Page Type: Customer Story

Light Sheet MicroscopyCustomer Stories

Dr. Jan Huisken, Director of Medical Engineering

Dr. Kurt Weiss, Postdoctoral Researcher

Morgridge Institute for Research, Wisconsin

Background

Dr. Jan Huisken, Director of Medical Engineering at the Morgridge Institute for Research has been instrumental in the invention and development of Light Sheet Microscopy. One of his current research interests is in the development of novel methods to image cleared tissues.

Dr. Kurt Weiss, postdoctoral researcher in the Huisken lab, told us, “We image entire organisms and organs that have been optically cleared. That includes samples from fish to mouse organs in the range of 1-20 mm. As a microscopy development lab with no eyepieces on our microscopes we depend exclusively on scientific CMOS cameras for feedback,” Dr. Weiss continued, “One of the common features of our samples is that they are fairly large, so we look for low magnification, and a moderate numerical aperture in our setup.”

Cleared zebrafish 8mm long x 2mm wide.
Imaged in 2 tiles using the full field of view of the Iris 15 Scientific CMOS camera. 1 µm per pixel resolution. Color coded for depth.

Challenge

For their light sheet imaging experiments, large fields of view are highly desired to minimize the number of images required to image a whole specimen. Dr. Weiss explained, “Most objectives have at least a 21 mm field number so the limiting factor is generally the camera.” Additionally, processing images can become challenging when smaller sensor sizes limit the field of view in a tiled image. “Smaller chips and larger pixels forced us to use a large number of image tiles, increasing imaging time and post-processing complexity.” Dr. Huisken adds, “At the same time we want to achieve diffraction limited resolution across the entire field of view. Hence, a large sensor with small pixels is ideal for light sheet imaging of cleared specimens.”

The Iris 15 camera has both a large chip and a small pixel size so we can optimize the numerical aperture of our lenses. The 4.25 µm pixel size on the Iris 15 allows us to use more of the 0.28NA at 4× than other cameras without zooming.

Solution

The large, 25 mm sensor coupled with small, 4.25 µm pixels make the Photometrics Iris 15 scientific CMOS camera a perfect fit for the light sheet imaging of cleared tissues at the Huisken lab.

Dr. Weiss told us, “The Iris 15 camera has both a large chip and a small pixel size so we can optimize the numerical aperture of our lenses. The 4.25 µm pixel size on the Iris 15 allows us to use more of the 0.28NA at 4× than other cameras without zooming. I was surprised with the sensitivity. I know that physically a small pixel will gather fewer photons per unit area. Thus, I expected to take a significant hit in sensitivity, but we can simply increase laser power slightly and get just as good of a signal as larger pixel cameras.” Additionally, the speed of the Iris 15 allows for extremely fast light sheet imaging experiments. Dr. Weiss shared, “We use this camera on a custom light sheet microscope to obtain 1-2 micron resolution across 10 mm samples in a matter of seconds.”

Light Sheet MicroscopyCustomer Stories

Christopher Yip, Ph.D.

Director of the Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto

Background

Currently the Associate Vice President, International Partnerships at the University of Toronto, Dr. Christopher Yip is the former Director of the Institute of Biomaterials and Biomedical Engineering, and a faculty member in IBBME, Chemical Engineering, and Biochemistry, where he and his team create a variety of experiment-specific microscopy tools.

Dr. Yip told us, “Our lab core research focus is in single-molecule biophysics and the development of combinatorial imaging platforms for the study of molecular and cellular dynamics. We are particularly interested in the integration and application of different imaging modalities to understand protein-protein interactions on a wide range of temporal and spatial scales. A key platform for our lab is light sheet microscopy, and, we have been actively developing a diverse suite of systems, each tailored to a specific experiment. Some of our efforts include tracking receptor association, protein-membrane interactions, and dynamics. Our most recent work has focused on understanding the mechanisms of coordinated cell migration during heart development using Drosophila as the model system.”

Figure 1.
Hand-GFP labeled Drosophila organoids, imaged at various angles with light sheet microscopy.
Figure 2.
The quantitative impact of hand-GFP signal intensity at varying imaging angles of Drosophila cardiac organoids.

