Page Type: Customer Story

Single Molecule DNA ImagingCustomer Stories

Jonathan Jeffet, Prof. Yuval Ebenstein

NanoBioPhotonix Lab, Department of Chemical Physics, Tel-Aviv University, Israel

Background

The NanoBioPhotonix Lab of Tel-Aviv University is led by Prof. Ebenstein and focuses on single-molecule genomics research and development of novel imaging techniques. Jonathan Jeffet is a PhD student and physicist from this highly multidisciplinary lab and works with their imaging systems performing optical genome mapping. This technique is complementary to typical DNA sequencing, where sequencing works by attempting to assemble short DNA segments, and optical mapping uses fluorescent tags with long fragments of DNA to create a ‘map’, this then helps to align sequences that would be problematic with other techniques.

As Mr Jeffet says: “We use fluorescence to tag DNA molecules, DNA damage, methylation, and we want to see all this genetic and epigenetic information on a single DNA molecule, this is the focus of the lab.”

Figure 1: Representative false color FOV of 100 nm silica beads labeled with five different fluorescent dyes (Alexa Fluor (AF) 405, 488, 568, 647 and IRDye 800CW) and imaged with RPA set to 174°.

Challenge

From Mr. Jeffet: “We try to image all these genetic and epigenetic modifications on a single DNA molecule together, if we sequentially image different colors then we lose information as the molecules move around while we switch filters.”

The lab uses a five-laser microscope, with the five light sources ranging from 405-785 nm meaning the experiments are working with the entire visible spectrum to acquire images for optical genome maps, resulting in a complex system requiring advanced triggering and illumination methods. Sensitivity is required across the visible spectrum in order for experimental flexibility. In addition, the scale of the samples means a high resolution is needed, and due to the type of flat-field excitation used, only 60x magnification was available.

The markers are single-molecule fluorescent tags that have a very low signal, meaning that imaging at higher speeds is limited by the need for a good signal-to-noise ratio, with longer exposures needed to collect enough signal for a good image.

The [Prime BSI] was really quick and easy to set up and worked well with MicroManager, and we’re happy because we’re seeing better results, the SNR is really better, it’s perfect for our needs

Solution

The high speed, low noise, and high sensitivity of the Prime BSI resulted in a big increase in signal-to-noise ratio with the single-molecule imaging for the NanoBioPhotonix Lab. The 6.5 um pixel of the Prime BSI is optimized for Nyquist at 60x magnification, which results in high-resolution images.

The multiple modes of the Prime BSI also allow for flexible imaging, offering both a high dynamic range mode and a high sensitivity CMS mode, making the Prime BSI a solution for a wide range of different single-molecule experiments.

The Prime BSI was an improvement on previous CMOS solutions, as outlined by Mr. Jeffet: “We previously used a front-illuminated sCMOS and the SNR was much worse at the same acquisition rates… the [Prime BSI] allowed us to see more of the sample and our readings were much better with this camera.”

References

Jeffet J., Michaeli Y., Torchinsky D., Israel-Elgali I., Shomrom N., Craggs T.D., Ebenstein Y. (2020) Multi-modal Single-molecule Imaging with Continuously Controlled Spectral-resolution (CoCoS) Microscopy, bioRxiv 2020.10.13.330910; doi: https://doi.org/10.1101/2020.10.13.330910

Voltage ImagingCustomer Stories

Dr. Miguel Aroso

Neuroengineering and Computational Neuroscience Lab, Institute for Research and Innovation in Health (i3s), University of Porto

Background

Dr. Miguel Aroso is a researcher involved in the functional assessment of neuronal populations using multi-electrode arrays (MEAs) and voltage imaging. This work is done in order to characterize neuronal circuits and how they communicate, how to control this activity, and how this can be used as a model for studying epilepsy and predicting seizures. The MEA electrodes can be used for recording as well as stimulation, allowing for both the recording of spontaneous and evoked activities across the network.

This, in combination with voltage imaging, allows a detailed picture of function across a network to be built up for research.

Figure 1: Voltage imaging with an MEA. The top image shows a graph with voltage from the MEA (blue) and fluorescence from voltages indicators (orange), showing activity from the neuronal circuits. The bottom image shows these neuronal circuits grown on an MEA and stained with a neuronal marker (green), with the MEA electrodes appearing as black circles.

