Theory

To assess the feasibility of this project, we performed a detailed photon budget, so as to determine if enough photons would be detected by the camera in each photoswitching cycle. This calculation is based on the work of Thompson et al. where a detailed analysis of single molecule localization is performed. This same method is used in STORM literature and it proved to be very close to the actual results. In short, and as shown below, the power delivered at the sample was calculated by determining the approximate area of the beam at the sample and then estimating the amount of power per area on the sample plane (a 0.5 factor was included to account for power losses in the laser path). Then this energy flux was converted to a photon flux by using Planck’s constant. The incoming photon flux was converted to an effective photon flux hitting the sample by using the sample’s cross section (obtained from the dye’s quantum yield). To calculate the approximate photon emission rate we used the quantum efficiency of the sample. Using the latter number, we took the reported parameters for 4 different cameras: the AVT Manta 032-B, the Hamamatsu Orca Flash 2.8, the Andor Neo-sCMOS and the Andor iXon3. These were picked since the first one is the standard camera in this lab, the third and fourth would be the ideal components and the second one seems to be the best price-quality compromise we can go with in this project. To approximate the number of photons actually detected with each camera, we estimated the light losses as 30% due to detection angle, 80% due to filters and 50% due to the objective (Thompson et al). Additionally, we accounted for the lower N.A. of our objective as 74% additional losses. With this, the quantum efficiency of the detector and the integration time we determined the effective amount of photons collected per cycle at the detector. To estimate the noise sources, we arbitrarily set a range of 1 to 10 electrons/pixel second as spurious light, and used the specified values for read and dark noise (the first and last weighted by the integration time). With the collected photons, the pixel sizes at the sample plane and the noise estimations we calculated the expected uncertainty in the localization of a single molecule per cycle, aside from the amount of photons counted and the uncertainty of this count. Finally, we calculated the Full Width at Half Maximum of the localization uncertainty as our resolution, and as a function of the number of cycles (1, 10 and 100). This is all summarized in the table below. It will be noted here though that the actual number of switching cycles is limited by the total number of photons that a dye molecule can emit before photobleaching, usually in the range of 250000 for an EM-CCD detector (less numbers for the less efficient detectors). (Thopmson et al.).