illumination system.

Challenge

The light source is in the early stages of development and so Ms Gilaberte-Basset is currently testing the performance of the light source in the visible ranges before moving on to the more challenging UV wavelengths.

Ms Gilaberte-Basset told us: “We require a camera to check the performance of our light source. To do this, one of the samples we use consists of an opaque sheet that contains holes to allow light through, and we then assess the contrast using the camera. We have several different patterns of sample with differing hole size so that we can check how good the source is and how good is the resolution is.”

The system inherently delivers extremely low light which is essential in order to not damage the samples when illuminating with UV light, and therefore sensitivity is very important. Ms Gilaberte-Basset told us, “We were previously using an EMCCD camera to test the light source as we thought it was necessary for the sensitivity, but after checking that the imaging system is efficient enough, we replaced the EMCCD with the sCMOS which offers higher resolution due to the smaller pixel size.”

Using the Prime BSI to image weakly illuminated and sparsely labeled fluorescent neurons, we are able to avoid uncontrolled activation of channelrhodopsin while imaging the signal in axon terminals using short exposure times

Solution

The group is now using the Teledyne Photometrics Prime BSI to check the efficiency of their light source. Ms Gilaberte-Basset shared, “We checked the system with the Prime BSI and it worked perfectly. One thing that is very important is that we want to bring the system to market. This means that if it can be more compact, robust and cost effective it is better for us.”

She went on to say: “The pixel size of the Prime BSI is great. It has much smaller pixels compared to an EMCCD so it can give us much better resolution which has been a huge benefit to us. The camera itself is also much smaller which is ideal for making the system more compact. The image we get out of the sCMOS is much better if we are checking small features, and we can get very fast acquisitions that allow us to record videos with the camera.”

Single Molecule ImagingCustomer Stories

Prof. Erwin Peterman
Dr. Andreas S. Biebricher

LaserLaB – Physics of Living Systems, Vrije Universiteit
Amsterdam, The Netherlands

Background

Prof. Peterman and team research protein structural and functional dynamics using combinations of optical tweezers and single molecule fluorescent microscopy. One branch of this research involves the study of proteins that bind to DNA and are involved in DNA repair and replication, in this case a human protein complex of topoisomerase IIIα, RMI1 and RMI2 that is related to DNA replication and a rare genetic disorder known as Bloom’s syndrome.

Using a custom-built inverted microscope that combines widefield fluorescence with dual-trap optical tweezers, a length of DNA (approx. 16 μm) can be stretched out between two points and imaged, observing the protein complexes bound to the length of DNA. These proteins are counted and potentially tracked using the mCherry red fluorescent protein, with an image taken every second at high magnification.

Figure 1: Protein complexes bound to a length of DNA held by dual-trap optical tweezers. The two large circles are the optically trapped beads, suspending and stretching between them a 16 um length of double-stranded DNA. While this DNA itself is invisible, the protein complexes bound to the DNA can be imaged with mCherry, with ~10 different proteins seen imaged in the space between the tweezer beads. Pixel size 120 nm.

Challenge

While the advanced experimental set-up does allow for images of proteins on captured DNA to be taken, it introduces several imaging challenges. The mCherry fluorophore gives a low signal and bleaches quickly, meaning that high sensitivity and low intensity/exposure times are required.

The imaging is also limited by a 1.2 numerical aperture water immersion objective with lower light collecting efficiency, resulting in multiple lenses required to reach a 120 nm pixel size for imaging DNA and proteins.

Using the Prime BSI to image weakly illuminated and sparsely labeled fluorescent neurons, we are able to avoid uncontrolled activation of channelrhodopsin while imaging the signal in axon terminals using short exposure times

Solution

The Prime BSI offers top-class sensitivity with a 6.5 μm optimized pixel size to maximize light collection even at high magnification. Prof. Peterman mentioned: “imaging with the mCherry is challenging due to intense laser light being filtered out, mCherry has low signal and bleaches fast, but the Prime BSI can still see this.”

Along with high sensitivity, the Prime BSI has a large field of view allowing for more capture per frame, especially vital when imaging at low speeds for high sensitivity as fewer frames are taken and need more information.

In addition, the Prime BSI is easy to use. As mentioned by Dr Biebricher: “it was straightforward to set up [the Prime BSI] and get it running… we had to play with the gain when using an EMCCD but this was not an issue on the CMOS as they are electronically different”.

The group are using Micro-Manager with the Prime BSI, which could be set up quickly and offered several perks for this research, including QuantView to allow for easier quantification of the proteins on the DNA, and the ability to tag frames for easy analysis.

