Nüvü Camēras’ innovative EMCCDs overcome research barriers to produce high quality images for a variety of biomedical applications including fluorescence and bioluminescence imaging, spectroscopy, super-resolution, clinical applications and much more.
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Fluorescence and bioluminescence
Spinning Disk Confocal Microscopy
Spinning disk confocal microscopy is an imaging technique that preserves the optical sectioning property of traditional confocal microscopy while being adapted for much higher frame rates. Between the illumination source and the sample, these microscopes include a pair of spinning disks separated by a dichroic mirror. Each disk is fitted with thousands of pinholes arranged in spiral arrays, with microlenses fitted into the pinholes of the first disk. Consequently, the excitation light is focused by the microlenses of the first disk and travels across the dichroic and second disk to be focused onto the sample. Next, the emission light from the focal plane travels back through the second disk’s pinholes and is reflected by the dichroic towards a detector. This technique thus allows a progressive excitation of the sample in rapidly moving arcs for quick and efficient image formation.
Emission light inherently lost due to spinning disks
Excitation in spinning disk confocal microscopy is achieved by the use of transient pinhole illumination on each point of the observed sample. While this limits light exposure to minimize photobleaching and other undesirable photogenic side-effects, the spinning disks also block a portion of the excitation and emission light. Thus, the signal strength in spinning disk confocal microscopy is inherently limited. Consequently, without a sufficiently low noise floor, the SNR of the produced images may very well be limited by the choice of detector.
Nüvü Camēras’ low light imaging solution for spinning disk confocal microscopy
In the spinning disk confocal microscope demonstration below, certain notable features including structures that were previously undetectable as well as a much more defined cell outline can be observed in the image acquired using Nüvü Camēras technology as compared to another high-end EMCCD camera manufacturer. By minimizing all sources of noise in EMCCD cameras including readout noise, clock-induced charges (CIC) and dark current while suppressing the excess noise factor (ENF) and maximizing charge transfer efficiency (CTE), Nüvü Camēras provides the most sensitive low-light imaging solution. With less than 0.001 ē/pixel/frame of total background noise, Nüvü Camēras offers microscopists the lowest noise floor to maximize SNR in applications such as spinning disk confocal microscopy.
The images below demonstrate the advantages of Nüvü Camēras’ EMCCD cameras using Quorum Technologies’ WaveFX-X1 spinning disk confocal microscope located at McGill University’s CIAN facility. The first image was produced using a typical high-end EMCCD camera (512 x 512 detector) while the second image shows the same sample as acquired by Nüvü Camēras’ EM N2 512. The observed sample was illuminated using a 561 nm laser beam at 3.5% of the maximum intensity.
Using a typical commercially available EMCCD camera, the detector temperature was set to -65ºC (the minimum for this camera model) with an EM gain of 1200, the maximum allowed gain. As a result, the image is mainly contaminated a much higher level of clock-induced charges (CIC) and pixels are strongly affected by the excess noise factor (ENF). The consequence of such an elevated noise floor is the deterioration of the image’s overall SNR. As such, details in the image are difficult to distinguish and the cell outline is hardly identifiable, especially in the lower portion of the image.
Using Nüvü Camēras’ Photon Counting mode, the effect of the excess noise factor (ENF) is completely eliminated. In addition, the EM N2′s colder temperature (-85°C) also reduces the dark current tenfold in comparison with a detector at -65°C. Finally, the CCCP controller contributes by significantly attenuating clock-induced charges (CIC) to produce the high quality image as seen here. These advantages, combined with a maximum EM gain of 5000, allow for a much greater SNR and thus, more distinguishable features including structures that were undetectable in the above image as well as a much clearer cell outline.
(Images courtesy of Quorum Technologies Inc.)
Bioluminescent reagents are often used in biological assays to assess and quantify the presence of a particular molecule of interest. A prime example is the detection of ATP in the context of biocompatibility and cytotoxicity tests. In such assays, a common pair of reagents used is the combination of luciferin and luciferase that produce a light-emitting product in the presence of ATP. If the ATP concentrations in play are inferior to those of the reagent pair, the signal produced via bioluminescence is linearly dependant on ATP quantity. As such, calibration curves can be generated in order to precisely quantify the amount of ATP in biological samples.
Traditionally limited to the picomole scale
Typical detection methods limit quantification of ATP quantities to the picomole scale. However, bioluminescence detection with Nüvü Camēras technology can push detection limits all the way down to a few femtomoles. These innovative EMCCD cameras thus provide researchers with unprecedented sensitivity and precision in such biological detection assays.
