Page Type: Customer Story

Multicolor Single Molecule FRETCustomer Stories

Professor Thorsten Hugel
PhD Candidate Leonie Vollmar

Institute of Physical Chemistry II, University of Freiburg, Germany

Background

The lab of Prof. Hugel is interested in obtaining real-time measurements of multi-protein complexes and their dynamic interaction. In living organisms, multi-protein complexes form intricate protein machines that regulate cellular processes. Of particular interest is the heat shock protein Hsp90 machinery, which consists of an Hsp90 dimer, co-chaperones, and client proteins. The complexes likely exist in multiple states that dynamically interchange and might be coordinated by utilizing the energy of ATP hydrolysis.

In the past, multicolor single-molecule FRET (Förster Resonance Energy Transfer) allowed Prof. Hugel and his team to study the Hsp90 machinery, determining the success of association and dissociation steps as well as large conformational changes of protein complexes simultaneously in real-time. Single-molecule FRET was also used to determine specific protein interfaces that interact as part of a complex. These studies impact the understanding of the Hsp90 machinery as well as general principles of multi-component protein systems, which is the basis for understanding cellular processes.

Challenge

Studying protein dynamics in real-time requires the ability to image on a wide range of timescales, meaning that any increase in imaging bandwidth would help to obtain a better understanding of protein interactions across all lengths of experiment.

Further challenges were outlined by Prof. Hugel: “For a proper dynamic analysis of several states of protein interaction many single-molecule FRET traces are needed, therefore an increased field of view (FOV) would help. As the analysis is carried out on a pixel-by-pixel basis a very high degree of stability, reliability and reproducibility are required, this can only be achieved by perfect noise characteristics of the imaging device”.

With a sensor diagonal of 18.6mm, the Prime 95B improved the FOV by a factor of >2.5x allowing for more data to be acquired at the same time

Solution

As Prof. Hugel states: “the Prime 95B seems to be a perfect answer to all questions asked, with a maximum speed of 82 fps it is significantly faster than standard EMCCD solutions, and without the need to crop the image.”

With a sensor diagonal of 18.6mm, the Prime 95B improved the FOV by a factor of >2.5x allowing for more data to be acquired at the same time. Prof Hugel mentioned that “the 11 x 11 μm pixel size enhances the resolution of the images compared to the previous EMCCD solution… this will accelerate the science by significantly strengthening the statistical validity.”

Prof. Hugel and his team are further optimizing the Prime 95B in their experiments, and have achieved a landmark experiment: “to our knowledge, this is the first time that dynamic single-molecule FRET traces of more than 10 seconds length were recorded on an sCMOS camera… there is a lot of potential, as a significantly higher time resolution is possible.”

Figure 1: A) Acceptor fluorescence upon donor excitation (FRET) and B) Donor fluorescence from single Holliday junctions labeled with Atto550 and Atto647N taken with the Prime 95B (cropped FOV).
Figure 2: Exemplary dynamic single-molecule FRET traces from
Holliday junctions recorded on the Prime 95B. Donor (green) and acceptor (red) fluorescence upon donor excitation in arbitrary digital units (ADU), uncorrected FRET efficiency (E, gray), Viterbi path by SMACKS1 (black). Exposure time 100 ms.

Additional Information

https://www.singlemolecule.uni-freiburg.de/

  1. S. Schmid, M. Götz, T. Hugel (2016) Single-molecule Analysis beyond Dwell Times: Demonstration and Assessment in and out of Equilibrium, Biophysical Journal 111, 1375-84

Confocal STORMCustomer Stories

Dr. Uri Manor, Biophotonics Core Director

Salk Institute for Biological Studies, Biophotonics Core Facility

Background

The Salk Institute is home to a highly collaborative cadre of scientists who delve into a broad range of research areas, from aging, cancer and immunology to diabetes, brain science and plant biology. The group is supported by on-campus research centers and core facilities that are equipped with cutting-edge technology.

The Institute embodies Jonas Salk’s mission to dare to make dreams into reality by exploring the very foundations of life, seeking new realities in neuroscience, genetics, immunology and more. The team lives to discover, be it cancer or Alzheimer’s, aging or diabetes, they understand that every cure has a starting point. Salk is where cures begin.

Uri Manor, biophotonics core director works with the Salk researchers to provide collaborative support for a wide variety of research projects that require scientific imaging. Manor also works with the faculty steering committee to incorporate new and advanced imaging technologies into the repertoire of resources offered through the Biophotonics Core Facility.

