Page Type: Customer Story

Single Molecule FRETCustomer Stories

Dr. Ehud Isacoff

Director, Helen Wills Neuroscience Institute, UC Berkeley

Background

Prof. Ehud Isacoff’s lab at the University of California, Berkeley, USA is interested in the mechanistic properties of metabotropic and ionotropic receptor subunits and the role the resultant protein complexes play in signal transduction. His group uses a combination of microscopy approaches including single molecule Föester resonance energy transfer (smFRET) in live cells, and in vivo imaging in a variety of species. They’ve developed a number of tools for the investigation of neuronal function. Having pioneered the manipulation of receptors with tethered ligands, more recently they’ve developed photoactivatable versions of sensors such as GCaMPs.

The effects of subunit composition on metabotropic receptor transduction can be subtle and impossible to separate in mixed populations of receptor subunits. In the brain of mammals, metabotropic glutamate receptor subunits have mixed and overlapping expression patterns, making it very difficult to understand the roles of particular subunits. Single molecule in vitro analysis of receptors with known subunit compositions is needed. This allows the full characterization of biophysical properties, such as conformation change kinetics, which can be measured by smFRET.

Figure 1: Donor and FRET signals separated to the top and bottom halves of the camera sensor. Comparison images were acquired on the Prime 95B sCMOS (left) and a 512×512 EMCCD (right) using the same exposure time. The increased field of view is obvious and subsequent image analysis revealed that the Prime 95B also had a higher signal-to-noise ratio. The Prime 95B is also capable of far higher speeds. This allows for higher quality data to be acquired and more protein complexes to be measured with faster sampling kinetics.

Challenge

To measure the conformation changes of single receptors, fluorescent receptor subunits expressed in bacteria are reconstituted into functional receptor types and immobilized on a surface for imaging. Specific manipulations like ligand applications can be performed to stimulate the receptors to change conformation, which is recorded in the FRET signal. The kinetics of the conformational change are accurately mapped by the temporal FRET response.

Single molecule FRET presents very specific problems for imaging. There are very few fluorophores present in each protein complex. Secondly, receptor kinetics can be extremely fast. High camera frame rates are needed to accurately sample fast events. This means exposure times are short and detecting the few photons being emitted is difficult. EMCCD cameras have traditionally been used due to their sensitivity, but they lack the speed to detect very fast events.

We have been using 10 ms exposure and getting 100 fps and I’m just as confident in the data at 100 fps with the Prime 95B as with 10 fps with the EMCCD.

Solution

Using the Prime 95B back-illuminated sCMOS camera, the Isacoff lab improved the speed at which they can record in smFRET experiments. Previously they achieved 10 fps with an EMCCD. They shared, “We have been using 10 ms exposure and getting 100 fps and I’m just as confident in the data at 100 fps with the Prime 95B as with 10 fps with the EMCCD.” This increase in frame rate, whilst still maintaining the high signal-to-noise ratio needed to reliably record FRET changes, has allowed detection of events of 10 fold shorter duration than before.

This has allowed the group to confirm the kinetics of slower moving protein complexes, but also determine faster kinetics of other complexes for the first time. The combination of large 11µm pixels with high 95% quantum efficiency and fast frame rates make the Prime 95B an excellent choice for smFRET where both sensitivity and speed are necessary to produce high-quality data.

Single Molecule TIRFCustomer Stories

Dr. Biswajit Pradhan

Cees Dekker Group, Department of Bionanoscience, Kavli Institute of Nanoscience at Delft University of Technology, The Netherlands.

Background

Dr. Pradhan’s research is concerned with understanding how DNA supercoils with a family of proteins called structural maintenance of chromosome (SMC) proteins.

When cells are not dividing, the chromosomes are not properly organized. However, during cell division DNA becomes organized. Just prior to cell division, following DNA replication, chromosomes condense and become tightly wound via supercoiling. Chromosomes then pair up along the center of the cell and are pulled to either side of the cell by fibers attached to the centromeres.