Challenge

In the development of light-sheet microscopy imaging for single molecule biophysics, many cameras require users to choose between a large field of view, sensitivity, and high spatial resolution.

Dr. Yip explains, “We face a number of key imaging challenges in our research. The first is low light levels as is often the case when studies are performed of low-expression proteins under endogenous promoter control. Additionally, the need for high spatial and temporal resolution is critical.”

The Iris 15 camera has both a large chip and a small pixel size so we can optimize the numerical aperture of our lenses. The 4.25 µm pixel size on the Iris 15 allows us to use more of the 0.28NA at 4× than other cameras without zooming.

Solution

For Dr. Yip’s work, the Iris 15 has provided the perfect solution for his light-sheet imaging applications. The 25 mm field of view and small framework of the camera body were particularly attractive and has made incorporation into their complex systems simple.

Dr. Yip shared, “Having the Iris 15’s high sensitivity, small pixels, and large field of view addresses a number of crucial experimental considerations for our studies of development, including the ability to acquire overlapping images from different imaging angles, which is key for multi-view SPIM.”

High Content ImagingCustomer Stories

Dr. Jonathan Rocheleau

Percy Edward Hart Professor in Biomedical Engineering & Associate Director, Research, University of Toronto

Background

Jonathan Rocheleau is an Associate Professor in the Institute of Biomaterials & Biomedical Engineering at the University of Toronto. His laboratory uses high content imaging to study pancreatic islet biology.

Dr. Rocheleau told us, “We do high content microscopy because we’re interested in the idea that cellular metabolism is heterogeneous – that some beta-cells are metabolically poised for proliferation or survival, while others are poised to secrete insulin.” Dr. Rocheleau continued, “We’ve created a high content microscope for measuring anisotropy sensors that we refer to as Apollo. We started with a sensor that responds to NADP+ but believe that there’s a lot of potential for the development of many more sensors using this design.

Three-color anisotropy measurements of AD293 cells expressing: mitochondrial Apollo-NADP+ (top row, mTurquoise2), Apollo-NADP+ (middle row, Venus) and mCherry (bottom row) at 63X magnification.

Challenge

The use of a camera with very stable readout and noise characteristics is critical to precise measurements in this application. Dr. Rocheleau explains, “Anisotropy imaging is very precise. It’s a photophysical measurement we’re making. With other sensors you often see data shown as representative traces. The precision of anisotropy imaging allows us to collect and collate data across many different cells with little to no drift between experiments”

Smaller fields of view on previously used cameras limited the number of events that could be recorded in a single experiment. “When we go to 63x with cameras with smaller chips, we were only imaging tens of cells at a time. We need to do populations of thousands of cells, and you’d have to tile a lot of images together. When you’re tiling and having to image, all of that takes time.”

The Iris 15 camera has both a large chip and a small pixel size so we can optimize the numerical aperture of our lenses. The 4.25 µm pixel size on the Iris 15 allows us to use more of the 0.28NA at 4× than other cameras without zooming.

Solution

The 25 mm field of view of the Iris 15 makes it a great camera for high content imaging applications, allowing Dr. Rocheleau’s team to image more cells and remove the need for tiling when projecting multiple images onto a single sensor. Dr. Rocheleau told us, “We like that the Iris 15 has such a big chip. In the future, we want to collect both parallel and perpendicular fluorescence simultaneously on the same chip. The camera gives us so much information, allowing us to collect tons of data.”

Additionally, the Iris 15’s noise stability and sensitivity across a wide spectrum of wavelengths allows the lab to use fluorescent sensors of a variety of emission wavelengths to tailor their imaging needs. Dr. Rocheleau concluded, “With this sensor we get very precise measurements. The precision of measurements is very high day to day, and we can make comparisons between experiments very well.”

Dynamics of Complex FluidsCustomer Stories

Alexandros Fragkopoulos, PhD
Oliver Bäumchen, PhD, Group Leader

Max Planck Institute for Dynamics and Self Organization
Physics of Soft and Living Matter, Bäumchen Lab, Göttingen

Background

The Bäumchen lab is researching the physics underlying interfaces of soft and biological matter. In particular, they want to understand how those interfaces can alter the dynamics of soft and living matter. To achieve this, a multitude of techniques including micro- and nanofluidics, lab-on-a-chip technologies and force spectroscopy methods are employed. One of the lab’s model systems is the microalgae Chlamydomonas reinhardtii, for which the lab is studying flagella-mediated cell adhesion and motility, at interfaces which can be controlled by light.

