Construction of a Scanning Laser Confocal Flourescence Microscope System for Single Molecule Detection and Spectroscopy

The Single Molecule Spectroscopy Group at the department of Chemical Physics in the Chemical Centre of Lund University conducts research involving spectroscopy on single molecules of organic dyes. Fluorescence microscopy using a wide-field scheme has been used and such a scheme has many advantages such as full image capture at once when using a high sensitivity CCD or CMOS camera. Using different techniques it may be possible to conduct more sensitive analysis on single points in such an image using a photo multiplier tube (PMT) or an avalanche photo diode (APD). It is desirable to have the possibility to analyse the spectral properties of single molecules of organic dyes, a sensitivity not attained by the wide-field strategies currently used at the SMS group. A confocal strategy has the advantage of a very shallow depth of field. The use of a pinhole for selecting a single plane of the sample, ideally diffraction limited, minimises the problem pertaining to stray light from the surroundings and effects of fluorescing sample carriers (glass plates). Thus the strategy can potentially greatly improve the signal to noise ratio in single molecule spectroscopy applications. The power intensity of the laser light normally used for excitation in fluorescence microscopy tend to be relatively high and problems with photo bleaching arise. Especially when analysing single points on the sample there is really no reason to illuminate the whole sample, risking bleaching of the entire sample, when only the analysed molecule needs illumination. To combine the benefits of a sensitive detector such as a PMT or APD with lowest possible total light dose and intermittent exposure of the sample a scanning confocal setup has been suggested and proven effective. A further advantage of a confocal strategy is the possibility to use high power pulsed lasers and two-photon processes to analyse samples. The use of two-photon processes would be, if not impossible, extremely impractical (and also dangerous if used in an open setup) in a wide-field setup as the light would be highly divergent and the optical power thus needs to be much higher to obtain the needed power density at the sample. This thesis covers the construction and optimisation of a scanning laser confocal fluorescence microscope system as well as a redesign and optimisation of a LabVIEW based control scheme presented by Giang Vu in his Masters thesis Software Control of a Confocal Microscope [1]