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Microscopy is one of the fun parts of working with yeast. If you fix your cells, you can get a snapshot of the structures. Live cells microscopy using fluorescent proteins tagged proteins is even better, as you can see the dynamics and cell machinery working before your own eyes. Light Microscopy to Check for Contamination…
Using fluorescent proteins as imaging probes is a widespread and versatile technique in microscopy. You can use them in a wide range of living systems, from single cultured cells to complete organisms and animals. Fluorescently tagged proteins can be used to track and examine real-time localization, interactions, and translocation of your protein of interest, as…
In part 1, “The Power of STED Microscopy: How Does it Work?” I covered the basics of STED Microscopy: how it works and why you might want to use it. If you now want to do STED Microscopy, the next step is to optimize your sample. In this article, I cover how you can get…
If you want to see in real time what is going on inside your cell then you should be performing live-cell imaging. Live-cell imaging techniques allow real-time examination of almost every aspect of cellular function under normal and experimental conditions. With all live-cell imaging experiments, the main challenges are to keep your cells alive and healthy…
You are probably familiar with fluorescent in situ hybridization (FISH) to detect and localize the presence or absence of specific DNA sequences on chromosomes. But did you know there are numerous FISH experiment variations? Including high-resolution FISH and quantitative FISH? Read here about Fiber-FISH, Q-FISH, and Flow-FISH and decide if you would like to undertake one of…
Would you eat your spaghetti dinner without a plate? No, of course not! It would make a big mess and be ugly to look at. Instead you NEED something to put that spaghetti on, to contain it, to keep it clean, to make it look nice – a bowl, a plate, SOMETHING! By the same…
Discover the different approaches to FRET quantification and get tips for selecting your FRET pairs.
There are about as many protocols to prepare coverslips as there are ways to make tuna casserole. You can spend from 5 seconds to 2 days, depending on what your lab prefers. But in the end, what’s really needed? I’ve tried many protocols over the years and I’ve questioned some steps. Here I share my…
If you want to make molecules stick together you need to know about streptavidin/biotin. This article follows on from Mike’s article looking at ‘sandwich’ and ‘amplification’ methods of immunohistochemistry (IHC) and covers how streptavidin-biotin works in IHC, including protocols. Streptavidin-Biotin What is it? Avidin is a natural biotin-binding protein found in egg whites. Streptavidin is similar…
Have you ever been shown how to use a microscope properly? Or do you just dive right onto the microscope with little or no training and scant knowledge of the basics, then twiddle knobs, snap photos and expect the publication-quality images to appear? If it’s the latter you are certainly not alone! If only there…
When you fix your tissue samples with paraformaldehyde (PFA) the proteins in your sample become covalently cross-linked. This is good to preserve the ‘architecture’ of your tissue sample. However, this cross-linking can become a problem when you carry out immunohistochemistry (IHC). Cross-linking can ‘mask’ or hide your antigens-of-interest and make them ‘invisible’ to your IHC…
Mercury burners are one of the most common light sources used in fluorescence microscopes, producing a wide spectrum of wavelengths making them a great light source for viewing your samples. But they do have their issues – one of which is their propensity to break or become damaged if not used and cared for correctly, which…
Do you suspect that your favourite protein is doing something really cool? But you cannot see it because your confocal microscope’s resolution is limited. Then Stimulated Emission Depletion (STED) microscopy is what you need! With the power to smash through the diffraction limit of confocal microscopy, STED opens up a whole new world of improved…
Colocalization blues (and reds and greens) Trying to find if and where two epitopes co-localize (or, to be more precise, where they are found in close proximity) may seem easy at first: 1) Bind your two epitopes with primary antibodies from two different species, 2) bind these primary antibodies with two secondary fluorescent antibodies, one…
Yup, we really are in the digital age…even the pathology is digital. Facebook, Instagram, Tumblr. It has never been easier to take pictures and share them. The digital revolution is upon us and nothing is safe, even your pathology samples. Digitizing your pathology samples can help you better organize and manage pathology for later. And…
Single-molecule localization techniques are another way to achieve super resolution. Discover how the different variants work, and get info on what researchers are doing with them.
You are finished with your super-arduous experiment. Now the fun part! To “read” your results with a microplate reader, but do you know which microplate reader to use? Or even that there are different types? Have no fear. I am here to help ye, scientific one! Read on for my pointers on picking the right…
The magnification and viewing of samples using a microscope relies on both the objectives and the eyepieces working harmoniously together. If you buy a ready-to-use microscope, then the objectives and the eyepieces which are fitted as standard will be designed to complement each other. On the other hand, if you are designing and building a…
Super resolution microscopy shattered the lower resolution limit offered by traditional confocal microscopy, enabling us to see more detail. Discover the different super resolution techniques, how they work, and what they are being used for.
