Abstract
The ability to detect single molecules over the electronic noise requires high performance detector systems. Electron Multiplying Charge-Coupled Device (EMCCD) cameras have been employed successfully to image single molecules. Recently, scientific Complementary Metal Oxide Semiconductor (sCMOS) based cameras have been introduced with very low read noise at faster read out rates, smaller pixel sizes and a lower price compared to EMCCD cameras. In this study, we have compared the two technologies using two EMCCD and three sCMOS cameras to detect single Cy5 molecules. Our findings indicate that the sCMOS cameras perform similar to EMCCD cameras for detecting and localizing single Cy5 molecules.
Highlights
The rapid development of single molecule (SM) microscopy techniques has significantly improved our understanding of biophysical systems through various methods, including diffusion analysis [1], energy transfer measurements [2] and more recently, super resolution microscopy [3,4,5]
We have demonstrated that single molecule detection is possible with the scientific Complementary Metal Oxide Semiconductor (sCMOS) cameras used in the study
Electron-Multiplication Charge Coupled Device (EMCCD) cameras have an advantage over the sCMOS cameras when it comes to peak signal to noise ratios
Summary
The rapid development of single molecule (SM) microscopy techniques has significantly improved our understanding of biophysical systems through various methods, including diffusion analysis [1], energy transfer measurements [2] and more recently, super resolution microscopy [3,4,5]. Most single molecule methodologies rely on sensitive charge coupled device (CCD) based detectors to convert photons into readable signal from a wide-field imaging system, allowing interrogation of many molecules simultaneously. Most wide-field single molecule experiments currently employ Electron-Multiplication Charge Coupled Device (EMCCD) based cameras. Single molecule detection typically requires exposure times of 10— 100 ms Such short exposure times require fast read-out rates with very low read noise. EMCCDs can provide an effective read noise substantially lower than the signal generated from a single photon. Scientific imaging sensors based on a Complementary Metal Oxide Semiconductor (CMOS) technology have been introduced with low read-noise, high read out rates, and small pixels, that may significantly improve and simplify single molecule detection systems [11]
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