25ms
Scientific CMOS
25ms, 400x EM gain
EMCCD
50ms
Scientific CMOS
50ms, 400x EM gain
EMCCD

Images depict the actin cytoskeleton as stained by AlexaFluor488-Phalloidin. EMCCD used was the Photometrics Evolve 512 and the Scientific CMOS was the Photometrics Prime 95B. Images were captured using Micro-Manager on a Zeiss Confocal microscope with a Yokogawa spinning disk scan head. 25ms and 50ms exposures were acquired with laser power set to the minimum of 5% with the EM Gain of the EMCCD set to 400.

Challenge

The Facility provides technical and logistical access to Salk faculty, enabling the integration of imaging tools into a variety of biological research programs. To maintain its ability to advance science, the Facility must maintain the latest, cutting-edge commercial imaging and data analysis technologies available. This is especially important given that most projects involve sophisticated and complex research techniques.

The primary instrumentation that core researchers must have access to include technologies that support confocal microscopy (both fixed and live cell), TIRF microscopy, two-photon microscopy, electron microscopy and super-resolution microscopy as well as in-vivo imaging modalities.

With a sensor diagonal of 18.6mm, the Prime 95B improved the FOV by a factor of >2.5x allowing for more data to be acquired at the same time

Solution

To continue supporting the diverse work of the Salk researchers, Manor must stay abreast of the advancements being made in scientific imaging. It is important that the team always have access to the newest and most advanced solutions.

Manor learned of the Prime 95B™ Scientific CMOS camera from Photometrics and was interested in learning more about the claim for 95 percent quantum efficiency. Of greater importance was how the camera was being touted as the most sensitive in the industry – sensitivity is always in high demand among Salk researchers.

After seeing the camera and testing it with his own samples, Manor realized the camera did stand up to the hype and would integrate well into the Facility’s imaging tools. The back illuminated technology and high QE make the camera exceptionally versatile. Manor shares, “The Prime 95B provides the speed, field of view and resolution of a CMOS camera, with the added sensitivity of an EMCCD camera for our more demanding experiments.”

The Prime 95B easily supports multiple scientific applications, this flexibility also makes it a very good investment for the Salk Institute.

Super-Resolution MicroscopyCustomer Stories

Prof. Ke Xu, Principal Investigator, Professor of Chemistry

University of California Berkeley, College of Chemistry

Background

The Ke Xu Group at the University of California Berkeley, College of Chemistry is an interdisciplinary lab that combines biophysics, physical chemistry and cell biology. Their goal is to understand how orders emerge in biological systems at the nano-meter scale from the interaction between biomolecules. They achieve this goal experimentally through the development and synergistic application of innovative quantitative methods such as super-resolution fluorescence microscopy.

Ke Xu, principal investigator and assistant professor of chemistry, successfully opened his lab in 2013 and today, leads a team that includes post-doctoral researchers and graduate students. The team recognizes and respects how living systems achieve versatile structural organizations at the nanoscale. Their dedication to gaining a greater understanding of this phenomenon has led to their ability to consistently achieve publication of their research findings.

Prime 95B Scientific CMOS Camera Test
β-tubulin-AF647 160 Hz 50k frames
Prime 95B Scientific CMOS vs EMCCD Camera Test
β-tubulin-AF647 110 Hz 50k frames

Challenge

Previously, EMCCD technology was the primary imaging solution in the Ke Xu Group. However, STORM experiments presented increasing demands on the existing imaging setup. The team decided to look at other technologies, specifically sCMOS solutions due to new advancements in CMOS sensors and more advanced capabilities becoming available.

Having reviewed available products, the team found the Prime 95B Scientific CMOS camera from Photometrics. The company was touting the first and only 95 percent quantum efficiency camera, which piqued their interest. Having the opportunity to fully test the camera, they discovered it offered comparable, if not better, results when compared to their existing EMCCD.

With a sensor diagonal of 18.6mm, the Prime 95B improved the FOV by a factor of >2.5x allowing for more data to be acquired at the same time

Solution

When comparing to the EMCCD camera that was used previously, the Prime 95B Scientific CMOS (sCMOS) camera provides many more benefits; faster imaging, comparable spatial resolution and a larger field of view. “The Prime 95B Scientific CMOS camera allows us to conduct our STORM experiments with higher frame rates and a larger field of view than with EMCCD technology,” Xu shares. “Plus, the 95 percent quantum efficiency allows for super-resolution imaging that’s not achievable with conventional sCMOS cameras,” he adds.