Figure shows representative images of a movie showing freely moving Chlamydomonas rheinhardtii in solution obtained with an IRIS 9 at maximum frame rate of 32 fps.
A) shows the entire full field of view. The insert (top right) is a minimum intensity projection showing the trajectories of individuals. B) Every 10th frame from this movie and C) a series of consecutive frames. Note that clearly all individuals can be made out and can thereby easily be tracked.

Challenge

To fully access the dynamics of the specimen of interest, a suspension of microalgae Chlamydomonas reinhardtii, Dr. Fragkopoulos, postdoc in the Bäumchen lab, looks into light-triggered behavior using high speed microscopy. The typical experiment includes a population of Chlamydomonas algae and documenting behavior over time. With their previous imaging solution, the lab did not have the camera sensitivity needed to record fast enough (>30 fps) at low-light conditions, nor did they have a suitable field of view (FOV) which meant having to produce extremely time-consuming imaging sessions.

The Iris 15 camera has both a large chip and a small pixel size so we can optimize the numerical aperture of our lenses. The 4.25 µm pixel size on the Iris 15 allows us to use more of the 0.28NA at 4× than other cameras without zooming.

Solution

Dr. Fragkopoulos is now using an Iris 9 Scientific CMOS (sCMOS) camera which enables the ability to achieve all targets that were not possible with the previous camera solution. The team can now image with a full FOV at very low magnification (10×/0.3NA), nearly three times more area at once. Moreover, they can now image at a higher speed of over 30 frames per second with a full field at a low light intensity of 0.2 µmol of photons/(m²s).

Ultimately, the team needs to segment individual alga and gain insight to their motility over time and light. To achieve this Dr. Fragkopoulos needs to collect approximately 5000 frames in stream acquisition. The Iris 9 camera, in conjunction with an SSD (write speed >1GB/sec), easily delivers. For segmentation of individuals in a densely packed FOV, resolving details is a must. The small pixels of the Iris 9 with its 4.25 µm x 4.25 µm provides more detailed information than previously obtainable with 6.5 µm x 6.5 µm pixels. In addition, the >73% quantum efficiency of the Iris 9 camera allows to maintain the exposure time of <20 ms, even in the lowest light conditions.

Calcium ImagingCustomer Stories

Prof. Michael J.M. Fischer, PhD

Institute for Molecular Physiology, Medical University of Vienna

Background

Dr. Fischer’s research addresses nociception, the nervous system’s response to harmful or potentially harmful stimuli, with focus on the peripheral nervous system. A particular interest is the role of transient receptor potential (TRP) channels, which play an important role in sensing pain.

Typically, imaging experiments in the Fischer lab involve the use of fluorescent indicators in cultured primary neurons or cell lines. Sensors for calcium ions (Ca2+) and voltage are used as activity readouts for spontaneous and induced signal transduction – with sensors being either ratiometric or non-ratiometric.

Figure 1. Montage of a sequence of grey scale images depicting a slow traveling calcium wave resolved at high temporal and spatial resolution with the Photometrics Prime BSI. Upon application of a receptor agonist, calcium influx started in a cellular extension (yellow arrowheads), followed by a spreading wave that reaches all cytosolic areas.
Cells were stained with Fluo-8 and excited at 455 nm using an Omicron LED HUB. The receptor distribution is compared to the start of the initiation of the calcium wave by subsequent confocal imaging of immunocytochemistry.

Challenge

A typical experiment performed by the group uses a chemical cue to generate rolling Ca2+ waves along a cellular extension which spread across the cell. Depending on the traveling speed, this requires a very high sampling rate. High-speed image acquisition, as well as structural-functional correlation, present important imaging challenges faced by the lab.

For conventional imaging techniques, observation of these Ca2+ waves requires a compromise between the acquisition speed necessary to obtain any meaningful kinetics and the low magnification needed to achieve substantial output. This is especially important when obtaining precise Ca2+-concentrations using ratiometric sensors such as Fura-2, which require switching between two excitation wavelengths.