Photon counting to reach the femtomole scale
Presented below are three images of the same microplate containing solutions of varying ATP concentration using Nüvü Camēras’ EM N2 1024. The quantities of ATP in each well vary from 5 to 156 femtomoles and are studied in triplicates. A constant amount of luciferin-luciferase solution in each well is used such that ATP is always the limiting reagent.
The first image is acquired in conventional mode over a 30-second exposure time and demonstrates what can be expected from a top-of-the-line CCD camera or even a high-quality sCMOS. As expected, the extreme low light generated as a result of the minuscule amounts of ATP is difficult to detect without electron multiplication.
In the second image, electron multiplication was used to suppress the readout noise inherent to the conventional mode of operation. The image results from the summation of 5 frames, each acquired over a 1-second exposure time. With a lower noise floor, all six ATP concentrations were detectable with SNR values varying from 1.9 to 14.1 (or equivalently, 2.8 dB to 11.5 dB).
The third image reveals the true potential of efficient photon counting imaging in the harshest of lighting conditions. Switching the camera acquisition to photon counting mode, the excess noise factor (ENF) was completely suppressed, thus allowing a strong improvement in image quality. Still combining 5 frames acquired over an exposure time of 1 second, the SNR then ranged from 6.6 to 51.3 (equivalent to a range of 8.2 dB to 17.1 dB). Using this acquisition method, the detection sensitivity more than tripled through the use of Nüvü Camēras’ photon counting mode. As a result, minute quantities as low as 5 femtomoles of ATP were detectable with a strong contrast with the image background in comparison to traditional detection methods using standard CCD or sCMOS technology.
(Images courtesy of the Université de Sherbrooke Hospital Centre)
FRET and BRET Imaging
Förster resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are techniques often used to study intermolecular reaction kinetics in biology and chemistry, yet certain drawbacks similar to those of single-molecule imaging exist.
Photobleaching and low signal intensity
Photobleaching (specifically in the case of FRET) as well as low intensity of observed specimens are the main obstacles of proper data acquisition for these imaging methods.
High acquisition speeds and superior SNR
Fortunately, both problems may be countered with the use of Nüvü Camēras’ EMCCD devices with the advantages of shorter exposure times, higher frame rates and higher SNR. Furthermore, the system’s high sensitivity may also allow the user to decrease the laser intensity in the case of FRET to minimize photobleaching effects.
It should be noted that certain FRET and BRET trials are undergone in multiwell plates with as many as 48, 96, 384 or even more individual wells. With so many simultaneous reactions in different wells, the use of Nüvü Camēras equipment can prove to be very advantageous for several aspects of the data acquisition. On one hand, certain wells may exhibit such high light intensity relative to neighbouring wells that they saturate the EMCCD detector. On the other hand, other wells may contain molecules that interact so weakly that the emitted signal is hardly distinguishable from the noise. By replacing the system’s camera with one of Nüvü Camēras’ own, superior acquisition speeds as well as higher SNR efficiently solve both issues simultaneously. Moreover, the system’s unrivalled charge transfer efficiency will further contribute to the virtually noiseless resulting images.
Small Animal Imaging
Small animals such as mice, rats and rabbits have been used throughout the past years to observe biological events in a non-invasive manner using light-emitting biomarkers via fluorescence or bioluminescence.
Harsh extinction of light
However, although the use of light-emitting molecules for diagnostic and research purposes is highly non-invasive, a major obstacle in these studies is poor light intensity. Unfortunately, a significant portion of the emitted light of interest is lost between the bioluminescent light source and the detector by absorption as well as scattering within the organism’s tissues, resulting in an extremely weak signal perceived by the camera.
Explore new detection limits
Nüvü Camēras’ research devices are ideal for such types of biomedical research thanks to unmatched sensitivity in ultra low light conditions, providing maximum EM gain and the highest SNR among all EMCCD cameras on the market. Furthermore, Nüvü Camēras’ EMCCD cameras not only allow the detection of substantially more elusive photons, but can reach higher detection speeds via greater frame rates on top of the superior reliability of the generated images.
