A particularly promising biomedical research tool is single-molecule imaging, which detects biomarker emissions with increased resolutions, reaching the molecular scale. However, such a method strongly depends on the stimulating laser intensity that triggers the fluorophore emission.
Limited by marker photobleaching
Fluorescence-based imaging is hindered by marker photobleaching, which, in turn, depends on the stimulating laser intensity as well as measurement duration. Optimizing exposure time and trigger intensity is key for achieving high-quality single-molecule imaging.
Adapted for in vivo single-molecule imaging
Nüvü™ offers EMCCD cameras that support greater frame rates and temporal resolutions as well as the lowest overall background level on the market. Nüvü Camēras technology elegantly resolves the photobleaching issue inherent to single-molecule imaging and allows the user to decrease the stimulating laser intensity. The result: Improved fluorophore lifetime and superior image quality at the same time.
Demonstration: Photon-counting in a single-molecule imaging
The Institute for Research in Immunology and Cancer (IRIC), along with the University of Montreal’s Department of Pathology and Cell Biology, confirm the increased performances of a single-molecule imaging microscopy system using Nüvü™’s EMCCD in photon-counting mode. Their poster is presented below.
Hyperspectral imaging for single molecules
Paul De Koninck from the Centre de recherche de l’Institut en santé mentale de Québec demonstrates the ability to detect and track 4 subtypes of receptors at synapses. Even if the number of photons detected in a single QD PSF was on the order of 100 to 150 photons, thereby limiting the localization error usually associated with these optical techniques, the low background signal provided by the Nüvü’s camera allowed for subpixel accuracy.
Bioluminescent reagents are often used in biological assays, such as sanitary evaluations, to assess and quantify the presence of a particular molecule. A prime example is adenosine triphosphate (ATP) detection in biocompatibility and cytotoxicity tests. In such assays, a combination of luciferin and luciferase produces a light-emitting compound in the presence of ATP. The bioluminescent signal varies linearly if ATP concentrations are inferior to those of the reagent pair. As such, calibration curves can be obtained to quantify precisely ATP amounts in biological samples.
Traditionally limited to the picomole scale
Typical detection methods inhibit ATP quantification lower than the picomole scale. Indeed, commercially available cameras do not reach the required sensitivity to observe smaller luciferin+luciferase concentrations.
Photon counting to reach the femtomole scale
Nüvü cameras enable better ATP detection as a result of their controller and packaging technology that significantly lower the overall sensor noise. Nüvü Camēras photon counting capabilities further push detection limits, pushing them down to the femtomole scale.
Demonstration: Detecting ATP femtomole-scale concentrations
Presented below are three images of the same microplate containing solutions of various ATP concentrations, ranging from 5 to 156 femtomoles and studied in triplicates. Each image was acquired with Nüvü™ EM N2 1024 camera.
Figure 1: Single 30-seconds acquisition in Conventional (CCD) mode. The image displays the expected performance of a top-of-the-line CCD camera or even a high-end sCMOS. Without the electron multiplication to boost the bioluminescent signal, ATP solutions are barely noticeable.
Figure 2: Superposition of five 1-second EM acquisition. With a lower noise floor due to the electron multiplication process, all six ATP concentrations are detectable. SNR values vary from 1.9 to 14.1 (or, equivalently, from 2.8 to 11.5 dB).
Figure 3: Superposition of five 1-second EM acquisition in photon-counting mode. Suppressing ENF, photon counting pushes the SNR, which ranges between 6.6 and 51.3 (8.2 to 17.1 dB). ATP quantities were as low as 5 femtomoles are detected with strong contrast. Images courtesy of the Université de Sherbrooke Hospital Centre.
The 3-B Technique
The 3-B technique takes advantage of the fluorophore blinking process caused by photobleaching, occurring at irregular intervals, and independent from one molecule to another. Combining photobleaching time-series recordings and the Bayesian analysis of the blinking and the bleaching, gave rise to the 3-B technique. This novel tool increases image resolution beyond that of the microscope and yields promising results for in vivo cellular studies.
High frame rates to harness blinking and bleaching
Transient intracellular events are studied in low light imaging conditions and require rapid acquisitions to expose their dynamics through the fluorophore blinking and bleaching. However, high frame rates increase CIC levels, thus noise, which leads to inferior image quality.
Better SNR with patented EMCCD noise reduction
Thanks to its efficient photon counting mode, Nüvü Camēras offers the ultimate solution for the 3-B technique. Nüvü™ EMCCD cameras can drastically reduce noise levels that previously prevented super-resolution microscopy studies. The EM gain accentuates the incoming photon flux while keeping background noise to a minimum. Furthermore, the patented Nüvü™ camera controller keeps CIC levels under 0.001 ē/pixel/frame even at 20 MHz frame rates.
Demonstration: Harnessing the EMCCD sensitivity for 3-B
Y. Zakharov and his team from the Lobachevsky University in Russia determined that ultra-low noise EMCCD cameras can specifically be used for 3-B measurements in the poster below, presented at the 2013 Optics in the Life Sciences, Optical Society of America Optics and Photonics Congress.
For more information, check out Dr. Susan Cox team’s publications entitled:
Bayesian localization microscopy reveals nanoscale podosome dynamics
Bayesian analysis of blinking and bleaching