Abstract
This review discusses the exploration of chromosome structure with a recently developed high-resolution microscopy technique, three-dimensional structured illumination microscopy (3dSIM). 3dSIM surpasses the diffraction limit of conventional widefield optical microscopy, increasing the level of detail in images by a factor of 2, while retaining the sample preparation methods, ease of use and flexibility of conventional microscopy. Special attention will be given to the ways in which imaging beyond the diffraction limit can shed light on the structural organization of meiotic chromosomes.
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Electronic supplementary material
The movies available as electronic supplementary material show Z stacks of the maize cells shown in Figure 9 under four different regimes.
Supplementary Movie 1
is the raw data collected by 3dSIM. The stripe pattern created by the diffraction grating is easily distinguished. At each Z section, five phases of the stripe pattern are collected; the grating moves by 1/5th of a period at each shift, so the stripes appear to move smoothly throughout the Z stack. Three concatenated stacks are shown, each with the grating in a different rotational position. (MOV 763 kb)
Supplementary Movie 2
shows the cells after averaging the 5 different phases of the stripe pattern in one rotational position, removing the stripes (and associated high-frequency information), and resulting in an image that is equivalent to conventional widefield illumination. (MOV 4.81 mb)
Supplementary Movie 3
shows the results of deconvolving the image data of Movie 2. While out-of-focus blur is removed and features are more visible, resolution is not noticeably increased. (MOV 1.80 mb)
Supplementary Movie 4
shows the results of reconstructing the raw data into a 3dSIM image stack. For all movies, the Z step distance is 0.125 μm; also see scale bar in Figure 9. (MOV 7.12 mb)
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Carlton, P.M. Three-dimensional structured illumination microscopy and its application to chromosome structure. Chromosome Res 16, 351–365 (2008). https://doi.org/10.1007/s10577-008-1231-9
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DOI: https://doi.org/10.1007/s10577-008-1231-9