Photometrics, a designer and manufacturer of high-performance CCD and EMCCD cameras for life sciences, will offer two educational presentations at the 2010 Biophysical Annual Meeting. The presentations will cover technologies in the Evolve camera line that markedly improve data quality for fluorescence correlation spectroscopy (FCS) experiments and, for the first time, make fluorescence imaging data reproducible between laboratories and cameras.
Researchers use FCS to track diffusion and other fast dynamic properties of molecules, which demand very low noise levels. Methods such as total internal reflectance fluorescence (TIRF) can be used to reduce image background noise and increase signal-to-noise ratio while maintaining good Z-resolution for FCS measurements. EMCCD cameras are often used in such experiments as they can provide higher signal-to-noise ratios than normal CCD cameras.
However, EMCCD cameras have an additional noise source often called “background events” , resulting from the multiplication of spurious events caused by clock induced charge and thermal generated charge. This noise produces what are referred to as “speckles” on the image, resulting in the image containing more noise.
A technology unique to Photometrics Evolve EMCCD cameras, “Background Event Reduction Technology” (BERT), helps improve FCS data clarity and consistency by reducing the “speckles” generated. Xiaotao Pan, Ph.D., will present data demonstrating how BERT can reduce noise, improve FCS data consistency, and make curve fitting more robust.
Reproducible, Quantitative Fluorescent Imaging Data
Deepak Sharma, Ph.D., senior product manager at Photometrics, will present the Evolve camera line’s advanced features, including those that enable researchers to measure and quantify data in photoelectrons, which allows data generated by any researcher, using any camera, at any time to be directly and quantitatively compared.
Currently, imaging data from EMCCD and CCD cameras is reported in non-standardized units with arbitrary meaning. Researchers cannot use these units (known by many names, including fluorescence units, gray scale values, and analog-to-digital units) to reliably compare or reproduce imaging data between labs—or even within their own.
The problem is that those units are merely electronic representations of incident photons. Photons that hit CCD and EMCCD sensors generate photoelectrons, the fundamental unit of measurement for these devices. These photoelectrons are processed and amplified by the camera in a variable manner, dependent on user-defined settings as well as sensor and camera characteristics. These characteristics differ even between the same make and model of camera. Certain characteristics, such as camera gain values, are in most cases not measured or revealed by camera manufacturers*.
Photometrics’ technology is the first to report imaging data in a camera’s fundamental unit, photoelectrons, not the variable units used by other cameras. Dr. Sharma will explain how this technology and unit of measurement will allow researchers seamlessly reproduce and compare imaging data—a previously impossible task.
The advanced features of the Evolve camera line that Dr. Sharma’s presentation will include are:
- Quant-View™ which provides a repeatable methodology to gather and interpret data by reading out pixel values in photoelectrons.
- Rapid-Cal™ provides the most accurate, simplest and fastest EM calibration technique in the industry.
- Background Event Reduction Technology™ (BERT) enables researchers to correct spurious event data.
- Top-Lock™ and Black-Lock™ are complementary intensity filtering tools, which allow researchers to narrow visualization to the intensity range of the image features under study.
The Evolve camera line’s advanced set of features are ideal for life science applications, including super-resolution fluorescence microscopy (e.g., Photo-Activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM)), and low light applications (e.g., spinning disk confocal microscopy, Total Internal Reflection Fluorescence (TIRF) microscopy, cell trafficking studies, Single Molecule Fluorescence (SMF), and live-cell fluorescent protein imaging).
*Photometrics has always provided exact gain values in a “certificate of performance” for every camera sold.