The data presented in this chapter demonstrate the successful installation of a custom-built PALM microscope in the Carr lab. The main aim of the work presented was to learn the specifics of how the PALM microscope and methodology work, and to successfully demonstrate photoactivation and localisation of mEos3 fluorophores expressed in fission yeast cells.
During the initial stages of this project, efforts were focussed on ensuring microscope operational stability and optimal preparation of samples. Axial stability was of great
Figure 3-6. The effect of a 580/14nm band pass on mEos3.1 localisation.
urg1-mEos3.1 cells (AW790) were grown in the presence of uracil to induce mEos3.1 expression. Cells were then fixed and PALM microscopy was performed. Data sets of 5000 localisations detected using either the 593/40nm or 580/14nm band-pass filter were compared for A) photon count per mEos3.1 localisation, B) mEos3.1 localisation precisions and C) Background photons calculated for fitting window.
importance for future experiments in this thesis due to the size of fission yeast cells. Any variation in the axial position would result in data being recorded from more than one focal plane during an experiment. This axial stability was achieved using the C-focus system, however as noted in the material and methods section this stability also relied on constant room temperature, magnets and a period of sample stabilisation. The accuracy of each experiment relied on the strict adherence to this procedure. As demonstrated in Figure 3-3C, sample drift also occurred in the lateral dimension during data acquisition.
This was caused mainly by fluctuations in air currents from conditioning units, and thus was substantially reduced by covering the microscope body (Figure 3-3D). However, localisation of fluorescent beads showed that drift still occurred at a sub-pixel level. Due to the type of future experiments described in the next chapters, this drift did not affect data collection or analysis.
Once a stable microscope system had been built, the most important factor in the success of each experiment is undoubtedly sample preparation. In order to detect single bursts of fluorescence in PALM experiments the background level of fluorescence needs to be much lower than that of a single fluorophore. The two main sources of background fluorescence were from the cell suspension and the agarose pad. Washing cells several times in filtered PBS and carefully removing the supernatant from the meniscus ensured that background from the suspension was negligible. The use of very clean glassware for agarose preparation and making up fresh molten agarose for each pad guaranteed very low background fluorescence. During the optimisation process, we found that ozonation of the coverslips used to make the agarose pad was also of upmost importance.
At the start of this project, the view was to use PALM for super-resolution based studies of protein localisation and oligomerisation. In order to ensure that localisations detected were from actual mEos3 fluorescent events and not autofluorescence, a narrow band pass filter was installed to filter out as much autofluorescence as possible. This successfully reduced the amount of false localisations that were detected in wild type cells, and will be important for future experiments in the Carr lab concerned with absolute quantification of protein number. However, as described in the next chapter, the experiments performed for the rest of the project did not aim to study protein localisation/oligomerisation and instead PALM was used as a semi-quantitative read-out of protein association with the chromatin. In this case the level of autofluorescence detected was not as crucial, but the
580/14nm bandpass was still utilised as it helped reduce the overall integrated autofluorescence from cells, which was beneficial at longer exposure times.
Chapter 4
Methodological development for the visualisation of DNA bound proteins in unfixed fission yeast
4.
4.1. Introduction
During the course of this project, the main aim was to develop ways of using photoactivated localisation microscopy to study proteins involved in DNA metabolism, specifically DNA replication. This field has particularly benefited from high-end microscopy platforms, including SMLM techniques (discussed in introduction, reviewed in Stracy 2014). However, these studies have largely been performed in prokaryotic model organisms exploiting their physical dimensions as the small axial sizes of bacterial cells match the focal depth of the microscope. This allows researchers to image the whole cell without the need to take multiple images from different z-planes. This permits the use of techniques such as Total Internal Reflection Fluorescence (TIRF) microscopy and single particle tracking techniques. Experiments in these systems have focussed on detecting individual replication events and attempting to quantify the number of individual proteins at replication forks (Reyes-Lamothe 2010, Su’etsugu and Errington 2011). More recently, DNA repair events have been detected in bacterial cells by using single particle tracking PALM, allowing researchers to probe residency times of repair factors on DNA (Uphoff 2013). There are clear advantages of using SMLM approaches within this field and as previously mentioned there is now a need to develop SMLM protocols that are applicable to larger eukaryotic model organisms, such as S. pombe.
PALM was designed to increase the resolution of fluorescence microscopy images, in order to visualise biological structures in detail greater than previously achieved.
Furthermore, it has the ability to provide quantitative data on protein stoichiometry as the method relies on seeing one fluorescent molecule at a time. To date many studies using single-molecule based super-resolution approaches have focussed on phenomena paralleled in structural biochemistry which have a repetitive structure or known stoichiometry such as microtubules (Heilemann 2008) or nuclear pores (Szymborska
2013). This acts as an internal control for such experiments as the researcher already knows what structure/stoichiometry to expect. Such ordered repeating protein structures, are not known to exist in the molecular pathways underlying the major DNA metabolism processes. This presented a challenge in that there was no positive control to certify that observations at this new improved level of resolution were not due to artefacts born from sample preparation or post-acquisition image processing.
Most proteins involved in DNA metabolism are typically localised within the nucleus and only transiently interact with the chromatin. These interactions may involve either direct binding to the DNA via specific domains or they may bind to other proteins that are involved in the same pathway. Moreover, the chromatin association of some proteins is often strictly controlled, occurring only during specific cell cycle stages or in response to DNA damage. Regardless of the mechanism of recruitment to the chromatin, it is this event that we are interested in studying during such processes. PALM microscopy presents opportunities to learn more about such recruitment at greater resolution as well as potentially extracting quantitative information on protein quantity.
Standard fluorescence microscopy protocols for visualising spatial organisation of proteins in fission yeast usually involve chemical fixation of the sample with either an aldehyde or methanol. However, in this chapter I will discuss how the use of such fixation methods when attempting to visualise only chromatin-associated proteins in S. pombe is not compatible with PALM. This incompatibility stems from the immobilisation of a freely diffusing sub-population of molecules, thus leading to an inability to distinguish between chromatin bound or un-bound molecules. The realisation of this unsuitability lead to the development of a PALM-based approach that enables the observation and relative quantification of chromatin-associated proteins inside unfixed fission yeast cells.
This technique uses motion blurring of fluorescence via increased detector exposure times as a way of filtering out the freely diffusing population of molecules in the nucleus. This adaptation facilitated the observation of chromatin-associated replication proteins and the quantification of levels of association in different cell cycle stages and distinct genetic backgrounds.