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Using ultrasound to image bacteria in vivo: acoustic reporter genes

Posted by Beth Kenkel on Jun 19, 2018 9:38:21 AM

Knowing where bacteria are located within their host is often key to understanding their role in both health and disease. To observe bacteria in action, researchers have developed in vivo bacterial reporters that use fluorophores and luciferases to track bacteria in real time, but each of these reporters has its drawbacks. Acoustic reporter genes (ARGs) overcome these limitations by using gas vesicle reporters that are detectable by an inexpensive and widely available imaging platform: ultrasound.

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Topics: Hot Plasmids, Microbiology, Imaging

Better Dyeing Through Chemistry & Small Molecule Fluorophores

Posted by Guest Blogger on Sep 8, 2016 10:30:00 AM

This post was contributed by guest blogger, Luke Lavis, a Group Leader at the Janelia Research Campus, Howard Hughes Medical Institute.

Chemistry is Dead, Long Live Chemistry!

The discovery of green fluorescent protein (GFP) sparked a renaissance in biological imaging. Suddenly, cell biologists were no longer beholden to chemists and (expensive) synthetic fluorophores. Add a dash of DNA with an electrical jolt and cells become perfectly capable of synthesizing fluorophore fusions on their own. Subsequent advances in fluorescent proteins have replicated many of the properties once exclusive to small-molecules: red-shifted spectra, ion sensitivity, photoactivation, etc. These impressive advances lead to an obvious question: In this age of GFP and its ilk, why should cell biologists talk to chemists?

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Topics: Fluorescent Proteins, Imaging

When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

Posted by Guest Blogger on Apr 19, 2016 10:30:00 AM

This post was contributed by guest blogger Erik L. Snapp.

Stop using EGFP/GFP for fusion proteins! Despite multiple studies in high profile journal articles, many researchers remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of EGFP can lead to significant artifacts.

If you're using green fluorescent protein or Enhanced Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label of cells, there's no problem. You're OK. However, if you fuse your protein of interest (POI) to GFP to study the protein's behavior in cells, in solution or something in between, you are using a tag with a serious drawback. The standard EGFP plasmid that used to be sold by Clontech and is in a freezer box in just about every lab in the world, is not inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently dimerize. That means that your POI fused to GFP or another fluorescent protein (FP) could be forming dimers in cells. Why should you care? Three simple ways a dimeric FP could ruin your day (and experiment) are listed below. Solutions to avoid these all too common issues follow.

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Topics: Imaging, Fluorescent Proteins

Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

Posted by Guest Blogger on Oct 27, 2015 11:00:00 AM

 This post was contributed by guest bloggers Erik L. Snapp and Lindsey M. Costantini.

"You underestimate the power of the Dark Side."

--Darth Vader in "Return of the Jedi"

While Vader was referring to the evil side of a mystical "Force," this quote is equally applicable to many microscopy experiments with fluorescent proteins (FPs) localized to compartments other than the cytoplasm. That is, unfortunately, some investigators realize too late that they have missed the impact of dark, non-fluorescent, and misfolded FP-fusions on quantitative imaging experiments and cell physiology in general.

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Topics: Imaging, Fluorescent Proteins

Mapping the 4D nucleome with CRISPR/Cas9

Posted by Mary Gearing on Aug 11, 2015 10:30:00 AM

It seems that there’s a new CRISPR advance or technique published every week! One of the newest applications is a colorful system that uses fluorescently labeled Cas9 to label multiple genomic loci in live cells. While other systems can be used to label loci, such as fluorescence in situ hybridization (FISH) or fluorescently labeled TALEs, CRISPR/Cas9’s ease of use and ability to label live cells make this system truly advantageous. This new technique, developed in Thoru Pederson’s lab, brings us one step closer to mapping the 4D nucleome, the organization of the nucleus in space and time, and to understanding how nuclear organization varies across the life of a cell, or how organization may be altered in disease states.

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Topics: CRISPR, Imaging, Fluorescent Proteins

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