In the best-case scenario, the Ca2+ signals will need to be imaged at close to 1000 frames per second (fps) and triggering through a software solution can be too slow to achieve this.

The Iris 15 camera has both a large chip and a small pixel size so we can optimize the numerical aperture of our lenses. The 4.25 µm pixel size on the Iris 15 allows us to use more of the 0.28NA at 4× than other cameras without zooming.

Solution

The Prime BSI enables the Fischer lab to image the progression of Ca2+ waves across neurites with unprecedented speed, accuracy, and precision.

Even ratiometric Fura-2 imaging can be achieved with an effective speed of up to 460 fps (>920fps, alternating 340/380 nm excitation) using the SMART (Sequenced Multiple Acquisition in Real Time) streaming feature implemented on Photometrics Prime cameras.

SMART streaming allows the camera to directly hardware-trigger up to 4 individual excitation channels with individually adjustable exposure times and intensities.

When imaging at these speeds, every single photon counts. The Prime BSI, with 95% quantum efficiency and very low read noise, produces results with an excellent signal-to-noise ratio and a very high dynamic range, which makes the camera the perfect match for the Fischer lab’s application.

Live Cell ImagingCustomer Stories

Jessica Kehrer, Dipl. Ing. & Prof. Friedrich Frischknecht

Frischknecht lab, Centre for Infectious Diseases, Parasitology

Heidelberg University Medical School

Background

The Frischknecht lab aims to understand transmission of the malaria parasite between host and mosquito. The lab focuses on the two motile stages of the life cycle – the ookinete and the sporozoite using the rodent model parasite Plasmodium berghei. The movement of this parasite is particularly interesting because the cells are able to move at very high speed without changing their shape.

For their standard experiments, they are using reverse genetics to either remove proteins or to label them with fluorescent markers followed by characterization of the resulting parasite line with state of the art microscopy techniques.

Figure 1. Maximum intensity projection of Plasmodia released from salivary glands. Cells were labelled with GFP. Imaging frequency was 250 fps.

Challenge

Motility can be studied using sporozoites freshly isolated from the salivary gland of infected mosquitoes. Wild-type sporozoites generally show a circular movement which proceeds at a speed of 1.5-2 µm/s for up to one hour.

The current imaging solution in the Frischknecht lab is a front illuminated CCD sensor with a very limited field of view (FOV) and low sensitivity. Sensitivity limitations result in long exposure times which in turn make the analysis of the obtained data – involving tracking of individuals and studying their motion – difficult. Typically, exposure times of 300-400 ms or more for the individual channels is common. Therefore, it is currently impossible to image the localization of weak fluorescent proteins.

The relatively low FOV also results in a lower sample throughput, reducing the quality of the statistical information – especially for mutants with low infection rates.

If sensitivity could be increased, the temporal resolution could also be improved. This would be crucial for improving tracking of the individual parasites, producing higher quality data that better represent physiological behaviour.

The Iris 15 camera has both a large chip and a small pixel size so we can optimize the numerical aperture of our lenses. The 4.25 µm pixel size on the Iris 15 allows us to use more of the 0.28NA at 4× than other cameras without zooming.

Solution

The Prime BSI back-illuminated sCMOS allows the Frischknecht lab to image up to 20x faster with the same signal to noise ratio. With the increased sensitivity, lower exposure times allow for an increase in speed which produces a more accurate image of multiple channels, enhancing the ability to track individual Plasmodium parasites more reliably. This also means that their movement can be studied more accurately. Jessica told us, “Weak fluorescent proteins, which had been impossible to observe with our previous camera, can now be readily visualized.”

In addition to the increase in speed, the Prime BSI has a much larger field of view resulting in the detection of nearly 3x more cells. This substantiates the produced data, solidifies statistics and makes the quality of scientific output much higher. In addition, this reduces the time needed to perform experiments, which reduces time at the microscope.