SMC proteins play a role in some of these events and Dr Pradhan is trying to study the interactions that occur between DNA and proteins at the single molecule level, to determine how the DNA becomes supercoiled by proteins during this process. Dr Pradhan is building a custom single molecule TIRF system to study these interactions.

Figure 1 Images of A) Single Molecules of ATOO647N and B) DNA Chains taken with the custom single molecule TIRF system using the Teledyne Photometrics Prime BSI.

Challenge

Dr Pradhan told us, “Our biggest challenge is the limited number of photons that a single molecule emits. Our molecules emit approximately 104-105 photons before they bleach so the molecules can only be observed for a limited amount of time. If we want to use lower exposure times, the excitation light intensity needs to be increased which means the molecules bleach even more quickly.”

Dr Pradhan was previously using an EMCCD camera with 120-150× magnification. As a result of this high magnification and relatively small field of view of the EMCCD camera, he was not able to image a very large sample area. He has since built a new system with 60× magnification to increase the sample area. However, the larger pixel size of the EMCCD does not allow for Nyquist sampling at 60×.

We have been using 10 ms exposure and getting 100 fps and I’m just as confident in the data at 100 fps with the Prime 95B as with 10 fps with the EMCCD.

Solution

Dr. Pradhan is now using the Teledyne Photometrics Prime BSI back-illuminated sCMOS with his home-built single molecule system.

Dr Pradhan shared, “The Prime BSI offers several advantages such as speed, field of view and price when compared to the EMCCD.” Dr Pradhan continued, “The service at Teledyne Photometrics is also very good, I was always updated, and my questions were answered quickly, Teledyne Photometrics were really willing to help out.”

Dr Pradhan went on to say, “The Prime BSI has the advantage of a small pixel size, so I don’t have to expand my beam. This makes the optical system much easier to work with. Without zooming I can get to the maximum resolution that I want. The high speed of the camera also really helps during my acquisitions.”

Super-Resolution Standing Wave MicroscopyCustomer Stories

Prof. Gail McConnell

Department of Physics, University of Strathclyde, Glasgow, UK

Background

The McConnell group are focused on the development of new optical microscopy methods for biomedical research and imaging.

Previously, the McConnell group were using mostly fixed cell imaging in their biological investigations, but they are now focusing on the development of techniques that will improve their live cell imaging capabilities. The group is currently working on the development of a new type of super-resolution microscopy based on the concept of standing wave microscopy.

Standing wave microscopy employs a wide field configuration that is combined with axial structured illumination to improve the resolution beyond the diffraction limit. The excitation light is composed of two beams which interfere giving rise to a standing wave, creating a sinusoidal excitation field in the axial plane. Only fluorescence molecules that coincide with the maxima of the standing wave will give rise to fluorescence.

Figure 1. MCF-7 breast cancer cell line stained with either a 10.7µM concentration of 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate (Invitrogen) [10µL of DiI to 1mL of Hepes Buffered Saline (Gibco)] or a 5.35µM concentration [5µL DiI to 1ml HBS]. Imaged with a 100ms exposure, excited with 525 nm CoolLED light source. Imaged using a 40X (air) or 60X (oil) objective on a Nikon Eclipse Ti2 with Teledyne Photometrics Prime BSI camera.
Cells plated in lbidi dishes by Gill Robb, imaged by Gill Robb and Shannan Foylan of the McConnell group.

Challenge

The McConnell group acquired a new Nikon Eclipse Ti2 widefield microscope as part of their tools and resources development project and so they started looking for a camera that would allow them to get the most out of their new system.

Professor McConnell told us, “We began to explore new options for suitable cameras to maximize the data quality from the new Ti2 instrument. We knew we wanted to use brightfield, fluorescence and DIC imaging. The microscope would also be used for live cell imaging using various magnification objectives, so we also required flexibility from the camera to be compatible with a range of objectives from 10X to 60X and compatibility with the new NIS-Elements software”.