Challenge

Voltage imaging records very fast events, meaning a highly sensitive, high speed system is needed in order to capture neuronal signaling. A previous solution using a typical sCMOS camera was not able to capture this information and could not be used for voltage imaging.

Dr. Aroso outlined how speed and sensitivity were most important for voltage imaging, with an aim to image at 1000 fps or 1 kHz in order to detect relevant changes in fluorescence.

The [Prime BSI] was really quick and easy to set up and worked well with MicroManager, and we’re happy because we’re seeing better results, the SNR is really better, it’s perfect for our needs

Solution

The Prime 95B is an ideal solution due to the high speed and high sensitivity, allowing Dr. Aroso and the team to see voltage imaging signals for the first time. Due to the larger pixel, the Prime 95B also allows high speed imaging over a larger region of interest, resulting in more cells and MEA electrodes within the field of view.

Dr. Aroso commented on the stability of the Prime 95B signal: “we have the high speeds and now the signal is stable; we can see less than 1% change in fluorescence related with the electrical activity of the cells.” Dr. Aroso went on to state the ease of setting up the Prime 95B in MicroManager.

High Content Multiplex FluorescenceCustomer Stories

Dr. Sonia Leonardelli, Prof. Michael Hölzel

Hölzel Lab, Institute of Experimental Oncology, University Hospital Bonn

Background

Dr Sonia Leonardelli of the Hölzel Lab performs fluorescent imaging of a range of different tumour tissue samples, one of the main projects being to study cell-cell interactions in adenocarcinoma.

The fluorescence imaging in this lab using the CODEX® system from Akoya Biosciences® with a Zeiss microscope in order to take multiple fluorescence images with multiple different fluorophores, which can then be collated and output with all the fluorophores in the same image, up to 60 different types. Images from these experiments typically have up to 8 different fluorescent markers and are dense, high content images that reveal details about the tumour microenvironment.

Imaging tumour samples with these multiplex fluorescence methods allows Dr Leonardelli to see what interactions are occurring, how the tissues respond to drugs, and how tumour malignancy can develop. This will further allow for the development of biomarkers to see how tumours develop and how they respond to different therapies.

Figure 1: Image of tissue taken from the tonsil, taken with the Prime BSI in 16-bit high dynamic range (HDR) mode in Zen imaging software, with the CODEX® system. Colours as follows: blue (DAPI), red (CD4), green (CD31), white (CD45), magenta (PA1), yellow (CD8), cyan (GZB).

Challenge

The CODEX® system has a 10x or 20x objective, meaning that while images can be acquired across a large field of view, they are limited on their resolution due to the lower objective numerical aperture. A high-resolution camera with smaller pixels will be needed in order to acquire details on the cellular level in order to see cell-cell interactions.

Due to the wide range of fluorophores used (including DAPI, Cy3, Cy5, and Cy7) any detector used with this system would need a high quantum efficiency across a wide range of wavelengths, from blue to near-infrared, in order to use the most fluorophores without spectral overlap. A high sensitivity camera would also allow for a reduction in illumination intensity to avoid bleaching.

The [Prime BSI] was really quick and easy to set up and worked well with MicroManager, and we’re happy because we’re seeing better results, the SNR is really better, it’s perfect for our needs

Solution

The Prime BSI is a great solution for this multiplex fluorescence imaging, as it offers both high sensitivity and high resolution. Dr Leonardelli stated that she was able to see cell-cell interactions due to the high resolution of the Prime BSI, across the multiple fluorophores available. The Prime BSI has 95% peak quantum efficiency, and high sensitivity across a wide range of wavelengths from UV to near-infrared, in order to acquire data from the various fluorophores in these multiplex experiments.

In addition, with CODEX® software optimization, Dr Leonardelli will also be able to reduce the illumination intensity and make use of the high sensitivity of the Prime BSI.

Liquid Beam Observation and FluorescenceCustomer Stories

Dr. Andreas Hans

Spectroscopy/Physics with Synchrotron Radiation Group, Experimental Physics, University of Kassel, Germany

Background

Dr. Andreas Hans is the subgroup leader of the Spectroscopy and Physics with Synchrotron Radiation group within Experimental Physics. This group is performing studies on x-ray interactions with samples and the effects on a molecular level, using x-rays to irradiate biomolecules in a vacuum and detecting the resulting emission of photons, electrons, and other subatomic particles.

