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Posted: Jul 23, 2013
Andor EMCCD Camera Delivers Ultrahigh Accuracy for Super Resolution Microscopy
(Nanowerk News) Super resolution localization microscopy techniques, which allow the capture of images with a higher resolution than the diffraction limit of approx. 250 nm, are breaking new ground in our understanding of subcellular structures and the movement of individual molecules within cells. However, the positional accuracy attained by these powerful techniques has been limited by the pixelation and noise generated by CCD and EMCCD detectors.
Now, a team from the University of Texas at Dallas led by Prof. Raimund J. Ober has discovered a method to almost eliminate these two image-deteriorating factors and improve parameter estimation accuracy by more than 200 percent. Publishing in Nature Methods, they used an Andor iXon3 897 EMCCD camera to present the Ultrahigh Accuracy Imaging Modality (UAIM).
Contrasting the appearance of a conventionally acquired EMCCD image with the appearance of an image acquired using UAIM. In each case, the image is that of an Atto647N molecule, and is visualized using standard and mesh representations. The mesh representation clearly contrasts the relatively smooth, Gaussian-like profile of the conventional image with the spiky appearance of the UAIM image. The conventional image was acquired at an effective pixel size of 253.97 nm using a standard 63x magnification, and in an experimental setting where, on average, ~183 photons per image were detected from the Atto647N molecule. It has an average photon count of 48.13 in its brightest pixel. The UAIM image was acquired at an effective pixel size of 16 nm using a 1000x magnification, and in an experimental setting where, on average, ~191 photons per image were detected from the Atto647N molecule. It has an average photon count of just 0.52 in its brightest pixel. Scale bars, 0.5 µm.
"UAIM uses the EMCCD camera to its fullest potential, beyond what is commonly believed to be possible by the scientific imaging community," says Prof. Ober. "Specifically, our results show that when an average of less than one photon is detected per pixel, the excess noise due to the camera's electron multiplication process is reduced to such an extent that an accuracy can be obtained that approaches the absolute best accuracy attainable only with an imaging detector that neither pixelates nor adds measurement noise to the image data.
According to Colin Coates of Andor, "UAIM uses the iXon3 EMCCD detector at a high level of signal amplification and in a highly unconventional and counterintuitive setting in which the number of photons detected in each pixel of an acquired image averages less than one. The resulting image does not give a visually clear representation of the object of interest as might be expected for obtaining high localization accuracies but, significantly, this new imaging method is applicable to all types of estimation problems. Although Prof. Ober and his team demonstrated UAIM using single molecule localization, it can be applied equally to other EMCCD imaging applications, for example in astronomy, surveillance and machine vision."
To learn more about the iXon camera series, please visit the Andor website iXon pages at http://www.andor.com/scientific_cameras/ixon_emccd_camera.
Jerry Chao, Sripad Ram, E Sally Ward and Raimund J Ober. "Ultrahigh accuracy imaging modality for super-localization microscopy" Nature Methods, Vol. 10 No. 4 335-338 (2013)
Andor is a world leader in Scientific Imaging, Spectroscopy Solutions and Microscopy Systems. Established in 1989 from Queen's University in Belfast, Northern Ireland, Andor Technology now employs over 340 people in 16 offices worldwide, distributing its portfolio of over 70 products to 10,000 customers in 55 countries.
Andorís digital cameras, designed and manufactured using pioneering techniques developed in-house, allow scientists around the world to measure light down to a single photon and capture events occurring within 1 billionth of a second. This unique capability is helping them push back the boundaries of knowledge in fields as diverse as drug discovery, toxicology analysis, medical diagnosis, food quality testing and solar energy research. More information about Andor Technology PLC (LSE: AND) is available at the company's website