Biomedical

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 microscopy, 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. The emission light from the focal plane then travels back through the second disk’s pinholes to be reflected by the dichroic towards a detector. This technique allows a progressive excitation of the sample in rapidly moving arcs for quick and efficient imaging.

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. It limits light exposure to minimize photobleaching and other undesirable photogenic side-effects, but the spinning disks block part of the excitation and emission light, thus decreasing signal strength. 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

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 Nüvü Camēras’ EMCCD cameras performances when coupled to Quorum Technologies’ WaveFX-X1 spinning disk confocal microscope at McGill University’s CIAN facility. The first image was produced using a typical high-end EMCCD camera (512 × 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. Certain notable features, including structures that were previously undetectable as well as a much more defined cell outline, are seen in the image acquired using Nüvü Camēras technology.

Using a typical commercially available EMCCD camera, the detector temperature was set to – 65ºC (the advertised minimum for this camera model) with an EM gain of 1200, the maximum allowed gain. As a result, the image is mainly contaminated by a much higher level of clock-induced charges (CIC). Pixels are also strongly affected by the excess noise factor (ENF). The image’s overall SNR is degraded due to such such elevated noise floor. As such, image details are difficult to distinguish and the cell outline is hardly identifiable, especially in the lower portion of the image.

With Nüvü Camēras’ Photon Counting mode, excess noise factor (ENF) is completely eliminated. In addition, the EM N2′s colder operating temperature (- 85°C) also reduces the dark current tenfold in comparison with a detector operating at – 65°C. Finally, the CCCP controller significantly attenuates clock-induced charges (CIC) to produce the high quality figure above. These advantages, combined with a maximum EM gain of 5000, allow for a much greater SNR, hence 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.)

Bioluminescence Analysis

Bioluminescent reagents are often used in biological assays to assess and quantify the presence of a particular molecule of interest. A prime example is adenosine triphosphate (ATP) detection in biocompatibility and cytotoxicity tests. In such assays, a combination of luciferin and luciferase produce a light-emitting product in the presence of ATP. If ATP concentrations are inferior to those of the reagent pair, the signal produced via bioluminescence varies linearly with ATP quantity. As such, calibration curves can be generated to precisely quantify ATP amounts 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 3 images of the same microplate containing solutions various ATP concentrations generated by Nüvü Camēras’ EM N2 1024. ATP amounts in each well vary from 5 to 156 femtomoles and are studied in triplicates. A constant amount of luciferin-luciferase solution in each well makes the ATP the limiting reagent.


The first image was acquired in conventional mode over a 30-second exposure time. It displays the expected from a top-of-the-line CCD camera or even a high-quality sCMOS performance for bioluminescence analysis. 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 is made 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 photon-counting imaging true potential in the harshest 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. Combining 5 1-second frames, the SNR now ranges from 6.6 to 51.3 (or, equivalently, 8.2 dB to 17.1 dB). Sensitivity has more than tripled through with Nüvü Camēras’ photon counting mode. As a result, minute ATP quantities as low as 5 femtomoles were detectable with a strong contrast compared 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 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) and the specimens low intensity are the main obstacles for proper data acquisition.

High acquisition speeds and superior SNR

Fortunately, both problems may be countered with Nüvü Camēras’ EMCCD devices, which provide shorter exposure times, higher frame rates and greater SNRs. Furthermore, for FRET imaging, a user may decrease the laser intensity due to the camera superior sensitivity , hence 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, Nüvü’s cameras can provide better data. 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 incoming signal is hardly distinguishable from the noise. With Nüvü Camēras’ EMMCD, 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 virtually noiseless images.

Small Animal Imaging

Small animals such as mice, rats, and rabbits have been used throughout the past years to study biological events in a non-invasive manner via fluorescing or luminescing biomarkers.

Harsh extinction of light

However, although using 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 their emission is lost between the bioluminescent light source and the detector through absorption and scattering within the organism’s tissues, resulting in an extremely weak incoming signal.

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, thus 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 to create superior 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. The group presented Ultrasound guided fluorescence tomography in which a “hybrid-model imaging system combining fluorescence and ultrasound (US) was investigated towards improvement of fluorescence reconstruction” at the 2012 Photonics North meeting.

Ultrasound Guided Fluorescence Tomography

Presented below are the latest developments of the fluorescence imaging portion of Dr. Lesage’s hybrid imaging system.

Mouse tests:

The CCy5.5 fluorophore was injected intravenously to a concentration of 0.1 mg/mL. The colored zone shows 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.

Hybrid FMT-MRI applied to in vivo atherosclerosis imaging

Spectroscopy

Raman Imaging

Raman spectroscopy studies vibrational, optical, and electronic material properties in a nondestructive manner.

Inherently weak Raman signals

Raman spectroscopy imaging is typically a very long process as Raman scattering generates extremely weak signals.

Inherently sensitive detection

With Nüvü Camēras’ unrivalled sensitivity, such processes can be accelerated up to 10 times without resorting to other more sensitive yet complex spectroscopic 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.

