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Microscopy and Imaging

Mouse Whole-Brain Volume Electron Microscopy for Cellular Connectomics – Enabling Large-Scale SEM Projects with ZEISS MultiSEM

Recent advances in high-throughput multi-beam scanning electron microscopy (EM) and mouse whole-brain EM preparation and collection on tape (“Brain-on-Tape”) have resulted in substantial progress towards a nano-scale map of the whole mouse brain. These maps can be used to determine how individual neurons are synaptically connected and can be used to reconstruct the precise “wiring…

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Enhance Your Imaging with Super-Resolution: Improve Spatial Resolution and Increase Signal to Noise Ratio in Difficult Samples

In these informative case studies, you will learn how: HyVolution 2 can improve the spatial resolution you get from your sample, whilst remaining gentle to the fluorophores HyVolution 2 overcomes issues experienced with other imaging modalities, even in difficult samples such as plant material, increasing resolution and signal to noise ratio, thereby facilitating downstream image…

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Harnessing the Power of Heterogeneity in Cryo-electron Microscopy

A major impediment in the determination of high resolution protein structures by single particle cryo-electron microscopy has been the presence of sample heterogeneity. Oftentimes, heterogeneity is due the dynamic nature of protein complexes, which can exist in multiple different conformational states in solution. Recent advancements in cryo-electron image processing have provided tools to accurately sort…

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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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Revealing Cellular Dynamics with Millisecond Precision – The New Tool That Turned Electron Micrographs into Motion Pictures of Neural Communication

Revealing Cellular Dynamics with Millisecond Precision – The New Tool That Turned Electron Micrographs into Motion Pictures of Neural Communication In this tutorial: What if you can dissect the cellular dynamics with millisecond precision? What if you can unravel the morphological transformation of a neuron millisecond by millisecond using electron microscopy? Could this be even…

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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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Tips for Peering into the Interior of Mice Using Intravital Microscopy

Techniques to study entire tissues, such as brain imaging microscopy, provide great insight into the biology of the whole tissue, rather than just individual cells.  Taking this one step further is intravital microscopy (IVM); a newer approach for the imaging of living tissues and organs in live animals. A wide variety of organs can be…

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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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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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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…

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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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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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Color Image Quality Control in Microscopy

In this webinar, you will learn about color reproduction in microscopy images—and how to fix it if your color reproduction goes awry. The main points we will cover are: The reasons that could color reproduction go wrong in your microscopy images How to correct your microscopy images with inappropriate color reproduction How to be ethical…

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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 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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Spectral Unmixing in Fluorescence Microscopy

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?

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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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What Lipid Bilayers Can Do For You

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.

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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…

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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…

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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…

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Introducing Aptamers for Super Resolution Microscopy

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.

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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…

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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,…

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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.…

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