Welcome to the BitesizeBio Microscopy and Imaging Channel!
Every week we will be publishing articles revolving around the central topics of light microscopy and imaging. We will cover aspects such as the fixation of specimens, sectioning and preparing slides, staining and immunohistochemistry, which method of microscopy to use (and how to ensure you get the best possible images), confocal microscopy, cameras and image analysis. This is the place where you’ll be able to find advice on the most essential basics right through to the latest developments in the field. It is our intention to build an invaluable web resource to help beginners and experienced microscopists alike to find their way through the complex world of light microscopy in order to build their own expertise.
The countless developments which have seen optical imaging advance over the last two hundred years highlight the great importance of this technology. From Leeuwenhoek’s studies of biological samples in the 17th Century, through to the development in optical imaging, and the wide use of related methods, means that we are now able to use the latest cutting-edge light microscopy systems to track single molecules in both space and time. Whereas many molecular technologies such as proteomics, molecular biology or biochemistry provide very limited or no spatial and temporal information, the ultimate goal for biomedical research is to understand how biological events take place in the context of the intact cell, organ or whole organism at high spatial and temporal resolution. Imaging techniques can deliver exactly this and enable biomedical researchers to address increasingly complex questions such as: where exactly do certain biological events happen in the cell or whole organism? What is the morphological structure of the cellular environment? How do these temporal and functional sequences of events occur in vitro and in situ?
Scientific imaging includes a wide range of technologies exploiting the use of light, electrons, X-rays and sound waves in order to analyse biological specimens from the size of large mammals down to the molecular or atomic level. Light microscopy is used to generate image data achieving a spatial resolution from millimetres down to the conventional diffraction limit of approximately 200 nm as described by Ernst Abbe in 1873. Technical developments in recent years have helped to circumnavigate this limit posed by the diffraction of light and to push the spatial resolution of optical systems down to approximately 20 nm and beyond. With the improvement of scanners and detectors, it is now possible to image specimens over a number of weeks and down to the temporal resolution of milliseconds or several hundred frames per second. The introduction of fluorescence labelling techniques, both in vitro and in vivo, marked another huge leap in light microscopy applications. By utilising specific chemicals, or fluorophores, which are activated by different wavelengths of light, we are now able to specifically detect single molecules at a greatly increased image contrast. The use of fluorophores which are excited by longer wavelengths of light has improved the limitation of sample penetration of light microscopes into biological samples by the order of a magnitude making it now possible to acquire optical sections of up to one millimetre into tissue samples. Further advances in all areas of light microscopy provide a constantly growing research tool kit, and within this Microscopy and Imaging Channel we will also highlight the most important new developments in this field.
To use optical imaging technologies efficiently and accurately, it is important to understand the whole sequence of necessary technical steps, which goes far beyond the ability to simply operate a light microscope and generate pretty pictures. This starts with a comprehensive experimental design and includes specimen maintenance and preparation, labelling techniques, the choice and operation of the suitable light microscope system(s), handling, storing and processing of image data and it ends with the quantitation, interpretation and visualisation of the generated image data. All these steps need in-depth expertise in a wide range of procedures- all of which are necessary to make complex imaging experiments into successful and robust experimental routines which allow the output of high content data. It is important, particularly for beginners, to describe how the wrong use of optical imaging techniques can quickly result in the generation of false data. For instance, just the use of a single inappropriate emission filter whilst recording data for a standard two-channel co-localisation analysis can lead to channel cross-talk and as a consequence to a wrong result. If you are new to microscopy and didn’t fully understand the last sentence, then stay tuned!
Finally, we will be providing information on where to find, and how to use, advanced imaging and sample preparation equipment which, although you might not have available in your own institute or campus, may be accessible via scientific collaborations or through using the many imaging facilities available worldwide. The general guidelines and advice which we will publish here each week are designed to help you to generate your own procedures and protocols, which should be optimised for use in your own laboratory. If procedures or techniques are being perceived differently or controversially in the light microscopy community, we will try to highlight this and give you all different views. Although there will be a general structure to this Channel, we are not limited by a rigid list of topics. Therefore, if you have any specific aspects of interest which you would like to learn more about, then please let us know and send us your feedback.