Microscopy – a Numbers Game

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…

The Art and Science of Figure Creation:  Think BIG to see Small

The Art and Science of Figure Creation: Think BIG to see Small

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…

Challenges of Autofluorescence in Neuroscience
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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…

How to Maintain Live Cells on a Microscope Stage

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…

Controlling Color Image Quality in Microscopy: Start at the Beginning
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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…

Three-Dimensional Scanning Electron Microscopy for Biology
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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…

Lasers for Confocal Microscopy

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…

A Simple Method for Measuring Intracellular Fluorescence

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…

Microscope Cameras: From SLR to CMOS Devices

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…

Resolving your Noise Complaints – the Basics of Signal/Noise Ratio in Microscopy

Resolving your Noise Complaints – the Basics of Signal/Noise Ratio in Microscopy

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…

Go For Gram! Staining Bacteria for Light Microscopy

Go For Gram! Staining Bacteria for Light Microscopy

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,…

Getting Started with Raman Spectroscopy: What You Need to Know

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…

Multifocal Structured Illumination Microscopy: The Fast Food of Super-Resolution Techniques

Multifocal Structured Illumination Microscopy: The Fast Food of Super-Resolution Techniques

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,…

Illustrated Optical Fiber Glossary (F –M)

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 (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…

Tips for Taking Immunofluorescent Images for Your Next Paper

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….

The Key to Unlocking DNA from FFPE Tissues

The Key to Unlocking DNA from FFPE Tissues

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…

How to See the Cell Cycle Through Your Microscope

How to See the Cell Cycle Through Your Microscope

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…

Immunoscience or Immunoalchemy?

Immunoscience or Immunoalchemy?

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…

How a Jellyfish Changed Biology: The Discovery and Development of GFP

How a Jellyfish Changed Biology: The Discovery and Development of GFP

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….

Free-Floating Versus Slide-Mounted Sections for Immunohistochemistry

Free-Floating Versus Slide-Mounted Sections for Immunohistochemistry

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…

Bright Minds: Overcoming Autofluorescence in Human Brain Samples

Bright Minds: Overcoming Autofluorescence in Human Brain Samples

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…

Let There Be Light! Microscope Maintenance Part 2: Köhler Illumination
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Let There Be Light! Microscope Maintenance Part 2: Köhler Illumination

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…

Let There Be Light! Microscope Maintenance Part 1: Routine Care and Replacing Bulbs
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Let There Be Light! Microscope Maintenance Part 1: Routine Care and Replacing Bulbs

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,…

Illustrated Optical Fiber Glossary (A – E)

Illustrated Optical Fiber Glossary (A – E)

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…