As Dr. Hans outlined: “We want to mimic what happens to real biological material after exposure to x-rays, but on a molecular level… we want to see the molecular mechanisms that cause the damage to the organism, such as sunburn or cancer.”

Figure 1: An image from the Prime BSI showing liquid beams forming ultrathin sheets, this is done in order to insert aqueous samples into a vacuum chamber for x-ray irradiation. Two liquid beams enter from the right (in the same plane) and combine into a sheet within the thicker section.

Challenge

Dr. Hans wishes to expand the repertoire of samples to include liquid samples: “The problem is bringing liquid samples into a vacuum… there is a technique that injects a high-pressure beam of liquid through a pinhole into the vacuum. A tiny beam of liquid forms in the vacuum and is very hard to characterize and stabilize.” This beam is tens of microns across, meaning a sensitive camera is needed to observe and measure the water beam. In addition, two water beams can be crossed to form a very thin sheet (order of nanometres) in the vacuum. These beams and sheets of water need to be imaged with a highly sensitive and high-resolution camera for effective characterization.

In addition, there are challenges with detection, as the irradiated samples produce very low signals (such as single photons). For detection, Dr. Hans uses an amplifying device: “It transfers the photons to electrons and amplifies it to a charge cloud, which hits a fluorescing screen”. The fluorescence from this screen has a very short lifetime and needs to be detected by a sensitive, high-speed camera

The [Prime BSI] was really quick and easy to set up and worked well with MicroManager, and we’re happy because we’re seeing better results, the SNR is really better, it’s perfect for our needs

Solution

The Prime BSI presents a highly sensitive, fast, and high-resolution imaging solution for this application, both for monitoring the properties of water beams and combined sheets and for detecting fluorescent events from the amplifier.

About the water beams, Dr. Hans said: “The water sheet is very thin, in the order of nanometres, and with the camera, we can very nicely see how this behaves.” As for detecting events from the amplifier, Dr. Hans mentioned: “Whenever a particle impinges on our detector we get a very short tiny flash, and due to the good contrast and sensitivity [of the Prime BSI], we can see these individual light spots and flashes.” Overall across the different applications, Dr. Hans was pleased with the performance of the Prime BSI: “I see that it works perfectly for what we are doing and what we plan to do further, the [Prime BSI] is robust and we can get all the information we want to…it is the best one for the combination of these two different applications.”

In addition, Dr. Hans and the team also have plans to implement the Prime BSI in a custom Python data acquisition system, which benefits from the excellent software support for Photometrics cameras.

Widefield Intrinsic Signal ImagingCustomer Stories

Dr. Daniel Hillier and Mr Abel Petik

Visual Systems Neuroscience Lab, Research Centre for Natural Sciences, Budapest, Hungary

Background

The lab of Dr. Daniel Hillier focuses on vision science and researching how vision works, using increasingly more complex model organisms and neuroscientific research methods. Organisms are stimulated with different visual stimuli and readings are taken from the primary visual cortex, in order to discover how these circuits link up, how visual information is processed, and how these systems are affected during visual impairment such as blindness.

The lab uses imaging techniques such as optogenetics, intrinsic signal imaging and fluorescence, in order to take physiological readings, target different cell types, and build cortical maps.

Figure 1: The imaging system in use at the lab of Dr. Hillier, featuring the Prime BSI Express scientific sCMOS camera.

Challenge

Research scientist Abel Petik performs both intrinsic signal imaging as well as fluorescence on a single imaging system, meaning a flexible camera solution is necessary. The system is a low-magnification macroscope for observing a large field of view with fluorescence widefield imaging. This is due to larger model organisms being used, such as cats and primates, meaning imaging methods are less established than rodents, and much larger brain diameters are being imaged, such as 10 mm.

While intrinsic signal imaging is slow (scale of seconds), being able to image at high speeds is still advantageous as more images can be acquired and then averaged to increase the signal-to-noise ratio. As stated by Dr. Hillier: “For intrinsic signal imaging the most important factor is the well depth of each camera pixel, but with fluorescence imaging the quantum efficiency and dark noise are important, meaning the ideal camera needs to have flexibility across a wide range of experiments.”

