Microscopy and Imaging

Ever wake up especially groggy in the morning, finding it takes a few minutes and a few eye rubs to be able to decipher the numbers on your alarm clock? Our eyes have the ability to resolve an image, so that you can observe separate objects and details. Similarly, microscopes have a parameter of resolution:…

In the same way that you should ‘Think Before You Fix’, the choice of embedding media should be dictated by your required end-point. The basic principle is that by processing tissue into an embedding medium you harden the tissue and provide support protecting it from the mechanical forces associated with sectioning. Parma ham and steak…

Fluorescent-based microscopy techniques are some of the most common ways to visualize biological structures. Almost any protein- or nucleic acid-based molecule can be tagged with a fluorescent marker or dye and subsequently detected by a light microscope. This fluorescence is seen as a bright object against a black background, allowing for intense and clear images. …

Dichroic Mirror/Filter This is a semi-reflective filter which can also be referred to as ‘dichromatic beam splitter’. Unlike the Longpass filters which absorb light which is not transmitted (see Part 1 of the Glossary), these filters reflect light at lower wavelengths and transmit light at wavelengths above the ‘cut-on’ wavelength. As beam splitters, they are…

Brightfield Illumination This defines the most basic method of optical microscopy using white light to illuminate the sample in the transmitted mode. Absorption and diffraction of the light by the molecules in the specimen generates the contrast in the image. Methods such as darkfield illumination, differential interference contrast and phase contrast help to increase the…

Don’t be left in the dark! Navigating through the sea of parameters that can aid or abet successful microscopy can perhaps seem daunting. It may be tempting to assume you can just sit down at a microscope, look at your sample, and obtain beautiful images simply by hoping for the best. However, even a basic…

It’s unclear exactly how the term ‘Special Stains’ first arose in the world of histology, but it refers to empirical and histochemical staining techniques that significantly contributed to the advancement of histology in the late 19th century. In a nutshell, these stains are ‘Special’ because they are not routine – simple as that. Therefore, Special…

You’ve spent days, perhaps weeks or months squirrelling away tubes of preserved tissue in the dark drawers under your laboratory bench like the trophies of a demented serial-killer. Hours have been spent in histology in the processing, embedding and sectioning onto slides. Finally, like a warrior victorious in battle, you hold aloft your thin glass…

I recently introduced you to the concept of polarising microscopy. Naturally, if evaluating refractile material is an everyday part of your research, it is definitely worth investing in a professional polariser modification for your microscope. But if you only use a polariser occasionally, this might not be the best use of your lab’s money. In…

When you stop to think about it, tissue slices for immunohistochemistry (IHC) undergo quite a lot of handling. From chemical reactions to washes – even manipulations and transfers between baskets and microtubes – final analysis is often hours away from the initial step of taking a tissue slice. Properly fixed tissue has to be robust…

Polarising microscopy involves the use of polarised light to investigate the optical properties of various specimens. Although originally used predominantly in the field of geology, it has recently become more widely used in medical and biological research fields too. Polarising light microscopy is a contrast-enhancing technique to allow you to evaluate the composition and three-dimensional…

Although the microscope is probably the most commonly used biological instrument, it is frequently used improperly. The rate-limiting step to getting high quality microscopic images is illumination of your specimen. When you examine a specimen under the microscope, the intensity and distribution of light must be clear and equal to enable you to evaluate all…

If you’re starting your PhD or post-doctoral work, chances are you’ll need to use a light microscope at some stage during your research. Some of you may be seasoned microscopists. For many of you though, this might be the first time you’ve ever plugged in a microscope, or at least the first time you’ve used…

For a long time we’ve been able to pinpoint the subcellular location of proteins, and the advent of FISH (Fluorescence in situ Hybridization) allowed us to locate the position of genes in the nucleus, but recent advances in RNA FISH are making it easier and easier to collect the same data about individual messenger RNAs….