Live Cell ImagingCustomer Stories

Dr. Philippe Bastin, Principle Investigator

Bastin Lab, Institut Pasteur, Paris

Background

The lab of Dr. Philippe Bastin at Institut Pasteur is primarily interested in improving understanding of the trypanosome parasites, which are significant in human health due to their role in sleeping sickness. The trypanosomes also offer a useful experimental model to increase understanding of cilia and flagella function. The lab therefore does a significant amount of cell biology investigations including live cell imaging studies, four color immunofluorescence (IF) and dSTORM imaging.

Figure 1. Snap shot of live imaging of protein trafficking through the flagella of the trypanosome. The kymograph analyses represent a quantification of the movement of proteins through the flagella, both anterograde (A – large trains, base to tip) and retrograde (R – small trains, tip to base) which can be detected using much lower exposure times using the Prime 95B.

Challenge

Dr. Bastin’s lab studies protein trafficking in the flagella of trypanosomes. The challenge is that these proteins move quickly, at a rate of 2-5 microns per second in a very narrow (300 nm) environment, which is very close to the resolution limit for light microscopy.

Imaging is a tradeoff between time resolution and sensitivity, even at 100 ms exposure the particle has moved by half a micron during the exposure time leading to smearing of the trains, so it is quite challenging. When the train moves to the end of the flagella they then split into 3 smaller trains making them more difficult to detect.

Dr. Bastin was looking for a camera that had good sensitivity to decrease the exposure time. If the camera was too slow, it wouldn’t capture the movement. The previous EMCCD was used for its sensitivity, but this was limiting the resolution due to the large pixels. Four-color immunofluorescence imaging was also challenging, as when imaging dyes with longer wavelengths, such as Cy5, quantum efficiency is low.

The Iris 15 camera has both a large chip and a small pixel size so we can optimize the numerical aperture of our lenses. The 4.25 µm pixel size on the Iris 15 allows us to use more of the 0.28NA at 4× than other cameras without zooming.

Solution

Dr. Bastin is now using the Prime 95B to reduce exposure time for better time resolution while imaging protein trafficking through the flagellum. “The combination of increased sensitivity and signal to noise ratio meant the 95B was the best camera choice. For us, one of the most important things was reducing the exposure time to get better time resolution, whilst maintaining sensitivity. Even with very short exposure times of 10 ms we could start to detect the protein trains with the 95B. As the diameter of the flagella is near the resolution limit the Prime 95B with a 100X objective was the best choice for us offering a combination of speed, signal to noise ratio and sensitivity making it ideal for our applications.”

Dr. Bastin also anticipates the use of the 95B for quadruple immunofluorescence in the future. He shared, “For the long wavelengths such as Cy5, quantum yield was previously an issue. We tested the Prime 95B with this technique and we were amazed at the quantum yield of the camera, which allows us to reduce the exposure time for this type of acquisitions.”

Dr. Bastin also mentioned that he anticipated making use of the large field of view in these types of experiments. He told us, “It is advantageous for scoring cell phenotypes, such as knock out models for understanding flagella construction and motility. More cells in the field of view gives more data for each acquisition maximizing data collection and minimizing acquisition time.”

Imaging Mitotic DynamicsCustomer Stories

Dr Gary Gorbsky, W.H. and Betty Phelps Chair in Developmental Biology
Program Chair, Cell Cycle & Cancer Biology Research Program

Oklahoma Medical Research Foundation

Background

Research in the Gorbsky lab focuses on the basic mechanisms of how chromosomes assemble and move during cell division in normal cells and in cancer cells, the process termed mitosis. A major emphasis is the mitotic spindle checkpoint pathway that makes sure that each copy of the 23 pairs of chromosomes is distributed equally to each of the daughter cells.

This checkpoint system detects chromosomes that have failed to align at the metaphase plate and delays chromatid separation at anaphase until alignment is complete. Failure of the checkpoint pathway may underlie chromosome abnormalities which are hallmarks of cancer and defects in embryo development.

Figure 1: Cultured Xenopus cell expressing GFP-tubulin undergoing mitosis.
Imaged every ten seconds for over 18 minutes using light sheet illumination and a high resolution oil immersion objective. Selected frames are shown depicting major changes in the mitotic spindle as the cell progressed from prometaphase through metaphase, and anaphase. The high sensitivity of the Prime BSI allowed for greater total number of samples at lower illumination without noticeable photobleaching or photodamage allowing us to track changes in the mitotic spindle microtubules.

