MICROSCOPY & IMAGING
APPLICATIONS OF SUPER-RESOLUTION MICROSCOPY
Lauren Gagnon describes how single-molecule localisation microscopy is advancing research across biological fi elds
T
he light microscope is the most common microscope found in the laboratory; it works using visible light and a system of lenses to
generate a magnifi ed image of an object. However, light microscopes can be limited by low resolution; this can produce poor images and can cause intricate and potentially critical details of the specimen to be overlooked. Super- resolution techniques, such as single molecule localisation (SML) microscopy, have been created to overcome some of the key challenges associated with light microscopy. T ese techniques can surpass the diff raction limit and generate higher quality images of biological specimens.
TYPES OF SUPER-RESOLUTION MICROSCOPY T ere are three main types of super- resolution microscopy; structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy and SML microscopy. SIM uses Moiré patterns and Fourier transforms to create a twofold
improvement on the resolution. STED reduces the size of the diff raction-limited spot by using point spread function engineering to achieve a fi ve- to tenfold resolution improvement. SML, on the other hand, off ers the best improvement in resolution along with quantitative and statistical analysis capabilities. SML microscopy takes a movie of an image, allowing information to be gathered from the image over time. T e data from SML allows spatial information to be abstracted to generate an image below the diff raction limit. SML microscopy can be used in a wide array of scientifi c fi elds, a few of which are discussed briefl y below.
SML IN GENOMICS In genomics, SML can be used to study the functional organisation of the genome. T e organisation of the genome within the nucleus can provide a lot of information on cellular pathology. Particularly it can help distinguish between pathological and non-pathological states. Understanding these diff erences is helping facilitate research toward future medical diagnoses
FIG. 1. In situ visualisation of the 3D structure of a chromosome. Each colour
represents a diff erent DNA labelled region on the maternal and paternal homologues
and treatment. However, work within this fi eld has been hampered by the resolution limits of conventional light microscopy. By enabling the generation of highly detailed images using fl uorescent methods, super-resolution approaches such as SML microscopy have overcome these limitations. Chromosomes are a collection of proteins and DNA that store genetic information. T e three-dimensional (3D) organisation of chromosomes regulates the expression of genes. Since the function of a chromosome depends on its 3D structure, it is important to image chromosomes as pathology can cause diff erences in the structure of chromosomes resulting in diff erences in gene expression. Using SML, a 3D image of a chromosome can be generated using specially designed oligonucleotides to label chromosomes. Unfortunately, the number of targets imaged is limited by the number of probes that can be labelled, therefore combining SML with techniques such as microfl uidics assays is allowing more targets to be sequentially labelled, and is thus becoming more widely used in research.
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