Novel studies conducted by Dr. Frédéric Lesage’s research group from the Montreal Heart Institute in collaboration with the Institute of Biomedical Engineering at École Polytechnique de Montréal efficiently demonstrates the potential of Nüvü Camēras EMCCD detectors in fluorescence imaging. In the 2012 edition of Photonics North, the group presented an abstract, entitled “Ultrasound guided fluorescence tomography” in which a “hybrid-model imaging system combining fluorescence and ultrasound (US) was investigated towards improvement of fluorescence reconstruction”.
Presented below are the latest developments of the fluorescence imaging portion of Dr. Lesage’s hybrid imaging system.
Fluorophore CCy5.5 was injected intravenously to a concentration of 0.1 mg/mL. The colored zone is the scanned area obtained by superposing fluorescence images to controls.
Results from the new hybrid FMT-MRI model were published by Dr. Lesage research group in the May 2014 issue of Biomedical Optics Express.
Raman spectroscopy studies the vibrational, optical, and electric properties of materials in a nondestructive manner.
Inherently weak Raman signals
Raman spectroscopy imaging is typically a very long process because of the extremely weak signals produced by the Raman scattering effect.
Inherently sensitive detection
With Nüvü Camēras’ unrivalled sensitivity, such processes can be accelerated up to 10× without using methods such as CARS (Coherent Anti-Stokes Raman Spectroscopy) or SERS (Surface-Enhanced Raman Spectroscopy), since weaker signals can be detected to obtain a full spectrum. The spectral or spatial scanning will benefit from great savings in acquisition time.
Another field of interest in biomedical sciences is the imaging of single molecules within cells that have been tagged with fluorescent proteins such as GFP. While bioluminescent molecules emit their own light, fluorescent markers need to be stimulated with a more energetic light source to emit photons.
Limited by marker photobleaching
As such, fluorescence-based imaging is limited by the photobleaching of the markers used, which in turn depends on the intensity of the stimulating laser as well as the exposure time.
Sensitivity adapted for in-vivo imaging
To counter this issue, Nüvü Camēras’ higher SNR may allow researchers specializing in single-molecule imaging to reduce the intensity of their light sources, decrease acquisition periods, increase frame rate and temporal resolution, all while providing superior image quality simply by replacing the camera component of their imaging systems.
The 3-B technique
The innovative 3-B Technique takes advantage of a particular characteristic of in vivo fluorescence: the fact that after some time fluorophores start blinking at irregular intervals, independently from one another. This phenomenon can be exploited using time series recordings on which a ’Bayesian analysis of blinking and bleaching’ (3-B) is performed. This novel technique allows resolution beyond the diffraction limit which is very promising for in vivo cellular studies.
High frame rates to harness blinking and bleaching of the fluorophores
Transient intracellular events need to be studied in low light imaging conditions and they need high frame rates because of their rapidly changing patterns. When using the 3B technique the factors strongly affecting the quality of the analysis are the number of frames, the level of out-of-focus background and the degree of overlap between fluorophores. The exposure time needs to be very short to keep the fluorophores fairly well separated in each frame however, higher frame rates increase the level of clock-induced charges (CIC). If satisfying levels of total background noise and overlapping can be achieved, the SNR becomes very important for better localization accuracy.
Better SNR with patented EMCCD noise reduction
Thanks to effective photon counting capabilities, EMCCD technology is able to drastically reduce noise levels that prevented super-resolution in the past. The EM gain greatly accentuates incoming photon flux while background noise is minimum. Thanks to its patented controller, Nüvü Caméras reach a level of CIC (dominant noise in low light flux conditions) that is kept under 0.001 e-/pixel/frame while frame rates can reach 20MHz. Dr Yuri Zakharov points out in his paper that new ultra low noise EMCCD cameras certainly hold the key to better performance levels of this already ground breaking technique.
Direct links to the poster and abstract presented by Dr. Zakharov at the Novel Techniques in Microscopy topical meeting of the 2013 Optics in the Life Sciences, Optical Society of America Optics and Photonics Congress held from April 14 to 18, 2013:
Ultra low noise EMCCD camera considered to test a novel superresolution microscopy technique – Abstract
Ultra low noise EMCCD camera considered to test a novel superresolution microscopy technique – Poster
Also check out the referenced article in Dr Zakharov’s abstract, published in Nature Methods on December 2011 by Dr Susan Cox and team: Bayesian localization microscopy reveals nanoscale podosome dynamics, and their following recent publication ImageJ plug-in for Bayesian analysis of blinking and bleaching.