Two different sensors are generally used in cameras for microscopy: Charge Coupled Devices (CCD) or Complementary Metal Oxide Semiconductors (CMOS or sCMOS). Although there are a number of similarities between the two sensors, differences in the way they function can have an effect on image capture time as well as signal-to-noise ratio. Let’s take a…
In the first part of this article (you can read it here), we looked at clipping and saturation in terms of microscope images, followed by a definition of Dynamic Range and an introduction to Bit Depth. Intrascene Dynamic Range The dynamic range which can be detected at the same time in the same field of…
Ever tried to turn the volume all the way up on a small radio or small stereo system? (Hopefully you have not tried it with earphones in!) Notice how, after some point, the sound didn’t get any louder- it just got more distorted? That’s because you’ve hit the ceiling of your machine’s dynamic range. It’s…
If you want to visualize elastic fibers in your sample, you need to use Verhoeff-van Gieson stain. Find out more about this stain, including how to use it.
“A two photon microscope has higher sensitivity than a normal confocal microscope, because it uses two photos instead of one!” Yes, I can bear witness that this phrase has actually been uttered, and it was not by an undergraduate student. No exception to the rule The condensation of various levels of misunderstandings in this statement…
If you remember from one of my previous articles (if not, you can read it here!), we introduced ‘fluorophores’. These are basically substances (natural or synthetic) which have the ability to absorb light at a low wavelength and re-emit at a higher wavelength. In other words- they fluoresce! In this article, I’ll introduce the three…
The issue of mouse-on-mouse background is only a cause for concern for the histotechnologist working within a research environment. Those working in a diagnostic setting will probably never experience this as they will be working with human tissue with antibodies raised in a variety of species- but one species that won’t be used is human!…
You may already use fluorescence as a tool in your microscopy and imaging work, but, do you know exactly what it is? Why are certain proteins and probes fluorescent? What causes this light emitting property? We’ll have a look at these and more questions in this article. Start with a definition We’ll start with a…
Like most things in this world, fluorophores are mortal, and eventually your once bright fluorescent image will inevitably fade to black. This fading or ‘photobleaching’ of fluorescent signal can make imaging difficult, especially if you are trying to take quantitative images. Read below to learn what causes photobleaching of your fluorophores and how best to…
We all know that stars are easier to see against the dark background of the night than they are to see against the bright sky of day. Well did you also know that the same may be true of your microscope specimen? Dark field microscopy is a popular microscope technique that makes your unstained transparent…
Although his name could fit in easily to the early 1980’s Hip-Hop Scene, Jerzy Nomarski (or ‘George’) was actually a Polish physicist with an interest in optical theory. Born in 1919, he eventually became a member of the Polish Resistance fighting in the Second World War. He was captured by enemy forces and held as…
While confocal microscopy seems to have become pervasive in cell biology, widefield microscopy techniques still have a special and important place. This month on the Microscopy and Imaging Channel, we’re focusing on widefield microscopy techniques: covering the basics of what these techniques are and when you should turn to them. What is this ‘Widefield’ you…
Phase contrast microscopy is a light microscopy technique which is primarily used to visualise live cells. Using various filters and condensers, the image produced by phase contrast allows us to see greater detail in live cells and can highlight aspects such as intracellular structures. Keep your cells alive! The best way to view cells is…
Discover the magic of toluidine blue – a polychromatic dye that changes color depending on which tissue component it is staining.
Get introduced to some of the special stains for histology and learn some top tips for getting great results.
It’s 11 PM…do you know where your samples are? If you dread the thought of having to hack through ice, snow and the bone-chilling depths of every freezer to locate them, flirt with frostbite only once. Better yet, never hunt for missing lab samples again! Here are some useful ways to keep track of every…
Looking for paraffin alternatives for tissue embedding? Find out the benefits of cryo and resin tissue embedding and how they work.
The lac operon is an amazing tool in molecular biology. It has been used for decades to turn on protein expression in an inducible manner with IPTG. The result is synthesis of vast amounts of protein to be used as you wish. While the lac operon is an amazing tool for protein production, it is…
Most of the microscopes you will encounter in your laboratories will be ‘upright’. In other words, they are assembled (from top to bottom) in the order of; eyepieces, objectives (on revolving nosepiece), stage, sub-stage condenser, diaphragm and base. However, there are two other types of light microscopes you will perhaps encounter (and use) and it…
Counterstaining can have a big impact on your histology result. This short guide will introduce you to some available counterstains providing you with a few more choices.
Achieving a good immunohistochemistry signal-to-noise ratio involves many factors, including a good blocking protocol. Read on to learn about blocking non-specific staining in IHC.
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