In order to allow for flexibility in experiment design, a custom microscope set-up was used. During this study two similar set ups were used that differ only slightly in their construction. The first set-up was designed by Dr Steven Lee (Cambridge University, UK) and built by Dr Remi Boulineau (Osborne lab, University of Sussex); this will be referred to as ‘SR-2.1’ (Figure3-1A). Further into the project a second microscope set-up was built, referred to from here on as ‘SR-2.2’ (Figure 3-1B). SR-2.2 was built by Dr Manisankar Maiti (Carr Lab, University of Sussex) and myself and was based on the original set-up, but included upgraded and dual colour imaging components. A comprehensive list of components and their manufacturers is given in the Materials and Methods chapter.
Both microscopes were fitted with four lasers spanning the visible spectrum: 405nm, 488nm, 561nm and 641nm. This allowed for the potential of single colour experiments with different fluorescent probes or dual colour experiments with two spectrally separated fluorophores. After each laser, two sets of neutral density filters were used to control the laser power that reached the sample. This is important in SMLM experiments as laser power can be used to control both the rate of photoconversion/photoswitching of fluorescent proteins as well their brightness. Thereafter, each laser line displayed a
Figure 3-1. Schematic representation of custom built PALM microscope.
Outline of A) SR2.1 and B) SR2.2 PALM microscopes. In both cases, all shutters are displayed as closed, except for the 561nm laser line, which depicts the light path into the microscope and onto the sample.
quarter-wave plate in order to convert the linearly polarised laser light into a circularly polarised beam. Circular polarisation of the laser beam is necessary in order to ensure that differentially orientated fluorophores within the sample are excited, rather than just those whose dipole orientations match that of the linearly polarised beam.
The circularly polarised beams were then passed through a band pass filter with bandwidths that match the specified output wavelengths of the lasers. This was to ensure that no wavelengths shorter or longer than that desired reached the sample, helping to prevent potential photobleaching, activation or autofluorescence from extraneous light.
Each laser beam was then expanded and collimated with a custom designed Galilean beam expander, using two (plano-concave/convex) matched lenses. Beams were expanded to increase the laser footprint on the sample, and thereby utilise the whole chip on the camera. Collimation was also important to prevent the beams from over expanding or focussing before they reached the sample.
The path of each expanded and collimated beam was opened or closed controlled by individual automated shutters. The beams were then coupled with the use of dichroic mirrors and were co-aligned to ensure spatial overlap of the beam footprints on the sample. Finally, the beams were focussed on to the back focal plane of an apochromatic TIRF objective lens using a plano-convex lens, thus ensuring a widefield illumination regime with a near-collimated beam emergent from the objective lens. The objective lens specification was 60x magnification, a high numerical aperture of 1.45 and was mounted on the objective turret of an Olympus IX71 inverted microscope body for SR-2.1, or in the case of SR-2.2 the upgraded IX-82 inverted body. In both cases a multi-band dichroic (405m/488nm/561nm/635-25nm) was used within the body of both microscopes in order to separate the fluorescence signal from reflected laser emission. Furthermore, both configurations were fitted with motorised stages that possessed custom-built steel inserts to increase microscope stability. Axial stability of the microscope during imaging was maintained by a constant focus device (C-focus, MCL – described later).
The emission path for the sample fluorescence was the major difference between the SR-2.1 and SR-2.2 configurations. Within SR-SR-2.1 the emission was reflected out of the camera side port of the microscope body, where it was expanded by a 2.5x Olympus photo-eyepiece and passed through a motorised filter wheel before reaching the detector.
One major advantage of the IX82 microscope body of SR-2.2 was the ability to insert optical components between the objective lens and the tube lens, in what is known as
‘infinity space’. This space in the microscope is where light is not focussed but propagates as a collimated beam of parallel rays Any optics that are inserted into this space will therefore not influence the focal plane of the final image. Thus we decided to insert the motorised filter wheel inside the microscope body so that there would be minimal chromatic aberrations when switching colours in future dual colour imaging. After passing through the filter wheel, fluorescence was directed out of the microscope side-port, expanded by a 2.5x photo-eyepiece and finally passed through a Photometrics DualView multichannel imaging system. In ‘bypass mode’, the fluorescence reached the detector without further filtering. The DualView system was installed into the SR-2.2 set-up for future two colour experiments that do not feature in this thesis.
In both configurations, the detectors were back-illuminated cooled EMCCD cameras from Photometrics. SR-2.1 was equipped with the Evolve 512, whereas SR-2.2 was fitted with the newer Evolve 512 Delta. The final image pixel size for both machines was optimised (by the use of the 2.5x photo-eyepiece) to 107nm and 110nm for SR-2.1 and SR-2.2 respectively. This was based on a study that highlighted the importance of having a final pixel size which was the equivalent to 1 standard deviation of the predicted point spread function of the microscope (Thompson 2002).
Finally, the shutters, emission filter wheel, camera and stage were controlled remotely using the open-source software Micro-manager (Edelstein 2010). For data acquisition that required alternating laser illumination, custom routines written for Micro-manager’s script panel were provided by Dr Steven Lee or Dr Remi Boulineau.