Calcium ImagingCustomer Stories

Professor Rod O’Connor
Assistant Professor David Moreau

Ecole des Mines de Saint-Etienne, France

Background

Professor Rod O’Connor and Dr. David Moreau work together to develop flexible, conductive polymer electrode devices to record electrical activity and optical measures of physiology from neurons to study the bioelectrical basis of diseases like epilepsy, Alzheimer’s disease and cancer. The electrical devices they create are ideal for imaging as they are nearly transparent and they can be used to both record the electrical behavior of cells and to stimulate.

At the moment, they are most interested in exploring new fluorescent probes that permit the simultaneous imaging of calcium signals and plasma membrane potential.

Figure 1 MDCK-II cells labeled with LifeAct- TagRFP (revealing Actin in Red) and Hoechst 33342 (revealing nuclei in blue).
Figure 2 U87 spheroid labelled with calceinAM (green) implanted on chorioallantoic membrane of Quail embryo with blood vessels labelled with Texas Red (red)

Challenge

One of the challenges the group face in their microscopy set up is that they need to use epifluorescence to image cell physiology and transmitted light to observe the semi-transparent plastic electrode devices. Switching between these two modes is not easy to manage within the conditions of their application.

To perform their experiments, they need to rapidly change between these two modes. Therefore, a high-speed camera with high sensitivity is crucial for their imaging needs.

Using the Prime BSI to image weakly illuminated and sparsely labeled fluorescent neurons, we are able to avoid uncontrolled activation of channelrhodopsin while imaging the signal in axon terminals using short exposure times

Solution

The group is now using the Teledyne Photometrics Prime 95B sCMOS camera for several projects using widefield fluorescence microscopy. With the Prime 95B, the group can take advantage of the almost perfect 95% quantum efficiency and high framerates to image with the high sensitivity with speed they need for their application.

Professor O’Connor told us, “The high speed and the high sensitivity of the Prime 95B were necessary to
improve our imaging methods”.

Light Sheet and Novel Spatial Frequency ImagingCustomer Stories

Professor John Girkin

Department of Physics and Biophysical Sciences Institute
Durham University, United Kingdom

Background

Prof. Girkin and his multidisciplinary team are interested in applying advanced photonics and optical technology to challenges within the life sciences, in this case imaging live zebrafish. By using selective plane illumination microscopy (SPIM), the beating hearts (1) and developing eyes (2) of live zebrafish can be imaged and analyzed.

Using a homebuilt SPIM imaging system (3) which incorporates a visual heart synchronization method; a variation on SPIM that includes beam scanning (digital SPIM or D-SPIM), and a novel spatial frequency domain imaging system (patent pending), Prof. Girkin and team have a wealth of imaging techniques used to interrogate live samples.

Figure 1 Left GFP expression in blood vessels within the developing zebrafish brain, maximum intensity projection. Right GFP expressing blood vessels near the developing kidney, maximum intensity projection.

Challenge

In order to visualize the zebrafish heartbeat, Prof. Girkin told us, “a high speed camera, operating in the near infrared, uses transmission images of the beating heart and real-time analysis software to synchronize the light sheet and fluorescence imaging camera to effectively ‘freeze’ the motion of the heart. The fluorescence imaging camera has to have accurate and repeatable shutter operation in combination with high sensitivity, to capture the image at the correct part of the heart’s cycle.”

The D-SPIM system has similar requirements, needing accurate camera control electronics and software to maintain synchronization, and a low noise sensitive camera to take advantage of the method. When imaging the developing zebrafish eye, a good signal is required even in low light conditions over a 24-48 hour study period.

For the novel spatial frequency domain imaging, Prof. Girkin informed us: “This is an excellent method of imaging through tissue, in particular, skin if near-infrared light is used. A highly sensitive camera is therefore required with, in our case, some capability in the near infrared (around 800 nm).”

Using the Prime BSI to image weakly illuminated and sparsely labeled fluorescent neurons, we are able to avoid uncontrolled activation of channelrhodopsin while imaging the signal in axon terminals using short exposure times

Solution

The Iris 9 meets all of the imaging requirements for these numerous microscopy and imaging techniques, as confirmed by Prof. Girkin, “We have now replaced our previous camera with the Iris 9 which is demonstrating high sensitivity to the low light levels required for rapid imaging and also the timing required to ensure the synchronization is correct. We have imaged hearts for over an hour without losing timing lock on the imaging, demonstrating the performance of the camera.”