Live Cell Spinning Disk ConfocalCustomer Stories

Dr. Jan Felix Evers

Centre for Organismal Studies at University of Heidelberg, Germany

Background

The Center for Organismal Studies (COS) Heidelberg has set the goal of researching organismal biology beyond the boundaries of the biological organizational stages. Research and teaching at the COS are devoted to the biology of organisms from the molecular basis to cell biology, developmental biology and physiology to evolution and biodiversity as well as system biology and biotechnology in plant and animal systems. Dr. Jan Felix Evers and his team’s primary interest is in how neuronal circuits form in the central nervous system, both on the cellular and molecular level. Their focus lies on investigating these issues with spinning disk live cell imaging, which requires very high sensitivity and resolution.

Image captured with the Prime 95B Scientific CMOS camera from Photometrics.

Challenge

Synaptogenesis is very sensitive to overexpression artifacts. To study synaptogenesis with minimal interference, the research team devised a system to visualize endogenous gene expression in single neurons. Their challenge lies in working with a low copy number of fluorophores down to the single molecule level and therefore low fluorescent yield.

With a sensor diagonal of 18.6mm, the Prime 95B improved the FOV by a factor of >2.5x allowing for more data to be acquired at the same time

Solution

The team wanted the best solution to cover all of their applications. The Prime 95B Scientific CMOS offers the largest usable field of view with the highest sensitivity of all available Scientific CMOS cameras, and an optimal sampling density with a 60x magnification. In addition, the Evolve 512 EMCCD provides the best sensitivity and signal-to-noise for detecting low endogenous gene expression at the single molecule level. “After carefully testing a larger range of cameras, we selected equipment from Photometrics. The Evolve provides the lowest noise floor and highest sensitivity for our most demanding samples while the Prime 95B provides a higher sampling density with signal-to-noise that is almost as good as an EMCCD” shares Dr. Evers. “Photometrics cameras are reliable with great performance,” Dr. Evers concludes. “We can now visualize things that we could not see before.”

Single Molecule ImagingCustomer Stories

Dr. Anders Kyrsting

The Linke Group, University of Lund, Sweden

Background

The Linke Group at the University of Lund, Sweden, creates artificial molecular motors and nanowires to better understand the role of biological motors in cellular processes such as cargo transportation, muscle contraction and cell division. The group tags the motors with quantum dots and images them using single molecule TIRF. They have further plans to expand their investigation with optical trapping and STORM super-resolution microscopy.

Alumina (Al2O3) coated gallium phosphide (GaP) nanowires functionalized with biotinylated BSA (bovine serum albumin). Streptavidin labelled with three fluorescent dyes (FITC, TRITC and Cy5), imaged under 100x magnification.

Challenge

Anders Kyrsting, post-doc with the Linke Group, explained, “Molecular motors move very fast so to track them, we need a camera with a very high frame rate.” The group was previously using an EMCCD camera but the slower frame rate of its architecture meant that they had very limited temporal resolution.

Kyrsting continued, “Investigating single molecules means working with very low fluorescence signal. So, a camera with high sensitivity is equally as important as our need for a fast camera.” For this reason, the group couldn’t afford to sacrifice sensitivity for speed.

With a sensor diagonal of 18.6mm, the Prime 95B improved the FOV by a factor of >2.5x allowing for more data to be acquired at the same time

Solution

The near-perfect 95% quantum efficiency and high speed of the Prime 95B Scientific CMOS camera made it a clear fit for their work and the group was excited to implement the camera into their system.

Kyrsting told Photometrics, “The Prime 95B enabled us to reach the 200 fps speed that we needed with a far larger field of view than would be possible on any EMCCD camera.”

Additionally, with the large 11 µm pixel of the Prime 95B, the group could achieve perfect diffraction limited resolution with a 100x objective, without using any additional optics.

Kyrsting continued, “The speed and sensitivity were exactly what we were looking for and the bonus of having such a large field of view has really helped our data throughput.” In conclusion, Kyrsting added, “I don’t think I’d use an EMCCD again, I don’t know why I’d use it with the performance we get out of the Prime 95B.”

STORM & PhotonicsCustomer Stories

Prof. Paul French

Photonics Group, Physics Department, Imperial College London

Background

The Photonics Group in the Physics Department at Imperial College London develops instrumentation for multidimensional fluorescence imaging – spanning a wide range of applications, from super-resolved microscopy through automated fluorescence lifetime imaging for high content assays to endoscopy and optical tomography.