The [Prime BSI] was really quick and easy to set up and worked well with MicroManager, and we’re happy because we’re seeing better results, the SNR is really better, it’s perfect for our needs

Solution

The Prime BSI Express offers the flexibility needed for these experiments, with high speeds, high signal-to-noise ratios and a deep well depth. The 16 bit HDR mode delivers a deep well depth for intrinsic signal imaging, and the low-noise CMS mode delivers a high signal-to-noise ratio for fluorescence imaging, meaning the Prime BSI Express can do it all.

Switching from intrinsic signal imaging to fluorescence imaging is easy and highly advantageous. As outlined by Mr Petik, camera setup out of the box was simple, the modern USB 3.2 Gen 2 cables were welcomed due to the high data rate, and Python package allowed for customisable software with the help of the Photometrics support team.

Super-Resolution Single Molecule LocalizationCustomer Stories

Prof. Pingyong Xu

Dr. Mingshu Zhang

Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences

Background

Single-molecule localization microscopy (SMLM) techniques, such as photoactivation localization microscopy (PALM), achieve some of the highest spatial resolutions among all the super-resolution imaging methods. However, image reconstruction in PALM requires a large number of raw images, which leads to low temporal resolution. Limited photo-controllable fluorescent protein probes (PCFPs) also restrict the widespread application of SMLM.

Prof. Pingyong Xu’s lab is engaged in the development of new super-resolution microscopy technologies. Previously, his group developed a new super-resolution method named single-molecule-guided Bayesian localization microscopy (SIMBA). It uses only 100 raw frames to calculate a super-resolution image with a 50 nm spatial resolution, which efficiently increases the temporal resolution of SMLM.

Figure 1: Quick-SIMBA imaging of the endoplasmic reticulum structures in live U2OS cells and COS-7 cells. (a) A diffraction-limited image represented by the sum of the first 50 raw frames. (b) Reconstructed images using the Quick-SIMBA algorithm.

Challenge

To achieve higher temporal resolution with SIMBA, prof. Xu intended to develop imaging techniques that improved on SIMBA and required even less raw frames for reconstruction. Another way to improve temporal resolution is to use a camera with a higher frame rate, with sufficient sensitivity to image with shorter exposure times. However, the slow readout speed of a previous EMCCD camera solution and the suboptimal properties of existing PCFPs hindered them from further increasing the temporal resolution of SIMBA.

The [Prime BSI] was really quick and easy to set up and worked well with MicroManager, and we’re happy because we’re seeing better results, the SNR is really better, it’s perfect for our needs

Solution

To overcome the shortcomings of their EMCCD, Prof. Xu’s lab decided to use the Teledyne Photometrics Prime 95B sCMOS. Combining the Prime 95B with pcStar, a new PCFP also developed by Xu lab, they created a new super-resolution method Quick-SIMBA. Quick-SIMBA only needs 50 raw frames (2-5 ms exposure) to reconstruct a final image. It could achieve the highest temporal resolution (0.1−0.25 s) with large FOV (from 76 × 9.4 μm2−76 × 31.4 μm2) among all SMLM methods.

With Quick-SIMBA, the dynamic movements of the endoplasmic reticulum dense tubular matrix were resolved, which are difficult to recognize by diffraction-limited microscopy. The combination of pcStar and Quick-SIMBA holds great potential for super-resolution imaging of short-term events, such as transcriptional bursts and cell mitosis, or for tracking short-lived proteins in living cells.

Reference

Fast Super-Resolution Imaging Technique and Immediate Early Nanostructure Capturing by a Photoconvertible Fluorescent Protein. M Zhang et.al. Nano Letters 2020 20 (4), 2197-2208

Live Cell DynamicsCustomer Stories

Prof. Cornelia Monzel

Faculty of Mathematics and Natural Sciences, Heinrich Heine University Düsseldorf, Germany.

Background

The group of Prof. Cornelia Monzel aims to understand the interplay of physical and biological mechanisms that give rise to relevant cellular functions for use in diagnostics and therapy. To do this they make use of label-free and fluorescent methods to investigate membrane fluctuation dynamics and cell signalling pathways.

