Nüvü Camēras’ innovative EMCCDs overcome research barriers to produce high quality images for imaging small animals, single molecules, FRET, BRET and more:
Spinning Disk Confocal Microscopy
(Images courtesy of Quorum Technologies Inc.)
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 the Université de Sherbrooke Hospital Centre)
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.
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 substantially more elusive photons, but can also reach higher detection speeds via greater frame rates on top of the superior reliability of the generated images.
(Courtesy of Dr. Frédéric Lesage’s research group at École Polytechnique de Montréal)
A novel study 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’s abstract, entitled “Ultrasound guided fluorescence tomography”, was presented at the 2012 edition of Photonics North, in which “a hybrid-model imaging system combining fluorescence and ultrasound (US) was investigated with the motivation of providing structural priors towards improvement of fluorescence reconstruction”. While the results presented in the paper are obtained from laboratory phantoms, future developments include an extension of the presented applications onto mouse models of cancer.
Presented below are the latest developments of the fluorescence imaging portion of Dr. Lesage’s hybrid imaging system.
As shown in the following figure, a continuous wave (CW) laser beam (658nm, HL6512MG, Thorlabs) was delivered through an optical fiber to illuminate the bottom of the sample. Before being coupled into the fiber, the laser beam was filtered by an optical bandpass filter D650/20 (Chroma Technology). On the opposite side, the emitted photons were selected by an optical filter (FF01-716/40, Semrock), then reflected by a mirror and eventually collected by Nüvü Camēras’ EM N2 camera. For optical imaging, the region of interest (ROI) was imaged in a raster-scanned fashion controlled by a pair of motorized linear stages (LSM100B, Zaber). For ultrasound imaging, a single-element transducer was used (10 MHz, diameter 0.25’’, F=10cm, Olympus) and the imaging was conducted in a water medium in order to couple ultrasonic pulse-echoes. A home-made electronic board drove the laser diode, pulsed the transducer, sampled ultrasonic signals and communicated with a computer via a USB link. In addition, a projector (PK101, Optoma) projected a black and white pattern on the sample to extract the boundary contour using a FFT profilometry method.
Geometry: rectangular parallelepiped shape (100×30×20 mm).
Optical properties: μa= 1.0 mm-1; μs = 0.2 mm-1.
Fluorescent inclusion: a tube filled with 10 nM Cy5.5 was inserted in the phantom at the position marked by the red rectangle at a depth of 10 mm.
Result: The maximum intensity of the fluorescence image is 13069, while being only 2183 for the control (H2O), thus indicating that 10nM Cy5.5 may be successfully detected in the phantom.
Subject: two mice; one is intravenously injected with fluorophore, another is used as a control.
Fluorophore: Cy5.5; 0.2ml; concentration: 0.1 mg/ml. The fluorophore was injected intravenously into the blood circulation of the mouse without labelling with any peptides or proteins.
Result: the colored areas shown on the pictures correspond to the scanned area. the fluorescence and control images are overlaid on the scanned area on the two mice separately on the pictures. The pixel intensities are normalized with 256 as the maximum amplitude.
Nüvü Camēras will be pleased to follow and present the future developments of Dr. Lesage’s research group.
(Poster courtesy of Dr. Paul S. Maddox from the Institute for Research in Immunology and Cancer (IRIC) & Université de Montréal)
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.
Raman Spectroscopy Imaging
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.
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.
Improved EMCCD gamma camera performance by SiPM pre-localization
(Courtesy of Dr. F. J. Beekman at Delft University of 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.
Direct link to the article: Improved E.M.C.C.D. gamma camera performance by SiPM pre-localization.
Also check out Nüvü Camēras’ referenced article in the publication:Characterization results of EMCCDs for extreme low light imaging
(Courtesy of Dr. Yves Bérubé-Lauzière from Centre d’Imagerie Moléculaire de Sherbrooke (CIMS) – Université Sherbrooke)
Multimodal imaging is quickly becoming a standard in pre-clinical studies, and new developments have already confirmed the strength of acquiring and analyzing parallel information obtained in a single imaging session. One such application is the introduction of an internal reference moiety (e.g. radioisotope) to an activatable fluorescent probe. One of the limitations of this approach consists of working at concentrations which are within the overlapping range of sensitivities of each modality. In the case of PET/Fluorescence imaging, this range is in the order of 10-9nM. Working in epi-illumination fluorescence imaging at such concentrations remains challenging. Here, we present in vitro and in vivo detection limits of a new fluorescent compound.
Conference #8574-22 presented at BiOS – Photonics West 2013 during Session 4 on Preclinical Imaging on February 2nd, 2013.
Higher SNR for Overcoming Research Frontiers
Innovative techniques involving light and living organisms are continually discovered in parallel with technological advancements in optics and imaging devices. Pioneering the exploration of uncharted areas of biomedical research, Nüvü Camēras’ innovative cameras provide solutions to the most complex detection challenges of the scientific community involved in ultra low light imaging. Reaching far beyond the limits of present day technology, Nüvü Camēras’ imaging systems are a significant contribution to the analytical work of modern scientists thanks to an especially higher SNR. This particular advantage among others allows scientists to improve obtained results simply by using a more sensitive camera.
In the field of biomedical studies, Nüvü Camēras’ EMCCD cameras may contribute to saving lives and minimizing hospitalization time. In fact, these cameras are more than capable of accomplishing any task a standard high-sensitivity camera can complete, but with a superior SNR. In fact, in spite of intense efforts to reduce the mortality rate due to cancer, it is still the second leading cause of death in the developed world. In addition, several types of cancer are diagnosed at a late stage due to limitations in detection methods, at which point treatments rapidly become invasive. As such, there exists an urgent need for innovation in the field of medical diagnostics — particularly in the case of cancer. The pursuit of imaging devices with superior sensitivity as proposed by Nüvü Camēras thus opens pathways to breakthrough discoveries and innovations in medical research, allowing sensitive and specific marker detection through in vivo tissues, which is but a single example of what new low light imaging limits may offer.
Among many possible applications, the detection of individual photons emitted by weak light sources such as biomarkers — while generating the lowest amount of noise in comparison with traditional EMCCD cameras — is of great technological interest. In fact, a significant amount of research is currently being conducted on various forms of disease using these biomarkers, as their use has the potential 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.
Moreover, Nüvü Camēras’ superior SNR yields a unique advantage in other growing areas of biomedical research such as techniques involving cancer research, spinning disk confocal microscopy, super-resolution imaging techniques and in vivo animal imaging, some of which are described above.
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 on one hand and an EM gain peaking at 5000 on the other, these EMCCD cameras can be used in several biomedical research fields to observe phenomena that have never before been examined with such precision, sensitivity and reliability, thus yielding potential benefits to a wide variety of fields of study.
The background noise in EMCCD cameras results from a combination of dark current and clock-induced charges, while 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 patented technology elegantly overcomes these obstacles. One important method is by allowing an amplification 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 using 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 floor for ultra low light imaging involving wavelengths between 250 nm and 1100 nm. Moreover, 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.