The availability of Scientific CMOS cameras has been transformative for their research because the technology provides unprecedented imaging performance with high resolution and high frame rates. The team particularly uses Scientific CMOS cameras for localization and light sheet microscopy.

NIH3T3 mouse embryonic fibroblast, starved overnight and treated with 1μm Trichostatin A for 4h prior to fixation. Cell is stained by anti-acetylated tubulin with an Alexa Fluor 647 secondary antibody. Using a Cairn OptoTIRF system, 5000 image frames were taken with a 30ms exposure time which composed 2768763 individual localisations. Mean uncertainty is 11.54 nm. (Scale bars are all 10μm)

Challenge

STORM [i, ii] for super-resolved microscopy is a particular interest and the team recently published an approach using low-cost diode lasers [iii]. “Our goal is to develop instruments that provide state-of-the-art performance while reducing the cost where possible so that more users are able to access such advanced imaging capabilities,” shared Professor Paul French.

With a sensor diagonal of 18.6mm, the Prime 95B improved the FOV by a factor of >2.5x allowing for more data to be acquired at the same time

Solution

Scientific CMOS cameras are relatively cost effective compared to EMCCD cameras and they had already been incorporated into the team’s STORM system. The group has since started using the Prime 95B.

French explained, “Since the achievable resolution is a function of the number of photons detected, we were excited to learn about the back-illuminated Scientific CMOS camera from Photometrics. The Prime 95B camera is specified to provide >95% quantum efficiency, giving us the advantages of Scientific CMOS with fantastic sensitivity.”

When the group made a comparison of images taken with the Prime 95B and a standard Scientific CMOS camera, they were immediately impressed with the increase in signal to noise.

French concluded, “We look forward to implementing the Prime 95B camera in the new STORM microscopy platform that we are currently developing.”

References

  • i M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793–796 (2006).
  • ii M. Heilemann, S. van de Linde, M. Schuttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, Angew. Chem. Int. Ed. 47, 6172–6176 (2008).
  • iii K. Kwakwa, A. Savell, T. Davies, I. Munro1 S. Parrinello, M.A. Purbhoo, C. Dunsby, M.A.A. Neil and P.M.W. French,J. Biophotonics 9 (2016) 948–95, DOI 10.1002/jbio.201500324

Microfluidics and Live Cell ImagingCustomer Stories

Simon Berger, Doctoral Student
Andrew deMello, Principal Investigator and Professor for Biochemical Engineering

deMello Group, ETH Zürich (Switzerland)
Department of Chemistry and Applied Biosciences

Background

The deMello Group at ETH Zürich is engaged in a broad range of activities in the general area of microfluidics and nanoscale science. Primary specializations include the development of microfluidic devices for high-throughput biological and chemical analysis, ultra-sensitive optical detection techniques, nanofluidic reaction systems for chemical synthesis, novel methods for nanoparticle synthesis, the exploitation of semiconducting materials in diagnostic applications, the development of intelligent microfluidics and the processing of living organisms.

In recent years the deMello group has developed a range of microfluidic tools for the long-term imaging of living organisms, specifically the nematode Caenorhabditis elegans. Currently, work is focused on the creation of novel microfluidic devices for worm manipulation and the study of a wide range of developmental processes, previously inaccessible.

Figure 1: Expression of DLG-1:eGFP, apical junctional protein in the seam cell epithelium. Each image represents a 10 ms exposure taken every 20 minutes for a full sequence time of 4.5 hours.
C. elegans strain courtesy of Dr. H. Ribeiro Pires and Dr. M. Boxem, Utrecht University.

Challenge

Live fluorescence imaging has seen a tremendous change over recent years. The development of sCMOS cameras has transformed image acquisition rates, fields of view and noise suppression, while also lowering unit costs. However, until recently the EMCCD has been the gold-standard for high sensitivity applications, significantly outperforming sCMOS devices with quantum efficiencies in excess of 95%, but lacking considerably with respect to sensor size and acquisition speed.

Simon Berger, doctoral student with the DeMello group, explains, “The primary challenge in live cell/organism imaging is the extraction of high quality images, both bright and with high contrast, while ensuring that phototoxicity and photobleaching are kept to a minimum. In this way, imaging does not affect sample viability and the biological processes under investigation.”

The need to ensure low photobleaching/phototoxicity often limits attainable image quality, as well as the frequency and detail with which images can be acquired. While the higher sensitivity associated with EMCCDs can remedy the effects of photobleaching/phototoxicity, the acquisition rates, the small fields of view and limited dynamic range, severely limit their usefulness for in vivo imaging.