We have been using 10 ms exposure and getting 100 fps and I’m just as confident in the data at 100 fps with the Prime 95B as with 10 fps with the EMCCD.

Solution

The McConnell group is now using the Teledyne Photometrics Prime BSI on the Nikon Eclipse Ti2 widefield system. Professor McConnell shared “Following the demo of the Prime BSI, we were very happy with the camera, and have been using it every day since.”

Professor McConnell told us, “The Prime BSI, with a 6.5 µm pixel, offers the most flexibility for use with the range of objectives we are using as well as improving the resolution”.
Professor McConnell went on to say, “The customer service from Teledyne Photometrics was excellent. All our queries were responded to quickly and Teledyne Photometrics were very available and always happy to help and to offer training. I was very happy to give them our custom.”

Live Vesicle TraffickingCustomer Stories

Dr George Sirinakis, Senior Research Associate

St Johnston Lab, part of the Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, UK

Background

The St Johnston Lab is primarily interested in how cells become asymmetric so that they can perform distinct functions on opposite sides of the cell. This is known as cell polarity.

In particular, the lab are interested in epithelial cells, which form the sheets that line our organs. Epithelial cells must have an apical side which contacts the lumen, for example the inside of your small intestine, lateral membranes where cell-cell contacts are made, and a basal surface that maintains contact with the extracellular matrix.

Loss of polarity, especially of epithelial cells, is a hallmark of cancer. In this project the lab aims to image the movement of vesicles with cargo proteins destined for these different membranes.

Figure 1 Drosophila epithelial cells. The arrows indicate vesicles containing a cargo protein, cell adhesion molecule Fasciclin III (FAS3) tagged with Halo and labeled with SiR dye.

Challenge

The St Johnston Lab are aiming to image fast moving, small vesicles that contain a limited number of cargo proteins which results in a very low fluorescence signal. What is more, the lab are imaging in tissue, which causes further reductions in brightness due to aberrations. Finally, the tissue needs to be kept alive for imaging so the laser power must be kept to a minimum to reduce any phototoxic effects.

We have been using 10 ms exposure and getting 100 fps and I’m just as confident in the data at 100 fps with the Prime 95B as with 10 fps with the EMCCD.

Solution

The Prime BSI back-illuminated sCMOS camera was a great solution for the St. Johnston Lab, combining high, 95% quantum efficiency and low read noise with the fast speed expected of an sCMOS device.

Dr Sirinakis shared with us, “The high quantum efficiency and dynamic range of the Prime BSI enables us to visualize even the dimmest vesicles that contain limited numbers of cargo molecules and track them with high speeds in live tissue.”

Dr Sirinakis went on to say, “The high sensitivity and low read noise of the Prime BSI is transformative in these experiments as it enables us to use low levels of excitation power and significantly reduce photo-damage to the tissue. With the Prime BSI we are now able to ask more meaningful questions about vesicle trafficking and better understand cell polarity.”

Single Molecule SpectroscopyCustomer Stories

Prof. Dirk-Peter Herten

Heidelberg University

Background

Professor Herten leads the Single Molecule Spectroscopy group at Heidelberg University aiming at quantitative analytical approaches in biology and chemistry based on single-molecule data acquired with advanced fluorescence microscopy.

The groups research interests range from the development of fluorescent probes for live-cell microscopy to the investigation of chemical reactions on a single-molecule level. The group successfully studies relatively simple reactions like complexation of Cu(II), Cu(I)/Cu(II) redox reactions and epoxidation of aliphatic double bonds.

Figure 1 Time series showing the activation of immobilized fluorogenic probes upon click-reaction to a reactive target molecule added during the experiment (50 ms exposure at 5 s intervals, going left to right, top to bottom.) Recorded field of view 50 × 50 µm, depicted region 6 x 6 µm.
The graph on the right shows the intensity changes in the ROI over time.

