Microscopy and Imaging

Following on from the first part of the H and E 101 articles, here are the materials and recipes you’ll need for your own H and E workstation (assuming you don’t have access to a histology lab). Many of the chemicals listed below are toxic and/or harmful. Use PPE when handling/storing, follow SOP’s in your…

Haematoxylin and Eosin staining is the most common staining in the modern (and old!) histology lab. This staining technique gives an overview of the structure of the tissue and can be used in pathological diagnosis. This article follows on from Nicola’s introduction, but we’ll take an in-depth look at the stains, chemistry and method to…

Fixing suspension cells for imaging can be trickier than fixing adherent cells, as they can’t be cultured on a coverslip. Discover how you can stick them down with the help of centrifugation.

You just can’t put raw tissue or cell samples on your slides and expect good histology results! Instead you must preserve or ‘fix’ your samples. Fixing ensures that your cell structures stay intact and that your antigens are immobilized. Ideally, fixation would also still permit unfettered access of your antibodies to your antigens. However, as…

The theory behind the idea of having shared microscopes is a good one, but, in reality, this can sometimes mean you have to put up with the dirty habits of your fellow scientists and researchers. And some of your lab mates turn out to be really mucky! Here’s my Top 10 of things which really…

“Do we use a monolayer or 3-D cell culture for our experiments?” A simple, yet puzzling question asked by my group leader while I was working on a drug development project at the University of Abertay Dundee. How do you answer such a question? Just read on to find out! Monolayer vs spheroid One of…

Have you ever isolated a great little population of cells after days or months of trying, got truly excited about doing some immunofluorescence with them only to find out (at the very end) that all your cells washed away?! If this has happened to you, then look no further; we will introduce you to some…

If you found our previous section on super resolution interesting, you may be curious for a more detailed explanation behind some of the techniques. Introduction to this counterintuitive method Of the super-resolution microscopy techniques, structured illumination microscopy (SIM) is arguably the most counterintuitive to grasp. Of course, that’s what makes it so much fun! To understand how…

Do you see what I see?  Maybe not, if the microscope is wrecked in one of these ten ways when you… 1. Carry the microscope incorrectly. A death-grip on anything but the arm and the base almost guarantees that it will slip away, crashing onto the floor to break in pieces. You don’t want a microscope which…

Do you know what your histology fixatives are really doing to your samples? Read on to learn what happens to tissue treated with two common fixatives.

Following on from Part 1, we’ll now take a look at the actual use of geometric probability in stereology, as well as the advantages and disadvantages of this technique. As you’ll know from the introduction (here), stereology looks at the relativity between an object and its sections, while geometric probability allows a researcher to expand…

The study of geometric probability in stereology may seem like a large, overwhelming area of focus but, don’t panic, it’s not! In fact, it is a very specific field that is likely to come easy to those that excel in mathematics or science (yes that means you). However, the concept is not one that most…

In Part 1, we looked at diffraction limits and how these can be overcome using Super-Resolution Microscopy techniques. We covered Single Molecule Localization Techniques, Structured Illumination Microscopy and Stimulated Emission Depletion. In this second part, we’ll take a look at Near-Field and Dual Objective Methods as well things to consider before buying a system. Near-field…

So you want to do meiotic spreads do you? Maybe it’s to check if meiosis is progressing the way it should, or even to look for sites of DNA damage. Whatever the case this technique can be a little tricky at first, but once you learn a few tricks it’s like riding a bicycle- so…

If you’re reading our Microscopy and Imaging Channel here on BitesizeBio, you might have heard about the new techniques which fall under the umbrella of ‘Super-Resolution Microscopy’. In her recent article, Cynthia introduced us to the limits of resolution and how this can be overcome. Question time You may have more questions; What are the…

Why do I see a cloud? When you are looking at protein localization within a cell, have you ever wondered why you see a cloud of fluorescence rather that several individual fluorescent points? Well, light microscopy has a theoretical resolution limit of 200 nm. This means that in theory, to resolve two points as being…

Gomori’s methenamine silver is a special histology stain for detecting fungi. Find out how and why you might want to use this stain in the lab.

In my previous article I covered different immunohistochemical staining techniques at a superficial level. In the following articles I will start to explain these technologies in a bit more detail and in which situations they should be applied. All of the following will involve additional stages when applying them, for example- serum blocking, protein blocking,…

According to the International Society for Stereology, the area of scientific study encompassed by this term is that which analyzes solids. If that all sounds a bit too much like materials science, then for us microscopists, it’s really about the review of three-dimensional objects (mainly tissues) by making horizontal and vertical incisions. Stereology can be…

The last two decades have seen a dramatic increase in the number of publications using immunohistochemistry (IHC) as a research tool to identify the spatial location of proteins of interest within cells, tissue sections and whole-mount preparations. Grinding and binding The advantages over ‘grind and bind’ methods are apparent, but the very best results will…

Need to stain Gram-negative organisms? You should consider the Warthin-Starry stain.

In Part 1 of this Guide, we learned about the importance of support, resources and objectives when choosing a fluorescence microscope. We continue this guide by looking at everything from filters to warranties. You should get these filters… Quality of the barrier filters and dichroic mirrors are the deciding factor on how well you can…

You’re a senior postgraduate student, a post doc or a junior PI with little knowledge on microscopes and someone between Senior PI /Dean level approaches you with this: “We have now the funds to buy the fluorescence microscope someone once told me we need. Can you handle this please? Oh, by the way, the funds…

Coating (or ‘subbing’) slides for immunohistochemistry can be the difference between having an organized set of tissue slices ready for microscopy- or watching them detach and float away during a wash. It takes a lot of time to place tissue slices in correct anatomical order, aligned right-side up and flat. To the naked eye, all…

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…