Ultra Fast Raman Hyperspectral Imaging Using Bragg Tunable Filters and a High Performance EMCCD Camera

Super-Resolution

Single-molecule Imaging

Another biomedical sciences field of interest is single molecules imaging within cells that have been tagged with fluorescent proteins like GFP. While bioluminescent molecules emit their own light, fluorescent markers require more energetic light stimulation to trigger emission.

Limited by marker photobleaching

As such, fluorescence-based imaging is limited by the markers photobleaching, which, in turn, depends on the stimulating laser intensity and exposure time.

Sensitivity adapted for in-vivo imaging

To counter this issue, Nüvü Camēras’ higher SNR allows reduced stimulated laser intensity, decreased acquisition periods, increased frame rates and temporal resolution, all while providing superior image quality.

Improved Single-Molecule Imaging Based on Photon Counting with an EMCCD Camera

The 3-B technique

The innovative 3-B technique takes advantage of a particular in vivo fluorescence characteristic: 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 a microscope diffraction limit and yields promising  results for in vivo cellular studies.

High frame rates to harness blinking and bleaching

Transient intracellular events need to be studied in low light imaging conditions, and they require high frame rates because due to rapidly changing patterns. The exposure time needs to be kept short so the fluorophores are fairly well separated in each frame. However, higher frame rates increase clock-induced charges (CIC) levels. If satisfying levels of total background noise and overlapping can be achieved, the SNR becomes very important and provides 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 keeping background noise to a minimum. Thanks to its patented controller, Nüvü cameras’ CIC (dominant noise in low light flux conditions) levels remain 0.001 e-/pixel/frame while their frame rates reaches up to 20MHz.

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. Dr Yuri Zakharov points out that new ultra low noise EMCCD cameras certainly hold the key to better 3-B performance levels.

Clinical Applications

Nuclear medicine

In nuclear medicine, the gamma and X-ray light emitted by radioactive tracers is much too energetic to be detected with conventional camera technology. As such, specialized gamma cameras fitted with scintillators are used to translate gamma and X-ray light into visible photons, which can adequately be detected by conventional silicon-based detectors.

Poor resolution in gamma ray detection

Current clinical gamma cameras have a poor resolution, typically in the millimetre scale. However, detector development with intrinsic resolutions of about 100 µm can would push forward the advancement of nuclear medicine. Such 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-EMCCD systems for 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. They have previously shown that false-positive events due to EMCCD noise can be rejected by using the summed SiPM signals. They now present enhancements made in SiPM signal processing that improve their system’ SNR and energy resolution. While they did not use Nüvü cameras for their research, 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.

Improved EMCCD gamma camera performance by SiPM pre-localization

Also take a look at Nüvü Camēras’ referenced article in the following publication.

Characterization results of EMCCDs for extreme low light imaging

Fluorescence-guided surgery

Fluorescence-guided surgery for brain tumor resection lies on the endogenous protoporphyrin IX (PpIX) molecule property to selectively accumulate in neoplastic tissues, revealing tumour boundaries. Improving this surgical technique will lead to better brain tumour resection prognostics regarding. The complexity of such procedure resides in the complete yet selective removal of the cancerous tissues. Failure to target malignant cell pockets can promote a recurrence of the pathology and compromise the patient’s remission.

Biological tissue fluorescence limits emitted photon flux

PpIX in vivo imaging results in faint, low incoming signal. In order to efficiently guide surgeons in the operating room, long exposure times are required for enough photons to accumulate in the sensor wells. Longer integration time means higher dark current noise levels, but also requires longer procedures, which directly affect the patient life expectancy.

Shorter exposure time thanks to more sensitive EMCCD cameras

Nüvü Camēras’ 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 readout noise negligible. These factors allow for better SNR, thus making accurate imaging possible in less time with low incoming flux.

In fact, using Nüvü cameras, fluorescence-guided surgery reduces the time, costs and fluctuations of neurosurgical procedures. This same technique could also be transfer to other  complex cancer resection surgeries like throat or prostate carcinomas.


Researchers have determined PpIX in vivo detection SNRs with respect to exposure time for Nüvü Camēras’ EMCCD EMN2 camera and a competitor’s CCD camera (details available below). With the EMCCD, they identified cancer cells with exposures up to 100 times shorter than those of a conventional CCD while obtaining superior images quality.

Below is a link to Yoann Gosselin’s poster at the Biophysical Society’s 57th annual meeting held from February 2 to 6 2013.

How can the enhanced sensitivity and favourable noise characteristics conferred by electron multiplication improve fluorescence-guided surgery

Multimodal imaging

Multimodal imaging allows for parallel information acquisition in a single exposure while using different probe modalities. Becoming a standard in the pre-clinical industry, it operates by injecting molecular markers 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 remains detectable and 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 when 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 case of fluorescence multimodal imaging, 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 while 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 vivo characterization of zinc- and copper-trapped phtalocyanines detection limits made with a Nüvü camera is available below (conference #8574-22 presented at BiOS – Photonics West 2013 during Session 4 on Preclinical Imaging on February 2nd, 2013).

Ultra-high sensitivity detection of bimodal probes at ultra-low noise for combined fluorescence and positron emission tomography imaging

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.

Weak signal

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. The latter radiation 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 taken by medical diagnostics. Numerous researches are currently being conducted using 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 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 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 signal amplification up to 5000 times via electron multiplication before digitization, 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.