One technique used by the group of Prof. Monzel is label-free reflection interference contrast microscopy (RICM) in order to look at the dynamics of membrane interactions [1-3]. This technique works by observing the light reflection at micro-structured interfaces, acquiring fast recordings (10 ms) of membrane fluctuations, and reconstructing the interference intensity pattern of bio-membranes into 2D height profiles, as seen in Figure 1. The membrane topography and its dynamics provide information about the strength and type of interaction governing adhesion.

Figure 1: Biomembrane adhesion to microstructured interface. Left) sketch of biomimetic membrane (vesicle) adhering to a microstructured substrate, mimicking adhesion to a limited number of binding sites. Middle) The contact area is visualized with the interferometric technique RICM. Right) The detected interference intensity I_int is converted into membrane topography with nanometric resolution. Images from [1-3].

Another interest of Prof. Monzel’s group is imaging the dynamics of cell signalling pathways involved in apoptosis (programmed cell death). To do this, they perform time-lapse imaging of live cells that are undergoing apoptosis, once the receptor CD95 in the plasma membrane gets activated by the CD95-Ligand. The group demonstrated that the process of apoptosis involves clustering of the CD95 receptors, as seen in Figure 2. The group is trying to decipher how the signal activation depends on receptor number and accumulation state, as well as on the dynamics of other molecules in the signalling pathway ultimately leading to apoptosis.

Figure 2: CD95 activation and accumulation triggers cell apoptosis. Left) sketch of CD95 receptors activated by CD95-Ligand. A mEGFP fluorophore is genetically fused to the cytoplasmic domain of the receptor. Right) Temporal dynamics of CD95 accumulation followed by cell apoptosis monitored in the fluorescent EGFP channel, taken with the Prime BSI.

Challenge

Interference microscopy and RICM provide a challenge in that the techniques requires a high contrast and signal to noise ratio. At the same time, the imaging needs to be fast in order to record membrane dynamics with 10 ms resolution.

When performing time-lapse imaging a large number of cells need to be recorded to ensure a representative sample of the population is captured. High and precise fluorescence signal acquisition is further required to enable ratiometric fluorescence analysis of molecular accumulation.

The [Prime BSI] was really quick and easy to set up and worked well with MicroManager, and we’re happy because we’re seeing better results, the SNR is really better, it’s perfect for our needs

Solution

The group of Prof. Monzel is now using the Teledyne Photometrics Prime BSI for acquiring both fast recordings of biomimetic membrane dynamics and time-lapse images of receptor clustering.

Prof Monzel told us, “there have been massive improvements in sCMOS technology over the past 5 years, especially due to the back illumination and 95% Quantum Efficiency, the sensitivity and efficiency was massively increased”. Prof Monzel went on to say that, “with the BSI we can now record a lot of cluster formation. I appreciate the large field of view with 4.2 Megapixel as it is good for getting high statistics during a short amount of time. The small 6.5 µm pixel size here enables resolving clusters at lower magnifications”.

References

  1. Monzel C., Schmidt D., Kleusch C. et al. (2015) Measuring fast stochastic displacements of bio-membranes with dynamic optical displacement spectroscopy. Nat Commun 6, 8162. https://doi.org/10.1038/ncomms9162
  2. Monzel C., Schmidt D., Seifert U., Smith A.S., Merkel R. and Sengupta K. (2016) Nanometric thermal fluctuations of weakly confined biomembranes measured with microsecond time-resolution. Soft Matter, 12, 4755-4768
  3. Zhang, Y., De Mets, R., Monzel, C. et al. (2020) Biomimetic niches reveal the minimal cues to trigger apical lumen formation in single hepatocytes. Nat. Mater. 19, 1026–1035. https://doi.org/10.1038/s41563-020-0662-3
  4. Gülce S. Gülcüler Balta, Cornelia Monzel, Susanne Kleber, et al. (2020) 3D Cellular Architecture Modulates Tyrosine Kinase Activity, Thereby Switching CD95-Mediated Apoptosis to Survival. Cell Reports 29, 8. 2295-2306. https://doi.org/10.1016/j.celrep.2019.10.054.