Challenge

In the area of single-molecule chemistry, the group have now turned to more complex catalytic reactions to try to identify individual reaction steps and alternative reaction pathways.

Here, the limited observation time due to photo-bleaching and low photon yield poses a major obstacle in the investigation of chemical transitions.

The precise identification of different molecular states occurring randomly during a chemical transformation requires excellent signal to noise ratio at high time-resolution in the millisecond range.

We have been using 10 ms exposure and getting 100 fps and I’m just as confident in the data at 100 fps with the Prime 95B as with 10 fps with the EMCCD.

Solution

The Herten group are now using the Teledyne Photometrics Prime 95B sCMOS camera with their home built single molecule system.

Professor Herten told us that, “The Prime 95B, with the combination of large pixels with back-illumination, achieves not only a very high quantum yield but also a low readout noise at very high frame rates. The high frame rates and the low readout noise brings us closer to our goal of observing individual reaction steps in single-molecule experiments.”

Professor Herten went on to say that, “The implementation of the Prime 95B into our existing self-build microscope setup was straight forward and super-easy. The programmable logical outputs enabled direct control of the camera during a running experiment and delivered excellent results already from the first experiment on.”

Single Molecule ImagingCustomer Stories

Redmar Vlieg

John van Noort Group, Leiden Institute of Physics, Leiden University, The Netherlands.

Background

Redmar Vlieg’s research, within the group of John van Noort, primarily involves the use of two-photon microscopy to investigate biological processes in zebrafish embryos and in vitro measurements on gold nanorods (GNRs).

Due to their localized surface plasmon resonance (LSPR), GNRs have unique optical properties which allow them to be used as single-molecule sensors or very bright luminescent markers. The resonance frequency is dependent on the refractive index in the near-field of the rod, hence perturbations by small molecules can be detected by measuring this frequency shift. By exciting the GNRs via the non-linear excitation mechanism of two-photon microscopy, sensitivity can be increased to detect even smaller molecules.

Besides detection of single molecules, GNRs are used as luminescent markers. The LSPR mediates a significant increase in absorption of the excitation light as it couples with the incoming EM waves, making them as bright as quantum dots. Moreover, GNRs have the added benefit that they do not bleach and blink, and their surface can be easily functionalized for biological applications. Hence, the group investigates the merits of using GNRs as two-photon contrast agents for in vivo measurements.

Figure 1 Multifocal two-photon image of gold nanorods randomly dispersed on a glass coverslip. Differences in signal intensity are explained by either exciting the rods at their LPSR, or rod aggregates.

Challenge

One issue that the group face when using GNRs is that when the temperature of the rods is increased the ends start to diffuse, even at relatively low temperatures. Although gold melts around 3000°C, the tips start diffusing at much lower temperatures. As gold nanorods start to lose their shape they begin to lose their aspect ratio, which causes the LSPR to shift and renders them useless for imaging.

The group initially were using an EMCCD camera with a 60× TIRF oil lens for their in vitro studies. However, oil objectives have a very short working distance which is not appropriate for in vivo imaging of thick samples such as Zebrafish embryos. For this reason, they have since changed the setup to have a single 25× objective to permit a longer working distance and larger field of view, which required a more suitable camera to maximize speed and resolution.

The group therefore want to image with the lowest excitation light possible to prevent destruction of their GNR markers, as well as imaging the in vivo samples with high speed to permit single molecule tracking. For this reason, the group was interested in back-illuminated sCMOS cameras as they have high QE combined with large sensors and fast readout speeds.

We have been using 10 ms exposure and getting 100 fps and I’m just as confident in the data at 100 fps with the Prime 95B as with 10 fps with the EMCCD.

