Building a Scanning PALM Microscope
The Application
Fluorescent proteins have low photon-yields, which limits localization precision, since the accuracy of the localization depends directly on the number of photons collected from a fluorescent sample [7]. Microscopy methods that decrease the photo-bleaching rate of fluorescent proteins, thereby increasing the photon-yield, are key to increasing the localization accuracy of such techniques. The instructors outlined the design of a scanning confocal system in an attempt to increase the photon-yield from individual fluorophores by allowing for fluorophore relaxation between rounds of excitation [6]. The undergraduates undertook the construction of the system, developed software algorithms and fluorescence constructs for calibration trials, all within the course of a semester.
Figure 1: Basic idea of the optical setup of such a system.