Challenge

The use of fluorescent proteins in live cell microscopy has revolutionized the study of mitosis. However, there are many pitfalls that stem from the tendency of the fluorescent proteins to be photobleached and from cellular damage due to phototoxicity. Mitosis is particularly sensitive to phototoxicity and substantial efforts are taken by the lab to minimize the amount of light reaching the sample.

At the same time, the lab is interested in mapping events at the highest temporal and spatial resolution. Measuring fluorescent protein location during mitosis is challenging, as structures assemble and disassemble quickly. High illumination and emission intensities perturb the mitotic process which requires them to use short exposure times to limit cell damage.

Low excitation light and short integration times result in low photon numbers arriving at the camera, so high camera detection efficiency and low camera noise is key to generating good data.

The Iris 15 camera has both a large chip and a small pixel size so we can optimize the numerical aperture of our lenses. The 4.25 µm pixel size on the Iris 15 allows us to use more of the 0.28NA at 4× than other cameras without zooming.

Solution

The Photometrics Prime BSI, with its small pixels and high quantum efficiency, allows the Gorbsky lab to sample at high spatial resolution with high sensitivity. This allows them to image events in mitosis with finer time resolution or over longer duration which gives them better sampling with less total light input and lower overall photobleaching and photodamage. This is critical with their new light sheet imaging system, where they might acquire through focus series at frequent intervals.

For example, where their previous high resolution microscope and camera could at most image cells at one to five minute intervals for the entire process of mitosis, the Prime BSI coupled to their light sheet microscope enables them to capture images every few seconds, with cells successfully completing mitosis with no ill effects.

Calcium ImagingCustomer Stories

Marc Freichel, PhD, Prof.

Volodymyr Tsvilovskyy, PhD.

Heidelberg University

Background

Dr. Freichel’s group are primarily interested in how ion channels regulate the influx of calcium ions (Ca2+) as messengers for cellular and systemic functions.

The group is mainly interested in understanding transient receptor potential (TRP) and Ca2+ channels and their role in calcium signaling in cells of the cardiovascular system (endothelial cells, smooth muscle cells, cardiac fibroblasts, platelets), epididymal cells and mast cells. This gives the group important information about the corresponding integrative body functions such as vascular and cardiac contractility, transmitter secretion, blood pressure regulation, blood vessel formation, fertility and mast cell activation as well as their role during maladaptive cardiac remodelling and for the development of diabetes mellitus associated long term sequelae.

The laboratory uses transgenic approaches such as gene targeting in embryonic stem cells for the generation of disease models and for the identification and validation of new drug targets. Within Dr Freichel’s lab, transgenic reporter lines (e.g. GCaMP6) are crossed with disease carrying lines to enable visualization of impaired signaling processes. In parallel, electrophysiological measurements via patch pipettes are also carried out.

Figure 1. Time lapse of Fura-2 imaging in mouse pancreatic acinar cells. Cells were stimulated with 2pM cholecystokinin (CCK) in physiological salt solution (PSS) supplemented with 0.25mM EGTA following 10µM Carbachol (Cch) in PSS containing 2mM Calcium. Figure 1A visualizes intracellular calcium concentrations immediately after (time point 4:00 min) and 4 minutes after application of CCK (time point 8:00 min). Representative F340/F380 ratios of 65 cells in response to 2pM CCK and 10µM Cch can be observed in Figure 1B.

Challenge

As with all reporters, individual light dose per image needs to be kept at a minimum to reduce potential side effects caused by phototoxic by-products. Moreover, the studied networks of cells exhibit spontaneous Ca2+ fluctuations which can be – dependent on cell type and disease model – very rare so many separate fields-of-view need to be imaged to reach a reliable statistical conclusion.

When transgenic reporters can’t be used, the lab applies indicator dyes such as AM-esters of Fura2 and Fluo-variants. Here, a further problem can occur when too high dye concentrations need to be used to reach a detectable signal level. This surplus of dye alters the kinetics of the physiological response, rendering it a non-physiological situation. This problem can only be overcome by reducing the concentration of dye, which means a scientific camera is needed that is sensitive enough to pick up very low signals.

