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Microscopy & Imaging

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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Lasers for Confocal Microscopy

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…

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A Biologist’s Guide to Choosing Your Fluorophore Palette

You may notice that nature is full of vibrant, even fluorescent, colors. The human eye detects wavelengths ranging from 390-700 nm and our perception of colors is actually a narrow part of the light spectrum. Other organisms can detect color from a wider spectrum. Why do colors exist? Arguably, colors are communicative, from tropical fish…

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A Simple Method for Measuring Intracellular Fluorescence

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…

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Microscope Cameras: From SLR to CMOS Devices

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…

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Immunohistochemistry: Getting The Stain You Want

Immunohistochemistry (IHC) is a vital tool, not just at your wet bench but also in clinical labs the world over. IHC is used extensively in hospitals and veterinary practices in grading and studying cancers, and the results may determine what treatment a patient gets – including the controversial Herceptin! But it’s also pretty useful in…

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Getting Started with Raman Spectroscopy: What You Need to Know

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…

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A Beginner’s Guide to Haematoxylin and Eosin Staining

Once a tissue specimen has been processed by a histology lab, and transferred onto a glass slide, it needs to be appropriately stained for microscopic evaluation. This is because unstained tissue lacks contrast: all of the fixed materials have a similar refractive index and a similar color. If you viewed an unstained tissue section under…

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Illustrated Optical Fiber Glossary (F –M)

Mastering a new topic cannot be done without mastering the vocabulary first. Last month in Illustrated Optical Fiber Glossary (F – M) 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…

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Tips for Taking Immunofluorescent Images for Your Next Paper

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.…

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Brightness and Contrast in Microscopy Imaging

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,…

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