Solution

The van Noort Group is now using the Teledyne Photometrics Prime BSI back-illuminated sCMOS with 25× magnification on their multifocal multiphoton custom microscope, using LabVIEW to control all components of the system. Redmar Vlieg told us that “The LabVIEW drivers provided with the Prime BSI allowed us to integrate the camera exactly the way we want in our in-house build microscope.”

Redmar Vlieg went on to say, “We decided on the Prime BSI as the smaller pixel size, compared to other back-illuminated cameras on the market, made it best suitable for high resolution tracking. Moreover, our EMCCD camera, the QuantEM:512SC, was also from Photometrics, which always performed very adequately”.

Having a high QE permits the reduction of exposure time which is ideal for his applications, permitting imaging of the gold nanorods without modifying their structure. Redmar Vlieg also stated that the camera offers great benefits such as no excess noise and no gain decay.

Live-Particle TrackingCustomer Stories

Dr. Adam Wexler, Post-Doctoral Researcher

Arie Zwijnenburg Laboratory for Advanced Microscopy and Optical Metrology,

Wetsus – European Centre of Excellence for Sustainable Water Technology, Leeuwarden, Netherlands

Background

Dr. Wexler’s research interest is to bring the power of photonics to the field of sustainable water technology. Currently he, along with collaborators at the University of Twente, are focused on preventing outbreaks of waterborne viruses by understanding and improving water filtration technologies.

The virus particles investigated are small, roughly 20-30 nm in size, and can sometimes pass through water filtration systems and remain infectious. There is currently no practical way to investigate the effectiveness of these filtration devices in real time.

Dr. Wexler is working towards building a microscope that permits tracking of these particles in real time to detect viable viruses in the water and assess the efficiency of these filtration processes. The ultimate aim is to couple the real-time imaging to automated imaging algorithms that can detect problems in the water and alert water production managers before an outbreak begins.

Fluorescent image of CCMV (cowpea chlorotic mottle virus) particles.
Figure 1 Fluorescent image of CCMV (cowpea chlorotic mottle virus) particles. The brightest and largest objects are aggregates of many viruses while the dimmer points are single virus particles while the dimmest are fragments of capsids.

Challenge

The primary challenge is the low fluorophore labelling densities, Dr. Wexler explains that they try not to modify the viruses too much so there are very few fluorophores (<40) per virus.

Dr. Wexler and his colleagues realized that when there are 40 fluorophores on a virus capsid they exhibit stable fluorescence but when the viruses are fragmented they blink. This allows differentiation between whole and broken virus particles. The combination of low fluorophore density, rapid blinking, and flow means that low noise, camera speed, and sensitivity are important.

Dr. Wexler looked at several sCMOS cameras and told us that one of the biggest factors for them was the quantum efficiency (QE). He sometimes uses far-red dyes so a high QE at these wavelengths is of additional importance.

We have been using 10 ms exposure and getting 100 fps and I’m just as confident in the data at 100 fps with the Prime 95B as with 10 fps with the EMCCD.

Solution

Dr. Wexler is now using the Teledyne Photometrics Prime BSI back-illuminated sCMOS camera on his widefield microscope to image these virus particles in real-time under flow at 40× magnification. He shared, “The increase in QE was huge, and really helped with our application.”

Dr. Wexler also mentioned that the larger field of view was a welcome improvement because they are tracking and counting particles, so the increased throughput really improves the quality of statistics from the population.

Dr. Wexler concluded, “So far the camera performs well and has a lot of potential for our future work.” They also hope that the high speed will be useful for quantification measurements using FRET to distinguish between whole and fragmented viral particles.

Widefield Fluorescence MicroscopyCustomer Stories

Dr. Steven Pittler

Director of the Vision Science Research Center,

University of Alabama at Birmingham

Background

Professor Steven Pittler, Director of the Vision Science Research Center at the University of Alabama at Birmingham, uses fluorescence imaging to study the diseases related to vision. Dr. Pittler explains, “Our Molecular and Cellular Analysis Core focuses on the investigation of a range of ocular diseases in the anterior and posterior segments of the eye. This includes common diseases like age-related macular degeneration and diabetic retinopathy, as well as less common diseases like retinitis pigmentosa. Our facility allows several investigators to image ocular tissues that are stained with different primary and secondary antibodies. Our goal is to develop a system that can cover the range of fluorescence wavelengths from 300 to 800 nm.”