With a sensor diagonal of 18.6mm, the Prime 95B improved the FOV by a factor of >2.5x allowing for more data to be acquired at the same time

Solution

Compared to its peers, the Prime 95B Scientific CMOS combines the features of sCMOS cameras (high acquisition rates, large sensor size, low noise and high dynamic range) with the exceptional sensitivity previously only available through EMCCDs.

Simon explains, “The Prime 95B allowed us to acquire high contrast fluorescence images using low excitation intensities, and subsequently allowed us to image over longer periods of time and at higher frequencies than previously possible. This allowed for intrusion-free study of many sensitive developmental processes.”

TIRF MicroscopyCustomer Stories

Gerry Hammond, Ph.D., Assistant Professor
Simon Watkins, PhD., Founder and Director of the Center for Biologic Imaging

University of Pittsburgh

Background

Gerry Hammond studies membrane trafficking and intracellular signaling and regulation in the Center for Biologic Imaging at University of Pittsburgh. Working with Simon Watkins, Director of the Center, they use gene editing technology to fuse GFP to endogenous alleles. They then use a combination of Total Internal Reflection Fluorescence Microscopy (TIRF) and other techniques to study the mechanisms of how inositol lipids effect membrane function and transport of nutrients in disease. Dr. Hammond shared, “We use TIRF because we are interested in what’s happening at the interface between the plasma membrane and the endoplasmic reticulum which sits very close to it.”

HEK 293 cells with GFP fused to Sec 61 at its endogenous allele.
Camera: Prime 95B
Magnification: 100x TIRF
Binning: 2×2

Challenge

When tagging genes at their endogenous loci, low expression levels are often a problem. This results in low levels of fluorescence and produces images with very low signal to noise. “The amount of fluorophore that you’ve got [in the sample] is much less than you’d normally have, but of course that’s the advantage because we get to see how the endogenous protein is behaving in a living cell,” Dr. Hammond explains.

With a sensor diagonal of 18.6mm, the Prime 95B improved the FOV by a factor of >2.5x allowing for more data to be acquired at the same time

Solution

Dr. Hammond told us, “The advantage of the Prime 95B [Scientific CMOS] for me was the improved sensitivity over other CMOS cameras but with all the advantages that come with CMOS technology like acquisition speed and a larger chip size.”

Dr. Watkins added, “The 11 micron pixel size with the 100x objective that Gerry and I are using gets us much closer to Nyquist sampling in XY… If you combine the back-thinning and the more appropriate pixel size you have much better resolution at higher sensitivity.”

Super-Resolution MicroscopyCustomer Stories

Dr. Sang-Hee Shim, Principal Investigator and Assistant Professor of Chemistry

Shim Group, Center for Molecular Spectroscopy and Dynamics, Institute for Basic Science, Korea University

Background

The Shim Group at Korea University is an interdisciplinary lab covering physical chemistry, biophysics and cell biology. Sang-Hee Shim, principal investigator and assistant professor of chemistry, leads a team composed of postdoctoral researchers and graduate students to develop new microscopic methods and apply them to answer complex biophysical questions.

Their core focus is to better understand life at the molecular scale by visualizing cell dynamics and the interactions of intracellular molecules. To do so, they explore the frontiers of optical microscopy with super-resolution fluorescence imaging.

Surface-immobilized DNA origami with 3 docking DNA strands with 80 nm gaps, imager DNA oligo transiently binding to the docking DNA strand, labelled with Atto 655
A. 1024×1024 EMCCD camera with a 130 nm effective pixel size 32×32 field. Acquired at 47 fps with 20 ms exposure, 30x EM gain. B. Prime 95B Scientific CMOS (sCMOS) camera with a 110 nm effective pixel size. Acquired at 50 fps with 20 ms exposure time. C. Prime 95B plus PrimeEnhance with a 110 nm effective pixel size. Acquired at 28 fps with 20 ms exposure time.
Localization accuracy analysis of the DNA origami
A. The 1024×1024 EMCCD camera shows a mean localization accuracy of 13.29 nm. B. The Prime 95B shows a mean localization accuracy of 9.25 nm. C. The Prime 95B plus PrimeEnhance shows a mean localization accuracy of 7.47 nm.

Challenge

The Shim Group previously used EMCCD technology for localization-based super-resolution fluorescence microscopy. However, although EMCCD offers better sensitivity than sCMOS technology, it suffers from excess noise generated by the process of electron multiplication. The precision and resolution of their experiments are highly dependent on the sensitivity and noise level of the camera so this presents an issue.

