Lauren Gagnon describes how single-molecule localisation microscopy is advancing research across biological fields
The light microscope is the most common microscope found in the laboratory; it works using visible light and a system of lenses to generate a magnified 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. These techniques can surpass the diffraction limit and generate higher quality images of biological specimens.
Types of super-resolution microscopy
There 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 diffraction-limited spot by using point spread function engineering to achieve a five- to tenfold resolution improvement. SML, on the other hand, offers 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. The data from SML allows spatial information to be abstracted to generate an image below the diffraction limit. SML microscopy can be used in a wide array of scientific fields, a few of which are discussed briefly below.
Single-molecule localisation microscopy in genomics
In genomics, SML can be used to study the functional organisation of the genome. The 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 differences is helping facilitate research toward future medical diagnoses and treatment. However, work within this field has been hampered by the resolution limits of conventional light microscopy. By enabling the generation of highly detailed images using fluorescent methods, super-resolution approaches such as SML microscopy have overcome these limitations.
Chromosomes are a collection of proteins and DNA that store genetic information. The 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 differences in the structure of chromosomes resulting in differences 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 microfluidics assays is allowing more targets to be sequentially labelled, and is thus becoming more widely used in research.
Single-molecule localisation microscopy in neuroscience
Neuroimaging tools, such as SML microscopy, can be used to answer complex questions regarding neurological and psychiatric disorders. Answers to these questions can lead to the development of innovative strategies to prevent the clinical manifestation of devastating conditions.
The high resolution and magnification used by SML microscopy can produce images of extremely small neurological structures; clear images of these structures would be impossible with any other microscope. One such example is the use of SML microscopy to label the synapses in Caenorhabditis elegans. This has allowed scientists to study and understand the relationship between the membrane-bound calcium sensor and endosomes in endocytosis. Only SML can distinguish these domains, since the synaptic structures themselves are often smaller than the diffraction limit of light. SML has also been used to determine the spatial distribution of the vesicular monoamine transporter 2 (VMAT2) pre- and post-drug treatment in the rat brain tissue sections.
Single-molecule localisation microscopy in oncology
Cancer research and treatment has advanced greatly over the past few decades. However, it is an extremely complex and heterogenous disease, and there are many different types of tumours – many we know little about. Preclinical and clinical research on potential drugs is needed to determine potential toxicities, confirm the efficacy and establish any off-target effects. Imaging technologies can be employed to study the effects of treatments in preclinical models and uncover key tumour signalling and progression mechanisms.
For example, SML has been used to investigate HuH7 – a hepatocellular carcinoma cell line – using markers for the endoplasmic reticulum via an RNA binding protein that labels stress granules. Furthermore, genomic imaging is a powerful tool to study cancer biology, as structural changes within the genome are associated with the heterogeneity of cancers.
Single-molecule localisation microscopy in cardiology
In the USA, approximately 647,000 people die from cardiovascular disease per year. Imaging technologies are used to provide detailed insights into the cardiovascular system, driving the development of diagnostic and treatment strategies. The high resolution and low magnification offered by SML microscopy has helped solve many complex questions within cardiology research.
40% of patients suffering from heart failure develop delays in ventricular electrical activation, which can result in ventricular dyssynchrony – the difference in timing or synchronisation of ventricle contraction. The downstream effect of this may be cardiac death. However, if detected, dyssynchronous heart failure can be treated with cardiac resynchronisation therapy (CRT). This therapy can resynchronise the ventricular mechanical and electrical activity, reducing mortality in patients. SML microscopy has been used to investigate the sarcomeric organisation pre- and post-CRT treatment using α-actinin as a marker to reveal the sarcomeric structures.
Single-molecule localisation microscopy in developmental and cell biology
Disease modelling and the development of future treatment resources relies on the fundamental knowledge gained from exploring the inner working of cells, along with understanding the intricacies of animal and plant development. Biological imaging produced by SML microscopy can be used to track individual biomolecules or observe biological processes. Samples are labelled with organic dyes, proteins or quantum dots, which enables the SML microscope to image single fluorophores at high speed and in three dimensions. This process is highly advantageous for any scientist wanting to capture live biological motion, which is difficult with many other microscopes.
This is just a short overview of some of the many emerging applications where super resolution microscopy in general, and single-molecule localisation in particular, are helping researchers to advance their understanding of complex structures.
Lauren Gagnon, PhD, is an application scientist at Bruker
FIG.2. Courtesy of Sean Merrill and Dr Erik Jorgensen, University of Utah.
FIG.3. Courtesy of Leremy Colf and Dr Wes Sundquist University of Utah