In nuclear medicine, the gamma and x-ray light that emanates from radioactive tracers is much too energetic to be detected with conventional camera technology. As such, specialized gamma cameras fitted with scintillation crystals are used in order to translate gamma and x-ray light into photons from the visible spectrum that can adequately be detected using conventional silicon-based detectors.
Poor resolution in gamma ray detection
Gamma cameras clinically used currently have a poor resolution when it comes to detection of gamma ray photons, typically limited to the millimetre scale. However, the development of detectors with an intrinsic resolution of around 100 µm can be very promising for the advancement of nuclear medicine, as such a technological improvement would yield superior tomographic resolution in addition to more compact device designs.
Increasing accuracy with EMCCD technology
High spatial resolution γ-imaging can be achieved with scintillator readout by EMCCDs for the development of future pre-clinical SPECT (Single-Photon Emission Computed Tomography) imagers. Dr. Beekman and his group present a novel hybrid gamma imaging detector combining an EMCCD camera and silicon photomultipliers (SiPMs) as side detectors for the scintillation readout. They have previously shown that false-positive events due to EMCCD noise can be rejected by using the sum signal from the SiPMs. They now present enhancements made in SiPM signal processing in order to improve the SNR and the energy resolution of their system. They did not use Nüvü Camēras’ products for the developments presented below, but they specify that further improvements can be expected by combining their method with EMCCDs that achieve less background noise such as those manufactured by Nüvü Camēras. This is why ultra low-noise EMCCD cameras, integrated into such hybrid devices, may enhance future high-resolution SPECT scanner technology.
Also take a look at Nüvü Camēras’ referenced article in the publication:Characterization results of EMCCDs for extreme low light imaging
The improvement of fluorescence-guided surgery allows for better prognostics regarding surgery such as brain tumour resection. The complexity of such procedures resides in the complete, but yet selective removal of the tumour. Failure to target malignant cell pockets can promote a recurrence of the pathology and compromise the patient’s remission. The concept behind fluorescence-guided surgery relies on the endogenous protoporphyrin IX (PpIX) molecule, which can selectively accumulate in neoplastic tissues, making the boundaries of the tumour identifiable through optical contrast.
Biological tissue fluorescence limits emitted photon flux
PpIX in vivo imaging results in faint, low incoming signal. In order to extract results that can effectively guide surgeons in their procedures, long exposure times are required for enough photons to accumulate in the sensor wells. Longer integration time not only means higher levels of noise related to dark current, but also requires longer procedures, which, in a context of brain surgery, can be too expensive to take.
Shorter exposure time thanks to more sensitive EMCCD cameras
Nüvü Camera’s cutting-edge EMCCD controller effectively reduces dark current noise as well as clock-induced charges with its patented electronics. Besides, it supports EM gains up to 5000, rendering the read-out noise negligible. These factors allow for better SNR making accurate imaging possible in less time with low incoming flux.
Researchers working on the detection of in vivo PpIX computed performance indexes (measuring SNR over exposure time) for an EMN2 EMCCD camera and a competitive CCD camera (details available in the study below). They improved their detection rate by a three orders of magnitude when using an EMCCD to identify cancer cells and discriminate them from healthy tissues. Thus, exploitation of fluorescence-guided surgery has the potential to reduce time, fluctuations and costs for neurosurgical procedures. Moreover, it could eventually be transposed to other types of difficultly operable cancers like throat or prostate carcinomas.
Direct link to the poster presented by researcher Yoann Gosselin at the Biophysical Society’s 57th annual meeting held from february 2 to 6 2013 showing the results above-mentioned:
Multimodal imaging allows for parallel information acquisition in a single exposure with the use of different probe modalities. Becoming a standard in the pre-clinical industry, it operates by injecting molecular markers that can be detected via multiple techniques. For example, one can resort to slightly radioactive and fluorescent probes, which emissions are then detected by a camera or other devices.
Low levels of fluorescence due to limited concentration of the probe
Each of the probe’s modality has a different threshold interval that needs to be met so it is detectable while remaining safe. But different modalities may have overlapping sensitivity ranges; some may be harmful at high enough concentrations, which would be wishful to generate enough fluorescence, and others are simply hindering fluorescence for increasing concentrations. Therefore, the probe’s emission is very faint and subject to noise contamination.
