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MICROSCOPY & IMAGING


of 10-20nm – a process that is repeated for thousands of frames to reconstruct the final super-resolved image,” he adds.


MOLECULAR DISCRIMINATION Amongst other key findings, Padiath explains that, using STORM, his teams’ studies revealed that the A and B type lamins form concentric but overlapping networks, with lamin B1 forming the outer concentric ring located adjacent to the INM. Tese results were consistent across multiple cells lines, such as mouse and human fibroblasts and human He La cell lines. For Padiath, the main advantage of using STORM is its ‘superior resolution,’ with the ‘most surprising’ discovery enabled by the technology being the ‘spatial separation between lamin B1 and lamin A/C at a scale of ~15-20nm.’ Such precise localisation of the two lamin species was achieved primarily by employing aberration-free two-colour super-resolution imaging. Padiath also explains that the use


of STORM microscopy was also important in enabling he and his research team to discover that the more peripheral localisation of lamin B1 is mediated by its carboxyl-terminal farnesyl group – and that ‘Lamin B1 localisation is also curvature- and strain-dependent,


while the localisation of lamin A/C is not.’ STORM was also used to demonstrate that lamin B1’s outer-facing localisation stabilises nuclear shape by restraining outward protrusions of the lamin A/C network. “In summary, we have identified a model for the spatial organisation of the nuclear lamina based on two critical principles: firstly, that lamin B1 forms a looser, outer meshwork facing the nuclear membrane, while lamin A/C forms a tighter, inner meshwork facing the nucleoplasm and, secondly, that lamin B1’s meshwork is more curvature and strain- responsive than lamin A/C’s meshwork, which affects its localisation in tightly curved structures,” he says. “Imaging at two different wavelengths often suffers from chromatic aberration and a complete correction of chromatic aberration is often difficult to achieve, especially when the imaging target is farther away from the coverslip surface. We used the same reporter dye in the activator- reporter dye pairs in two-colour STORM imaging to eliminate any chromatic aberration,” he adds. In the future, Padiath reveals that he and his team plan to make use of STORM to further elucidate the organisation and structure of the nuclear


The STORM microscope used at the University of Pittsburgh


lamina in numerous diseases that are associated with this structure and to ‘understand how the nuclear lamina is altered with age.’ “STORM is best suited for applications


where high-resolution imaging needs to be coupled with molecular discrimination. Te ability to distinguish multiple molecular species by labelling them with fluorophores of different ‘colours’ makes this technique critical in visualiing complex cellular structures at the nanometre scale,” he adds.


STRUCTURED ILLUMINATION MICROSCOPY I


n addition to STORM, another increasingly popular form of super resolution optical microscopy is


structured illumination microscopy (SIM) – a novel imaging technique that uses the patterns of interference – also known as moiré patterns – that emerge when two grids are overlaid at an angle to increase resolution. According to SIM technology provider Nikon, the technique is suitable for used in ‘both fixed and live cell imaging’ – moreover it ‘effectively doubles the resolution of traditional light microscopy and is of great value in structural, developmental, and intracellular communication studies.’


Another company at the forefront


of SIM technology development is German outfit Zeiss, which has recently released the Zeiss Elyra 7 with Lattice SIM, billed as a ‘new platform for fast and gentle 3D super-resolution microscopy.’ As Klaus Weisshart, product manager at Zeiss Research Microscopy Solutions, explains, Lattice SIM expands the possibilities of structured illumination microscopy by ‘illuminating the sample with a lattice pattern rather than grid lines’ – in the process giving higher contrast and allowing a ‘more robust image reconstruction.’ “With Elyra 7 with Lattice SIM, scientists can use a two times’


higher sampling efficiency to lower the illumination dosage to observe fast cellular processes in super-resolution. High image quality is maintained even at high frame rates,” Weisshart adds.


The sample is illuminated with a lattice pattern


www.scientistlive.com 55


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