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There are those of us who began our careers literally in the dark. Yes, there was a time and not that long ago, that all figures had to be on film. Slide presentations were slides. Micrographs were, well, micrographs on film. Figure creation involved several steps: figures for publications had to be mocked up; then…
When it comes to light sources for microscopy, there is really no such thing as the “best.” The type of light source you use depends on the system you are working with and the type of result that you want. Digital systems are usually designed to work with either a halogen or a LED light…
If you have ever imaged biological samples, you have likely encountered autofluorescence. That pesky background coloration you see under the microscope, which can make it difficult to distinguish your actual signal from the noise.1 When you are trying to look for something as delicate as RNA, you don’t want to be hunting for your signal…
Are you preparing to set up live cell imaging experiments? You’ve got all your cell lines, antibodies, reagents, and protocol ready. You just want to wake up in the morning and enter into that dark room. Well, think again!! As we (I mean the cell biologists) always say, happy cells mean happy life. You have…
Image analysis can be biased. Discover the three main steps to image quantification and learn how to quantify images in an unbiased way.
The only constant with microscopy imaging is variability in both color and image quality. You only need to look at images in journal articles, posters, around your laboratory, or compare your images with a colleague’s—the evidence is staggering. Interestingly, variability doesn’t generally come from the digital camera, rather it comes from our use of imaging…
Scanning electron microscopy (SEM) is a powerful technique, traditionally used for imaging the surface of cells, tissues and whole multicellular organisms (see An Introduction to Electron Microscopy for Biologists)(Fig. 1). While the resultant images appear to be three dimensional (3D), they actually contain no depth information. However, there are several SEM techniques that can obtain…
Do you use biotinylated secondary antibodies in your immunohistochemistry? You could use polymers instead. They are a great time-saving reagent.
Lasers were once called “a solution looking for a problem.” The word—which is an acronym for Light Amplification by Stimulated Emission of Radiation—used to conjure up images of deadly weapons from Sci-Fi movies and TV series. However, their increasing use in everyday life, first in CD players and then in barcode scanners and pointers, have…
Selecting fluorophores can be a tricky business, but we’ve got you covered in this handy how-to guide.
Fortunately for microscopy users, measuring intracellular fluorescence has been made relatively simple through an ImageJ plugin called the Cell Magic Wand. For those of you unfamiliar with ImageJ, it’s a popular image processing program that runs on Mac, Windows, and Linux. How to use ImageJ for measuring intracellular fluorescence First of all, to begin measuring…
Photography has undergone great improvements in the last few decades. In times gone past, photographic film was used. Now most researchers use digital means to capture their images. But not all digital cameras are the same. For optimal results you need to know the different types of microscope cameras and how they work. Before the…
Need a simple, error-proof protocol for using immunohistochemistry to stain your slides? Here’s a protocol to try – from dewaxing to mounting.
It doesn’t matter how excited you are about the research or how intriguing the biology is, if you cannot record it – and record it well – it won’t matter. Here I will tell you how to take the perfect electron micrograph.
Even in the most basic applications, fluorescence microscopy can be a very powerful technique. Simply put, the ability to actually see the biology you are interested in cannot be matched in directness. Often, the aim of fluorescence microscopy is to observe the effect of an experimental manipulation. Ultimately, you would like to know that the…
The concepts of brightness and contrast are so general, and the issues related to them so many, that it may seem strange to have a single brief article with such a title. Indeed, when we speak about brightness, we can think about the brightness of the light source, the aperture and magnification of the lens,…
The Gram stain is another commonly used special stain in the histology lab. Why use a Gram stain? The Gram stain is a type of differential staining technique which represents an important initial step in the characterization and classification of bacteria using a light microscope. It is named after a Danish scientist, Hans Christian Gram,…
IMS is a rapidly advancing technique that nicely blends the best bits of mass spectrometry with microscopic imaging. Learn more about it and how it could be applied to your work.
After countless immunos with free-floating sections – troubleshooting, testing antibodies, and finally doing the actual experiments – I felt like an expert on immunohistochemistry. I knew everything there is to know, right? Well, of course not – it does not work like this in science! For my next project, I would need to perform immunohistochemistry…
Formalin fixed paraffin embedded (FFPE) tissues are valuable samples that typically come from human specimens collected for examination of the histology of biopsies for the detection of cancer. But each sample contains much more information just waiting to be unlocked. Despite the tiny sample size, DNA can be extracted from the tissue sections and used…
When we are doing a double labelling, it makes good sense to choose two fluorophores with spectra widely spaced apart to avoid mixing. Why combine GFP with Rhodamine, when you can combine it with Texas Red?