Mouse retina displaying the layer stratification of cell types and subregions

Photoreceptor cells (outer retina), bipolar, horizontal, and amacrine cells (inner retina) and ganglion cells layers. Image acquired with Micro-Manager 2.0 beta software on a Zeiss Axioplan 2, with DAPI (blue) and Cy5 (Red) filters at 20× magnification taken with the Iris 9 Scientific CMOS camera.

The primary antibody recognizes a channel protein in the photoreceptor outer segments and in the inner plexiform layer of the inner retina. Nuclear layers are stained with DAPI.

Challenge

Dr. Pittler and his colleagues must gather structural information from ocular tissues. For the Vision Science Center, this means distinguishing between various tissues and layers of the eye to learn about normal and disease processes in the eye. A high degree of spatial resolution is required. Additionally, sensitivity to detect low emitting areas of the sample is key to determining exactly which cells are expressing genetic abnormalities. Dr. Pittler told us, “The Iris 9 has outstanding sensitivity of detection and helped to minimize issues with autofluorescence.”

At 9MP resolution, the Iris 9 image quality is spectacular and allows for the capture of very fine detail

Solution

For the Pittler lab and the Vision Science Research Center molecular core, having the 4.25 µm pixel size and sensitivity over a wide range of visible wavelengths on the Iris 9 proved to be crucial to their retinal studies. “The resolution and level of sensitivity has made a huge difference. At 9MP resolution, the Iris 9 image quality is spectacular and allows for the capture of very fine detail,” Dr. Pittler continued, “Additionally, the fact that we can use this camera with Fiji and micro-manager was also an added plus.”

Single Molecule Micromirror TIRFCustomer Stories

Prof. Karl Duderstadt

Duderstadt Group
Max-Planck Institute of Biochemistry, Germany

Background

The Duderstadt Group are interested in understanding the organization and dynamics of macromolecular complexes, such as the replisome which is responsible for DNA replication.

Successful DNA replication is critical for cell survival, and errors within this process have been implicated in many disease pathologies. The Duderstadt Group studies this process by introducing fluorescently labeled enzymes, such as DNA polymerase and RNA polymerase, into bacterial model systems. This allows them to visualize and track the polymerase enzymes within the replisome as they move along a DNA substrate during replication. They can then use kymograph analyses to quantify the movement along the DNA strands to better understand these processes.

The group primarily use a home built single molecule micromirror TIRF imaging system in their investigations. The micromirrors remove the need for a dichroic mirror and permit switching colors quickly, also removing the need for a filter wheel, making it an ideal system for live imaging.

Figure 1: The field of view shows linear DNA stretches which are used to study DNA replication in real time.
One end of the DNA substrate attaches to the glass coverslip by a streptavidin-biotin interaction so when flow is applied the DNA molecule stretches and the other end attaches to the surface via the same interaction. These linear DNA stretches are used as the basis for the replication assay. One end mimics a preprimed replication fork where the replication machinery can assemble.
To observe replication in real time, the polymerases are labeled with single dyes while the DNA itself is labeled with a different color. The image shows only the DNA channel. The position of the polymerases can be correlated with the DNA and the replication rate can be estimated. Because of the single dyes on the polymerases, a highly sensitive camera is needed to image them over the course of the experiment without bleaching.

Challenge

Professor Duderstadt told us, “Our biggest challenge is how many photons to collect and the corresponding lifetime of the fluorophore we are looking at. This limits our ability to track sometimes.” For this reason, the group needs the highest camera sensitivity to ensure the dyes do not bleach and to permit the collection of as much light as possible to allow tracking.

