Materials 2001). This can be seen in the

Materials and MethodsHeLa cells placed on glass coverslips suitable for wide-field and confocal microscopy. Cells fixed and stained with Anti-tubulin-Alexa 546, Phalloidin-Alexa 488 and DAPI. For confocal microscopy, a Zeiss LSM880 microscope was used to obtain 3D data set by capturing sequential images at a range of focal depths. For deconvolution microscopy, a DeltaVision Widefield Deconvolution system was employed to collect a z-stack of images. A deconvolution algorithm was utilized iteratively to restore out-of-focus light signal and distortion created whilst imaging. Analysis using ImageJ (FIJI) was then conducted on both data sets. Analysis includes adjustment of brightness/contrast, colour optimisation, RGB image formation, scale bar addition and threshold adjustment. Results and DiscussionThe diffraction of light along the z-axis, required for magnification of sub-resolution objects, leads to the incorporation of out-of-focus light from each image plane being incorporated into the final image. The resultant blur dramatically reduces the contrast and signal: noise (S:N) ratio of the image. Reduced S:N ratio jeopardises resolution which impedes the distinguishability of sub-cellular features. Deconvolution of the data reverses this process and improves these features by reassigning the blurred light (Swedlow, Sedat, & Agard, 1993; Wallace, Schaefer, & Swedlow, 2001). This can be seen in the figure 2 below. Here we see two images of a HeLa cell during metaphase. The image on the left is a widefield fluorescent image whilst the image on the right is the same image with an applied deconvolution algorithm. The deconvolved image has increased contrast allowing the increased visualisation of the mitotic spindle fibres which are much more difficult to observe in the widefield fluorescence image. Moreover, with improved S:N ratio the individual phalloidin fibres within the cell are more distinct and observable. LSCM utilises a pinhole to eliminate out-of-focus light leading to superior axial resolution. This has the advantage of increasing the preciseness of focal sectioning and enhancing contrast (Shaw, 2006). An example of superior lateral resolution can be seen in figure 3 by comparison of the deconvolved (figure 3B) and laser scanning confocal image (figure 3D) of a HeLa cell during metaphase. The spindle fibres are more clearly visible with greater fluorescence in figure 3D, despite being overlaid by the DAPI stain.However, this comes at a price as the detection sensitivity is diminished leading to reduced lateral resolution, a problem especially when dealing with small amounts of fluorescence (Le Puil et al., 2006). Moreover, scanning a confocal laser across the sample pixel by pixel is a slow process and the high intensity of the laser can lead to bleaching of dyes and phototoxicity of live cells (Shaw, 2006). Despite this, the software required for confocal microscopy allows the undertaking of 3D, 4D and even 5D analysis as well as spectral deconvolution and other techniques including FRET and FRAP. Furthermore, a variable pinhole size allows a range of optical section thickness values to be exploited, a feature beneficial to thicker samples. Additionally, the incorporation of a greater number of fluorophores allows more features of a cell to