The Iris 9 is working well with the D-SPIM system, and the high sensitivity is utilized when studying the movement of individual cells within the developing zebrafish eye (1), with the Iris 9 “demonstrating significant advantages over our previous camera in this regard.”

References

  1. Taylor, J. M., Saunter, C. D., Love, G. D., Girkin, J. M., Henderson, D. J., & Chaudhry, B. (2011). Real-time optical gating for three-dimensional beating heart imaging. Journal of Biomedical Optics, 16(11), 116021.
  2. Young, L. K., Jarrin, M., Saunter, C. D., Quinlan, R. A., & Girkin, J. M. (2018). Non-invasive in vivo quantification of the developing optical properties and graded index of the embryonic eye lens using SPIM. Biomedical Optics Express, 9(5), 3947–3958.
  3. Girkin, J. M., & Carvalho, M. T. (2018). The light-sheet microscopy revolution. Journal of Optics, 20, 053002.

Single Molecule DynamicsCustomer Stories

Dr Siddharth Deshpande

Prof. Cees Dekker Group, Delft University of Technology

Background

Dr. Deshpande’s research focuses on the use of synthetic biology approaches to study real-time dynamics of biomolecules.

While Cees Dekker’s group deals with diverse research projects, Dr. Deshpande’s work is focused on looking at biomolecular processes in vitro by applying bottom-up approaches to improve understanding of how living cells work. Specifically, the group studies key individual components of biological relevance within a confined space, such as liposomes or droplets, which are excellent scaffolds for creating synthetic cells. The size range of these containers is in the 5-50 micrometer range.

Dr. Deshpande mainly uses high-resolution time-lapse imaging to look at the dynamics of fluorescently labeled components, such as lipids, proteins, polymers, and DNA molecules, confined within microcontainers, to understand the self-organization and emerging behavior of these molecules.

Figure 1 Unilamellar liposomes (red circles) showing
phase separated membraneless organelles (green areas)
formed within their lumen.

Challenge

Dr. Deshpande was previously using a front-illuminated sCMOS camera but wanted to improve upon the sensitivity to push the exposure time as low as possible whilst maintaining enough signal for imaging.

One challenge they faced with this type of dynamic imaging of synthetic cells was poor signal-to-noise ratio (SNR). Dr. Deshpande shared, “We struggled with SNR due to the low quantum efficiency of our previous camera and the low exposure times of 10 ms, which are necessary to prevent photo-bleaching during imaging. We want to image with the lowest exposure time possible. We also want to image at 100 or 1000 fps, so sensitivity is really important.”

Dr. Deshpande told us that “Our previous camera also had a split sensor design which leads to degradation of image quality and image artefacts.”

Using the Prime BSI to image weakly illuminated and sparsely labeled fluorescent neurons, we are able to avoid uncontrolled activation of channelrhodopsin while imaging the signal in axon terminals using short exposure times

Solution

Dr. Deshpande is now using the Teledyne Photometrics Prime BSI camera with a Ti2 widefield system.

Dr. Deshpande shared, “The main reason we wanted to buy this camera was that we wanted something really sensitive that can give high frame rates. One of our colleagues had purchased the Teledyne Photometrics Prime BSI and we were impressed by the sensitivity offered by the back-illumination.”

He went on to say, “We are really happy with the camera. We previously used to image in a single plane but now we can take z-stacks through the liposome with high enough frame rates to visualize the internal dynamics. The additional quantum efficiency also allows us to use lower exposure times and not photobleach the sample.”

The camera is also being used for experiments where the high sensitivity and speed are less important but the large field of view (FOV) plays an important role.

Dr. Desphande told us, “When we are looking at slower processes we want to have the larger FOV to collect as much information as possible. This allows us to visualize multiple objects at once and get more data for better statistics. The pixel is 6.5 microns which makes the resolution ideal, this was another major factor when choosing the camera.”

Light Sheet MicroscopyCustomer Stories

Greg Perry

Image Resource Facility, St Georges, University of London

Dr. Ferran Valderrama

Academic Director of the Image Resource Facility

Dr. Dan Osborn

Molecular and Clinical Sciences Research Institution

Background

Greg Perry, from the Image Resource Facility at the University of London, works closely alongside academics such as Dr. Osborn and Dr. Valderrama, to improve imaging across a variety of research applications including long-term live imaging of zebrafish development and 3D organization of prostate cancer cell structures.