Highest SNR and optimal frame rates for optical tomography
While using the same EMCCD sensors as all of its competitors, Nüvü Camēras offers “very attractive characteristics for ultra low light and ultra-sensitive optical biomedical imaging with the best SNR in low light applications” (Bérubé-Lauzière, 2013). In the came of multimodal imaging resorting to fluorescence, it effectively solves the issue of the diminished probe’s emission at low concentration. Furthermore, with pixel readout rates reaching up to 20 MHz, acquisition of the diffuse emission can be done in real-time the camera rotates around the subject, supporting a 360° acquisition live optical tomography setup.
A direct link to Dr. Bérubé-Lauzière’s article presenting results for in vitro and in vivocharacterization of zinc- and copper-trapped phtalocyanines detection limits using Nüvü Camēras is available below (conference #8574-22 presented at BiOS – Photonics West 2013 during Session 4 on Preclinical Imaging on February 2nd, 2013).
Multiple sensor optical systems for multimodal imaging is a very interesting avenue for OEM prospects.
Cerenkov luminescence imaging
When travelling faster than light in a given medium, high-energy particles emit what is known as Cerenkov radiation. Such radiation, the source behind the notorious nuclear reactor pools blue hue, reveals itself as a promising tool for biomedical imaging, whether for biomedical studies or cancer detection.
Injecting a radioactive dye in biomolecules or bodies will inevitably produce Cerenkov radiation. As the dye decays, it creates alpha (helium core) and beta (electron) radiation that will travel through the material, mostly composed of water, and generates optical and near infrared emission. This signal is however quite faint and calls for high sensitivity cameras to be detected.
Detecting every Cerenkov photons
Nüvü Camēras EMCCDs such as the HNü camera series are perfectly suited for Cerenkov luminescence imaging. With their high quantum efficiency, outstanding sensitivity over the visible and NIR spectra, and ultra low noise even at high EM gain, they can detect every Cerenkov photons, thus providing the precision required for this cutting edge imaging technique.
Higher SNR for Overcoming Research Frontiers
Cutting-edge techniques manipulating light sources to study living organisms are continually discovered in parallel with technological progress in optical and imaging devices. Pioneering the exploration of uncharted areas of biomedical research, Nüvü Camēras’ innovative cameras provide solutions to the most complex detection challenges in ultra low light imaging. Reaching far beyond the limits of present day technology, Nüvü Camēras’ technology enhances the analytical work of modern scientists, significantly increasing their outcomes with superior SNR data.
Nüvü Camēras’ EMCCD cameras may contribute to save lives and minimize hospitalization time. Indeed, the detection of individual photons emitted by weak light sources such as biomarkers, a field in which Nüvü Camēras technology shines, is among the avenue followed for medical diagnostics. Numerous researches are currently being conducted using these biomarkers for diagnostic purposes; they may help to identify and even aid in the treatment of various illnesses. For example, such specific and precise detection may allow the non-radioactive identification of specific types of cancer in their early stages of development, earlier than traditional detection methods would allow. Treatment time and complexity would therefore be greatly reduced, and hospitalization too.
Moreover, Nüvü Camēras’ superior SNR yields a unique advantage in other growing areas of biomedical research by enabling high-resolution optical detection with spinning disk confocal microscopy, super-resolution or in vivo animal imaging, to name only a few applications.
Photon Counting for Life Sciences
The ability to observe individual photons at high frame rates with an astoundingly high SNR can now be achieved with Nüvü Camēras’ EMCCD cameras. With background noise averaging below 0.001 ē/pixel/frame as well as an EM gain peaking at 5000, these EMCCD cameras can be used in several biomedical research fields to study phenomena that have never before been examined with such precision, sensitivity and reliability.
An EMCCD background noise results from a combination of dark current and clock-induced charges. It is also being influenced by readout noise, the excess noise factor and improper charge transfer. In spite of these various parameters that affect the total background noise, Nüvü Camēras’ state-of-the-art and patented technology elegantly overcomes these obstacles. It allows the amplification of the observed signal up to 5000 times via electron multiplication before subjecting it to conversion from charge to electric potential, while typical EMCCDs are limited to an EM gain of 1000. Combined with the multitude of other noise-countering methods made available with CCCP technology (CCD Controller for Counting Photons) explained in further detail in the EMCCD Tutorial, Nüvü Camēras provides today’s researchers with the highest SNR and lowest noise level for ultra low light imaging in the visible and near-infrared spectra. Additionally, this advanced technology sets these new standards using creative and innovative engineering to physically reduce the system’s noise; not a single computer-based filtering algorithm is employed.