Calcium ImagingCustomer Stories

Dr. Wiktor Phillips

Department of Women’s and Children’s Health, Karolinska Institutet, Sweden

Background

Dr. Wiktor Phillips is primarily interested in observing the pattern generation of respiratory rhythms in mammals through the study of the pre-Bötzinger complex in the medulla of the brainstem, and whether these patterns are affected by birth and related to respiratory dysfunction in newborns.

This group investigates pattern generation in these complexes through in vitro study of mouse brain slices, either taken acutely or organotypically cultured for several weeks. These slices are subject to both single-cell patch clamping and whole network calcium imaging, often over long experiments where drugs can be administered, onset of activity can be monitored, and sub-populations of neurons and astrocytes can be functionally compared.

A variety of fluorophores are used for calcium imaging, including Cal-590™ AM for red and Fluo 8® AM/Cal-520® AM for green, in combination with patch clamping.

Figure 1: An image taken with the Prime 95B, showing neural cells labeled with Cal-590AM (a red calcium dye) in an acute slice of brainstem tissue.

Challenge

When imaging sub-cellular functional dynamics via patch clamping as well as whole-network calcium imaging with numerous functional populations of neurons and astrocytes, it is necessary to have highly sensitive imaging equipment with high framerates and a wide field of view. In addition, when calcium imaging brain slices there can be issues with light scattering depending on the age of the slice, and time lag while the calcium fluorophore dyes bind to the sites of interest.

As Dr. Phillips uses a combination of different electrophysiological and imaging hardware it became necessary to use more customizable operating systems than Windows, namely Linux. Some imaging and electrophysiological software are locked to certain operating systems and not customizable, but in Linux it is possible to write custom scripts to support specific applications, even synchronizing between entirely different pieces of hardware. By using Linux experiments become far more flexible and optimizable, but as many products are only supported in Windows, the switch to Linux can be inconvenient.

The [Prime BSI] was really quick and easy to set up and worked well with MicroManager, and we’re happy because we’re seeing better results, the SNR is really better, it’s perfect for our needs

Solution

The Prime 95B offers both a hardware and software solution, being fully supported in Linux and featuring the optimal hardware specifications for imaging both sub-cellular and whole-network brain slices.

The large sensor of the 95B allows for high-speed images to be acquired using a region of interest (ROI) without sacrificing the required field of view when working with single cell patch clamping, as well as the use of the full-frame sensor for whole network calcium imaging. The Prime 95B offers a flexible solution for any scale or length of experiment and excels in low light conditions. As Dr. Philips told us “We barely need any light and we still get a great image”.

The 95B is well supported in Linux which allows for flexible software customization, with the PVCAM Linux driver also tested frequently with Ubuntu: “some software is locked to Windows but with Photometrics, I could write custom scripts and interfaces for the Prime 95B camera in Linux and pair recordings from whole-network calcium imaging and patch clamping simultaneously”.

Calcium Imaging and ElectrophysiologyCustomer Stories

Prof. Dieter Bruns

Dr. Yvonne Schwarz

Molecular Neurophysiology, Center for Integrative Physiology and Molecuar Medicine (CIPMM), University of Saarland, Germany

Background

The CIPMM Molecular Neurophysiology Lab studies the relationships between astrocytes and neurons, and their communication via the release of vesicles of neurotransmitters, including calcium (Ca2+). Prof. Dieter Bruns heads the lab, and researcher Dr. Yvonne Schwarz spoke about the research: “There is not much known in how astrocytes release neurotransmitters… they are prime candidates for governing neuronal function and even diminishing epileptic seizures. We are interested in how they release these transmitters in vesicles, and how the neurons will react.” Calcium homeostasis is important for synaptic plasticity and learning, making this an active area of research.

Dr. Schwarz works with a combination of different samples, studying calcium signaling cascades with basic cell line models, then extrapolating into primary hippocampal co-cultures of astrocytes and neurons. These samples are stimulated, recorded, and imaged, combining calcium imaging and electrophysiology.

Figure 1: An image taken with the Prime 95B of glutamatergic neurons stained with the glutamate sensitive fluorescent reporter iGluSnFR.

Challenge

The Molecular Neurophysiology Lab works with different neuroscientific applications, from optogenetics to calcium imaging, meaning that flexible imaging solutions are needed, such as high acquisition speeds needed when calcium imaging, in order to capture dynamic activity within the cells.