The group investigated potential solutions and found the Prime 95B Scientific CMOS (sCMOS) camera from Photometrics – the first and only 95 percent quantum efficient CMOS device. The camera affords comparable sensitivity to EMCCD, yet offers far higher imaging speed and a larger field of view. After testing the camera, the group also found some cases in which it produced even better spatial resolution when compared to their existing EMCCD.

With a sensor diagonal of 18.6mm, the Prime 95B improved the FOV by a factor of >2.5x allowing for more data to be acquired at the same time

Solution

The team compared the Prime 95B to their EMCCD camera using single-molecule DNA-PAINT imaging and found that the camera gave improved localization precision. This is suggested to be because the Prime 95B does not rely on electron multiplication to increase sensitivity. By removing the excess noise factor generated by the electron multiplication process, the Prime 95B Scientific CMOS can achieve a higher signal to noise ratio than an EMCCD.

The team also investigated PrimeEnhance™, the active denoising algorithm that accompanies the Prime 95B camera. They found that PrimeEnhance can amplify some noise and produced false localizations. When the localization software was optimized for PrimeEnhance, the localization precision was further improved and gave the best results among all tested conditions.

Shim explains, “For single-molecule images like in DNA-PAINT, the Prime 95B combined with PrimeEnhance allows us to conduct super-resolution imaging with higher spatial resolution than that of EMCCD technology.” Shim adds, “Plus, the Prime 95B offers the additional benefits of higher frame rate and a larger field of view.”

STORM Super-ResolutionCustomer Stories

Dr. Yandong Yin, Postdoctoral Fellow
Prof. Eli Rothenberg, PhD, Associate Professor

New York University, School of Medicine

Background

The laboratory of Dr. Rothenberg at the New York University School of Medicine focuses on new optical methods to study biological molecules and processes at real time and nanometer scale. The Rothenberg research team studies the mechanisms of enzymes and proteins that participate in repair of DNA damage leading to cancer, and develops new imaging methods that will enable them to visualize the behavior of individual biological molecules. STORM Microscopy is used to localize and track DNA as it replicates in the cell. “We try to look at the nucleus of cancer cells as they are replicating the DNA. The DNA and proteins involved in DNA replication are labelled so we can understand what is going on when replication happens,” Yandong Yin, PhD. Postdoctoral fellow states.

Cell Type: U2OS cells
Exposure time: 30 ms
Magnification: 150 times (the configured pixel size is ~ 73 nm)
Reconstruction Algorithm Used: Maximum Likelihood Estimation (MLE) method for single PSF fitting

Challenge

One of the challenges of imaging replicating DNA is that inside the nucleus of the cell there are many labeled components crowded together, as well as very small components that need to be clearly resolved. To determine how each component is organized spatially, the lab often performs STORM imaging using three or four colors sequentially, which makes resolution, sensitivity, and localization accuracy a great concern. “The DNA replication fork is very small. We can’t image it without super resolution,” says Yin. The laboratory calibrates their STORM post-processing conditions based on the variances of each pixel in the chip of the camera, correcting for any major variations, in order to better fit the point spread function of each fluorophore. Because of this, pixel to pixel variations, like those seen in patterned noise on CMOS cameras becomes a major problem.

With a sensor diagonal of 18.6mm, the Prime 95B improved the FOV by a factor of >2.5x allowing for more data to be acquired at the same time

Solution

The move away from EMCCD technology to the Prime 95B back-thinned CMOS was made easier because of the improved sensitivity in the detection of low-emission fluorophores, and the reduction in pattern noise when compared to other CMOS cameras. “For single-molecule localization imaging, the most important thing is the reconstruction process in how we fit each single-molecule point spread function into its centroid coordinate,” Yin noted on his use of the Prime 95B. The post-processing of images collected with the Prime 95B are made significantly easier because of the reduction in pixel to pixel pattern noise, which makes the localization and reconstruction of fluorophores of multiple colors easier to do. Yin continued, “The variance for each pixel is much smaller than what is reported on other sCMOS cameras. We have found that more than 90% of the pixels fall within a very tight noise distribution.”

Additionally, the large field of view coupled with the improved sensitivity allows the team to get an image that contains useful information more often. Since they often work with samples that are dark at the start of acquisition, it was an issue that they couldn’t guarantee seeing something with the smaller field of view of an EMCCD. Yin says “Previously we used an EMCCD, but the EMCCD has a smaller chip. With a bigger chip we can see multiple cells simultaneously.”