Faster frame rates and lower costs were also strong factors in the decision to purchase a new camera. Professor Duderstadt told us, “Ideally, we want to put the least amount of excitation light in to allow just enough signal to track the polymerase over time. That’s why, in the past, we used an EMCCD camera, but it appeared to us that the Prime BSI could match the EMCCD for sensitivity.”

We have been using 10 ms exposure and getting 100 fps and I’m just as confident in the data at 100 fps with the Prime 95B as with 10 fps with the EMCCD.

Solution

The Duderstadt group are now using the Teledyne Photometrics Prime BSI on their custom single molecule micro-mirror TIRF system. Professor Duderstadt told us, “We compared specifications from various manufacturers, but the Teledyne Photometrics Prime BSI offered the QE, chip specifications and multi-ROI features that we required”.

He also told us, “The chip size is large, giving us a big field of view, which is really fantastic, and the pixel size is small at 6.5 microns, which permits Nyquist matching on our 60X lens, maximizing resolution in our experiments. The multi-ROI feature will also permit higher frame rates, and we have some experiments in mind that will make use of this.”

He went on to say that, “Although most of our applications so far haven’t been at exceedingly high frame rates, in the future we do want to look at the rotation of DNA molecules using gold nanoparticles, and these might have faster kinetics, this was a further motivation for choosing the Prime BSI.”

Live Yeast Vacuole ImagingCustomer Stories

Professor Andreas Mayer

Department of Biochemistry, Université de Lausanne, Switzerland

Background

Prof. Mayer and group work to study the cell membrane, especially the processes of membrane fusion and fission, exploring the molecular machinery that drives these reactions. Of particular interest are endosomes, lysosomes and other components of the endolysosomal system, which routinely fuse amongst each other to transfer cargo proteins or with the cell membrane to initiate repair and to release their contents out of the cell.

Most of this study is performed on the model organism yeast rather than mammalian cells, as yeast vacuoles are similar to mammalian lysosomes but are very big (2-4 μm), making them easier to image. By tracking these yeast vacuoles they can be observed to fuse together or undergo fission into smaller pieces. These studies involve live cell imaging and long time-lapse imaging using spinning disk confocal microscopy.

Figure 1 Yeast cells imaged with spinning disk confocal fluorescence microscopy. The yeast cell wall is stained in blue, the vacuoles in red and septins (proteins involved in mitosis and cell splitting) in yellow.
This represents a 3D reconstruction of a stack of confocal images taken on a spinning disk system.

Challenge

As Prof. Mayer states, “For us, a major challenge is photobleaching and phototoxicity because we want to look at the cells and organelles for a long time with repeated exposures… if we have low sensitivity we see many artifacts inside the cells, we get artificial fusion of organelles, purely light induced. These we can minimize if we have a microscope with maximum sensitivity.”

Most of these images are taken as stacks to build into 3D images over time. When imaging up to an hour, taking 10-20 slices per minute, there is an accumulation of exposure and phototoxicity, meaning that the exposure needs to be as short as possible, and the camera as sensitive as possible. A combination of speed and sensitivity would allow for minimal phototoxicity.

We have been using 10 ms exposure and getting 100 fps and I’m just as confident in the data at 100 fps with the Prime 95B as with 10 fps with the EMCCD.

Solution

Based on the need for sensitivity the Prime BSI is a clear choice, as outlined by Prof. Mayer, “We have mounted two Prime BSIs on our spinning disk system, and we appreciate the high sensitivity of the camera while it still gives a good framerate, which is more than sufficient for our purposes.”

The high sensitivity and speed of the BSI means minimal exposure times are an option, reducing the phototoxicity and allowing for long experiments. Prof. Mayer shared, “Now we can definitely detect signals that we had a hard time detecting before, signals now come out much clearer. We can do time courses now that were not doable before as the cells would be bleached or dying from phototoxicity by the end of the experiment.”