The Osborn lab is interested in translational research using zebrafish embryos as a model organism for tissue regeneration. The group manipulates an enzyme called nitrogen reductase that can be used to convert a pro-drug into a cytotoxic agent, permitting temporal control of apoptosis in these model organisms. Once apoptosis has been initiated, the regeneration of tissue can be observed using microscopy to elucidate mechanisms of repair.

Dr Valderrama’s group are interested in improving the understanding of prostate cancer progression. Prostate cancer originates from lesions in the glandular structures (acini) of the prostate. The group has developed a 3D cellular model that recapitulates the morphogenesis of these acini and serves as a model for cancer formation. The group uses this model in conjunction with microscopy methods to understand the molecular mechanisms of prostate cancer progression

Figure 1 Single frame of an OpenSPIM acquisition of a zebrafish embryo expressing GFP in vascular endothelial cells, allowing visualization of the heart and the vasculature.

Challenge

Cleared samples are typically very large which means that low magnification is often necessary to image the entire sample. However, this comes at the cost of resolution.

The Image Resource Facility recently acquired a new OpenSPIM (T-SPIM) system, which will be used to further advance the research areas of the Osborn Group and Valderrama Group. The new OpenSPIM setup permits live imaging of whole organisms such as transgenic zebrafish to enable the visualization of tissue repair over the course of days, as well as allowing the imaging of 3D acini cellular structures.

The facility required a camera to use alongside this new system to maximize the information gained during acquisitions. The OpenSPIM system requires a camera that can offer high speed, sensitivity and a large FOV to facilitate fast image capture of these large 3D samples. Sensitivity is important not only for imaging, but also for alignment which can be tricky with dim samples.

Using the Prime BSI to image weakly illuminated and sparsely labeled fluorescent neurons, we are able to avoid uncontrolled activation of channelrhodopsin while imaging the signal in axon terminals using short exposure times

Solution

Greg Perry is now using the Teledyne Photometrics Iris 9 on their new OpenSPIM system to collect time-lapse images of 3D model systems alongside collaborating groups such as the Osborn Group and Valderrama Group.

Mr Perry told us that, “[The Teledyne Photometrics Iris 9] is a really good camera that is clearly very flexible in application”. He also shared that, “The camera is ideal for our OpenSPIM system due to the sensitivity and the large field of view.” Mr Perry also noted that the camera was very fast which is ideal for their applications.

smFRET, STORM and TIRFCustomer Stories

Dr. Johannes Hohlbein

Laboratory of Biophysics, Department of Agrotechnology and Food Sciences
Wageningen, The Netherlands

Background

The Hohlbein Lab uses techniques such as single molecule imaging, FRET and super-resolution PALM/STORM to investigate a variety of topics. These include increasing the throughput of single-molecule measurements by using fluidic devices in combination with TIRF microscopy, using single-molecule FRET to study binding DNA-protein interactions and probing anisotropic food structures using single particle diffusometry.

Figure 1 Widefield and STORM images of Cos-7 cells labelled with Anti-Tubulin and Alexa 647. A) Widefield microscopy (low intensity, average frames) and B) miCube (dSTORM) images of the same region of the cell.

Challenge

The Hohlbein Lab typically struggles with the limited number of emitted photons in their single-molecule imaging and smFRET experiments, therefore a camera with a high quantum efficiency is advantageous for these applications.

In addition to this, Dr. Hohlbein would like to image the largest field of view as possible with the system when using a 100x objective to achieve the maximum throughput in data acquisition.

Using the Prime BSI to image weakly illuminated and sparsely labeled fluorescent neurons, we are able to avoid uncontrolled activation of channelrhodopsin while imaging the signal in axon terminals using short exposure times

Solution

Dr. Hohlbein chose the Teledyne Photometrics Prime 95B back-illuminated sCMOS for a number of reasons. He told us, “Firstly, the 95% quantum efficiency of the back-illuminated sensor ensures that almost all photons arriving at the camera are collected which is of great benefit for our lowest light applications.”

He went on to say that, “The pixel size of 11 × 11 µm ensures that with the 100× magnification of the optical system, we have an ideal 110×110 nm mapped from the sample plane onto each camera pixel, fulfilling the Nyquist sampling criterion and maximizing the field of view. With the camera sensor size of the Prime 95B which consists of 1200×1200 pixels, our usable area is around 5 times higher than with a conventional 512×512 pixel EMCCD camera.”

The lab is planning to make use of PrimeLocate™ in the future, which automatically detects the brightest pixels in the image and only transfers this data to the computer. Dr. Hohlbein told us, “With PrimeLocate, we will have an intriguing option to reduce the storage requirement of our raw data as ROIs are automatically detected and selected for saving.”