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Microscopy and Imaging

Detecting Cell Apoptosis on Tissue Slides

Apoptosis, or programmed cell death, is a physiological process in which individual cells are set to die without harming their environment. It involves a cascade of complex and tightly regulated cellular events. Detecting apoptosis on tissue slides, involves either detecting the molecular participants of these events, or the morphological changes that occur on the cellular…

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Automated Microscopy

The traditional microscope that you know and love is operated manually. Picture the scene: the microscopist chooses the light source, gently places the sample the moveable stage, selects the objective lens, and scans to select the field of view. This process is perfect for processing and analyzing a small number of samples per day. But…

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Sample Preparation for Scanning Electron Microscopy

Proper sample preparation plays an important role in obtaining the required information when using scanning electron microscopy (SEM). You need to consider the sample’s size, shape, state, and conductive properties prior to sample preparation. Ideally, the smallest representative sample size is the one to use. The microscope’s detection capacity is as much as 1µm from…

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In Situ Zymography: Let’s Catch that Enzyme in Action!

In situ zymography (ISZ) is the best choice to study proteases. Proteases are a challenge to study as proteases are extremely potent enzymes. As such, they need to be controlled at multiple levels to prevent them from being unleashed and making a cellular mess. Regulation of their activity occurs at virtually all levels: transcriptional control,…

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Automated Cell Counting with a Fluorescent Twist

Cell counting is the bane of existence of many researchers. Countless hours spent in front of the microscope with a haemocytometer on the stand and a manual tally (or “clicker”) in hand can be really daunting. Not to mention that no one will ever double check your count if you don’t take a picture. Those…

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Analyze Immunostained Slides with Semiquantitative Scoring

A  routine task in the lab is to investigate the presence of your favorite protein in a range of histological samples. No doubt, staining your tissue sections using good old immunohistochemistry (IHC) would be your first choice. You just got to love a technique that has celebrated its 70th birthday, and is still used in…

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Data Analysis for Three-dimensional Volume Scanning Electron Microscopy

In recent years, three-dimensional (3D) scanning electron microscopy techniques have gained recognition in the biological sciences. In particular, array tomography, serial block face scanning electron microscopy (SBFSEM) and focused ion beam scanning electron microscopy (FIBSEM) (described in Three-Dimensional Scanning Electron Microscopy for Biology) have shown an increase in biological applications, elucidating ultrastructural details of cells…

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Can You Stand the Cold? Cryosectioning for Beginners

Before you can perform histology on your tissue samples – you need to prep them. This means you must fix them, embed them and section them into thin slices for analysis. A great way to slice your tissues is cryosectioning. But cryosectioning is not so great when it your tissues melt, fold, curl, wrinkle, tear,…

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Microscopy – a Numbers Game

While the microscope is synonymous with biology, it is a child of physics and technology. When we learn about the microscope we learn physics—specifically, we learn about optics. Many great resources are available that explain the inner working of microscopy. And, like most things in physics, the inner working of microscopes comes down to a numbers…

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Halogen vs LED Lighting in Digital Microscopy

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…

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Challenges of Autofluorescence in Neuroscience

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…

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How to Maintain Live Cells on a Microscope Stage

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…

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How to Quantify Images in an Unbiased Way

Microscopists like to say that seeing is believing. The so-called belief in this context is a product of highly experienced imagination, which raises a concern about the objectivity. To remain objective, we try to use analytical approaches to quantify images and turn phenotypes into numbers. Many image analysis approaches exist nowadays, from simple manual measurements…

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Controlling Color Image Quality in Microscopy: Start at the Beginning

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…

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Quick and Easy Automatic Cell Counting

Are you wondering how on earth you’re going to count thousands of cells across a stack of images? Well, I’m going to show you a simple method for automatic cell counting with ImageJ. For those of you unfamiliar with ImageJ, it’s a popular image processing program that runs on Mac, Windows, and Linux. Assuming you…

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Three-Dimensional Scanning Electron Microscopy for Biology

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…

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Polymers as Secondary Antibodies for Immunohistochemistry

Have you ever thought about using polymers for your immunohistochemistry experiments? Take this simple quiz and find out if polymers may be right for you: I order biotinylated secondary antibodies for immunohistochemistry. Yes/No I use avidin/biotin complexes in my staining protocol. Yes/No I have to block endogenous biotin in my tissue samples to avoid heavy…

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