Their current imaging solutions are either state-of-the-art CCD sensors or EMCCDs. Both require a very long exposure time and provide images with a suboptimal signal-to-noise ratio. The small field of view of CCD-based sensors also requires many imaging sessions to acquire statistically significant data.

The Iris 15 camera has both a large chip and a small pixel size so we can optimize the numerical aperture of our lenses. The 4.25 µm pixel size on the Iris 15 allows us to use more of the 0.28NA at 4× than other cameras without zooming.

Solution

The Photometrics Prime 95B back-illuminated sCMOS is the perfect match for the imaging demands of the Freichel lab.

The increased sensitivity achieved by the 11 µm pixel and the peak-quantum efficiency of 95% at a wavelength range which matters mostly for their reporters dramatically reduces the exposure time for individual images. This carries two benefits. Firstly, kinetics can be determined more accurately as more data points can be acquired in the same amount of time. Secondly, less light needs to be used to detect their cells which allows the group to obtain more physiologically relevant data.

The field of view of the Prime 95B is >2.5x bigger than their previous camera solution, allowing the group to image more than twice the amount of data in a single acquisition, further improving their statistics and their time spent at the microscope.

The Prime 95B is also fully supported in Zeiss’ ZEN software which is convenient as the group is already familiar with the interface.

In brief, the Prime 95B enhances the Freichel lab’s scientific output and opens up new ways for them to study their samples of interest, bringing them closer to finding answers to their scientific questions.

Single Molecule ImagingCustomer Stories

Dr. Alexander Carr

Dr. Steven Lee, Principal Investigator

The Lee Lab, Department of Chemistry, University of Cambridge

Background

TheLeeLab, at the University of Cambridge, focuses its research on developing biophysical tools to answer fundamental biological questions, primarily using single-molecule fluorescence imaging techniques. Recently the group has established single-molecule spectroscopic imaging, facilitating local hydrophobicity mapping (1), as well as implemented cutting-edge point-spread function engineering for large-volume single-particle tracking in live T cells (2).

Fast single-particle tracking in multiple live T cells. Top Six Jurkat T cells were imaged in a single field-of-view at 100Hz. The cytoplasmic protein Zap70 was fluorescently labeled with Tetramethylrhodamine (TMR) using the HaloTag enzyme. Bottom Sequential images of a single Zap70 molecule diffusing within the cytoplasm of a Jurkat T cell (highlighted region from top).

Challenge

As with all single-molecule techniques, sensitivity is vital as the photon budget of individual fluorophores can be limited. For single-particle tracking applications, detecting more photons allows for longer trajectories to be recorded and more robust statistical analysis to be conducted.

Additionally, the motion of a fluorophore must be adequately sampled in order to accurately record its behaviour. For fast-moving targets, such as many cytoplasmic and nucleic proteins, short exposure times are required and thus the emission is spread over a greater number of frames.

In order to achieve live single-particle tracking of fast-moving targets, both high sensitivity and fast acquisition rates are vital. Although EMCCDs have previously been used to achieve both of these factors, high speed came at the cost of a much reduced field of view, which made data collection inefficient.

The Iris 15 camera has both a large chip and a small pixel size so we can optimize the numerical aperture of our lenses. The 4.25 µm pixel size on the Iris 15 allows us to use more of the 0.28NA at 4× than other cameras without zooming.

Solution

The high quantum efficiency and low noise of the Prime BSI sCMOS combines EMCCD-level sensitivity with a fast acquisition rate and much larger field of view. The Prime BSI speeds up data collection as multiple cells can be imaged within the same field of view at >100 fps with high contrast.

  1. Bongiovanni, M.N., J. Godet, M.H. Horrocks, L. Tosatto, A.R. Carr, D.C. Wirthensohn, R.T. Ranasinghe, J.-E. Lee, A. Ponjavic, J. V. Fritz, C.M. Dobson, D. Klenerman, and S.F. Lee. 2016. Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping. Nat. Commun. 7: 13544.
  2. Carr, A.R., A. Ponjavic, S. Basu, J. McColl, A.M. Santos, S. Davis, E.D. Laue, D. Klenerman, and S.F. Lee. 2017. Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function. Biophys. J. 112: 1444–1454.