As Dr. Schwarz mentions: “We are trying to image with high speeds, the most challenging part is really combining electrophysiology with imaging, with electrophysiology coming from patch-clamping the neuron and calcium imaging coming from the GCaMP-transfected astrocytes, so it can be tough to combine the two and get them synced.”

Dr. Schwarz has also been using an EMCCD camera for other experiments but was interested in sCMOS technology for a new imaging system.

The [Prime BSI] was really quick and easy to set up and worked well with MicroManager, and we’re happy because we’re seeing better results, the SNR is really better, it’s perfect for our needs

Solution

The high speed, high sensitivity, and large field of view of the Prime 95B presents a highly-flexible solution to the range of experiments occurring in the Molecular Neurophysiology Lab. As Dr. Schwarz said: “I have integrated [the Prime 95B] for the first time with an LED, and now I am running experiments and they work nicely, and I am actually pretty happy with it, I have to say… I haven’t done any year-long experiments yet but it is a very good camera.”

In terms of the Prime 95B field of view compared to EMCCD: “It is larger and it has a much better signal to noise than the EMCCD, and that helps us a lot actually, as I can image more different cell populations within my astrocyte network, which is nice.” The low read noise, clean bias, and 95% QE of the Prime 95B results in a highly sensitive camera that improves on EMCCD technologies.

This application needs a combination of speed and sensitivity depending on the specific experiment, meaning the flexibility of the Prime 95B is very useful, using the high speed for calcium imaging in neurons, the high resolution for transmitter release in astrocytes, and the large field of view for general cell population experiments.

Cryo Super-Resolution ImagingCustomer Stories

Dr. Arjun Sharma

Dr. Mario Brameshuber

Biophysics Group, Institute Of Applied Physics, TU Wien, Austria

Background

The Biophysics Group at TU Wien is interested in super-resolution imaging in order to study the dynamics of biological systems such as T cells. One approach to this is cryo super-resolution imaging, which aims to produce highly-resolved images by imaging at extremely low cryogenic temperatures of -180 °C. At this temperature fluorophores are far more stable: while samples imaged at room temperature may bleach within minutes, the same sample would last hours at cryogenic temperatures, allowing for long exposures and high light intensities with little risk of bleaching the sample.

Dr. Arjun Sharma uses the cryogenic imaging system, and stated: “with blinking fluorophores and cryogenic temperatures, we can differentiate one molecule from another and increase the resolution up to 1 nm, this is our target.” This sub-cellular resolution allows for a detailed analysis of protein oligomers and aggregates within T cells and other samples.

Figure 1: A super-resolution image of Alexa Fluor 647 dye molecules captured at cryogenic temperatures (-145 °C) using a 60x 0.7 NA air objective with the Prime BSI.

Challenge

Due to the extremely low temperatures, the cryogenic system avoids standard imaging challenges concerned with photodamage and bleaching. However, the system introduces some unique challenges, as outlined by Dr. Sharma: “We are using a 60x low 0.7 NA air objective, the photon collection efficiency is not great so we really need a camera that is very sensitive and with minimum noise.” Only air objectives can be used with the cryogenic imaging system as water or oil would freeze, consequently, higher-NA immersion objectives cannot be used.

The cold also limits fluorophore blinking, meaning that a large field of view is needed in order to capture blinking and achieve super-resolution acquisition. The high exposure times mean that speed is not a factor, but a suitable camera should provide high sensitivity over a large field.

The [Prime BSI] was really quick and easy to set up and worked well with MicroManager, and we’re happy because we’re seeing better results, the SNR is really better, it’s perfect for our needs

Solution

The Prime BSI is an ideal fit for the cryogenic super-resolution system, with the balanced 6.5 μm pixel providing Nyquist-optimized resolutions at 60x. In addition, the low noise and 95% quantum efficiency result in a high signal to noise ratio when using the Prime BSI.

Dr. Sharma was also impressed by the Prime BSI: “From the specifications on your website it said your noise levels are better compared to normal CMOS camera and EMCCD, we checked that and also compared the noise level to another EMCCD we were using, and the images taken [with the Prime BSI] were much better.”