First of all let me say the technique of labeling tissues (immunohistochemistry, IHC), and cells (immunocytochemistry, ICC) is indeed immunoscience NOT alchemy, though at times it may certainly seem like alchemy! But to scientists inexperienced in this technique, who typically see the results of IHC/ICC experiments in the form of pretty pictures, it can certainly…
We have to rely on artificial systems to test our hypotheses and often have to come up with original set-ups to investigate specific problems. One of these creative inventions is the use of supported lipid bilayers.
Fluorescent tags are widely used for microscopy and expression studies – but it wasn’t so long ago that this everyday tool was unheard of. In this article we’ll talk about how GFP came to be, and what it means for you. Green fluorescent protein, or GFP, was first identified in a fluorescent jellyfish, Aequorea victoria….
The human brain autofluoresces—a funny thought next time you see a cartoon character with a bright idea and a light bulb over his head—but not so funny if you are attempting immunofluorescence analysis. But there are some significant advantages to using fluorescence detection over chromogenic methods. In this article, I will cover the advantages of…
What do you use if antibodies are too large for super resolution microscopy? Aptamers. These are small affinity reagents (~ 2 nm!) that interact with their target in the same way as an antibody, but without the hefty backbone attached.
When you first start out using a microscope, you might only adjust the eye pieces, objectives, and the focus controls. However, you shouldn’t overlook the microscope condensers as they are an important part of the whole optical system of a microscope.
We can, however, use special dyes or engineered proteins to bind to calcium, indicating where in the cell it is. The trick is determining which probe is most suitable to your cells and application.
The resolution of any microscope is related to the numerical aperture of the lens and the wavelength of light used to form the image, and can be calculated using Abbe’s law. This, however, is the ideal situation – the best case scenario. In real life, resolution must be defined in terms of contrast, and there…
Supported lipid bilayers are a very useful tool in many fields of cell and membrane biology. But how easy is it to make them? Bilayers can be made quite reproducibly, once you have found a reliable protocol! However, it can take some time to optimize your technique, so to increase your chances, make sure you…
Are you an assiduous biologist who prefers label-free imaging methods for biological samples analysis? Raman spectroscopy offers you a wonderland of imaging technique with unlimited benefits. To start with, Raman Spectroscopy is a spectroscopic technique based on inelastic scattering of monochromatic light usually from a laser in the visible or near infra-red part of electromagnetic…
While most of us have heard of super resolution microscopy, many of you may not have heard of MSIM, or Multifocal Structured Illumination Microscopy. This under-the-radar imaging technique is relatively quick, cheap (by comparison) and will allow you to get a lot of data, fast. So What is MSIM Anyway? MSIM, as I mentioned earlier,…
Mastering a new topic cannot be done without mastering the vocabulary first. Last month in Illustrated Optical Fiber Glossary (A – E) I got you started. This month I will cover F through M in my Illustrated Optical Fiber Glossary. Fabry-Perot (FP) Generally refers to any device (e.g., laser diode) that uses mirrors in an…
Taking publication quality immunofluorescent images of can be a very time intensive, and frustrating process with hours spent capturing, processing, and putting the images into final figure format. And, if you aren’t careful, you can do a lot of work only to realize later that you need to re-image something for one reason or another….
In Part 1 of these articles, you’ll have learnt about common microscope light sources and how to replace and align these correctly. In this article, we will discuss the importance of Köhler illumination and how to set up the microscope to achieve optimal imaging results. What is Köhler illumination? Before discussing this technique, let us…
Do you want the best imaging experience each time you use a microscope? Well, this is a rhetorical question, as we all desire that these delicate optical instruments are clean, free from immersion oil and correctly aligned. From the routine checking of slides, capturing images for presentations and publications, to diagnosing diseases using point-of-care microscopes,…
Sometimes the biggest hurdle to mastering a new topic is mastering the vocabulary. To help you master optical fibers I put together this illustrated glossary of Optical Fiber Vocabulary. Read this along with my first article “Introduction to Optical Fibers” and you will be well on your way to mastering optical fibers. Acceptance Angle The…
So far in this series I have given you an overview of how STED works and how to design your STED experiments. In this final article, I will tell you how to do two color STED and go over some tips and tricks to acquire the best images from your sample. Dual Color STED Fluorophore…
Through our eyes, seeing is not always believing. Under different lighting conditions, we tend to see the same objects as having the same color. For example, an apple will appear red whether it is lit by daylight or candlelight and a white sheet of paper will be perceived as being white regardless of the light…
Think of the very first time you looked at cells under a table-top microscope. Here’s what you would have done in that experiment: Step 1: Grow cells. Step 2: Plate cells on to a glass/quartz slide. Step 3: Insert the slide under a microscope and look. The protocol for performing single-cell microscopy has a similar…
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