Database of fluorescent protein sequences

A paper recently came out on a web server for predicting the oligomeric state of fluorescent proteins [1]. It’s only about 80% accurate, which isn’t so nice, but they have a github page with sequences for hundreds of fluorescent proteins in a csv file. Tracking down all these sequences can be tedious, so this is a nice resource.

References

  1. S. Simeon, W. Shoombuatong, N. Anuwongcharoen, L. Preeyanon, V. Prachayasittikul, J.E.S. Wikberg, and C. Nantasenamat, "osFP: a web server for predicting the oligomeric states of fluorescent proteins", Journal of Cheminformatics, vol. 8, 2016. http://dx.doi.org/10.1186/s13321-016-0185-8

Michael Davidson and Roger Tsien Commemorative Travel Awards from Addgene

Addgene is offering travel awards for students or postdocs using fluorescent proteins in their research. The award is named in honor of Michael Davidson and Roger Tsien – two giants in the fluorescent protein field. While Roger Tsien is well known, Michael Davidson is one of the unsung heroes of fluorescent protein research – his lab built thousands of fluorescent protein fusions to test the performance of fluorescent proteins in a wide variety of contexts. He then distributed these plasmids to many labs, and eventually deposited nearly all of them in Addgene.

Fluorescence lifetime and quantum yield

Two months ago I saw a tweet noting the linear relationship between quantum yield and fluorescence lifetime in fluorescent proteins. I hadn’t seen this before, so I wanted to see if it held on a wider range of fluorescent proteins, so I added the ability to plot lifetimes on my fluorescent protein visualization and added lifetimes for all the proteins I could find (37 in total).

Quantum yield vs. fluorescent lifetime (ns) for 37 fluorescent proteins, colored by emission wavelength and brightness. Click for full size image.

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Github pages

Github pages is awesome. I’ve been dimly aware of it for some time, but only just tried it. It’s really simple – if you have a Github repo that is a webpage, just tell Github that it should serve it as such, and it will become a live webpage. For instance, a few mouse clicks made my FPvisualization repository visible as a live webpage. Commits pushed to the repository automatically go live on the web.

Fluorobrite DMEM and sptPALM

A number of our users have been testing out the low-fluorescence DMEM from Life Technologies, Fluorobrite DMEM, and many have found that it substantially improves the signal-to-background of their experiments, particularly for imaging dim samples. Recently we tried it when doing sptPALM of PA-GFP, and found that it made a substantial difference in image quality. Below are two movies, taken under identical conditions, and scaled identically, of single PA-GFP labeled receptors diffusing in the membrane of a cell. They are illuminated with modest 488 nm excitation and weak 405 nm excitation to switch PA-GFP on. The movies were acquired by TIRF, and are shown in real time. The top movie is of cells in conventional DMEM-based media; the bottom, cells in Fluorobrite DMEM. The Fluorobrite media substantially improves the image contrast; there’s also a noticeable reduction in background events outside of the cell.

If you’re doing low-signal imaging, particularly in the GFP channel, I think the Fluorobrite media is well worth trying out.

Nice GCaMP movie

One of the labs we did in this year’s QB3/UCSF Microscopy course was to image cells transfected with a labeled β2-adrenergic receptor and with the calcium reporter GCaMP. Addition of a β2-receptor agonist triggers signaling, leading to calcium influx, reported by GCaMP, and receptor internalization, which can be seen as clustering of the labelled receptors.  This experiment generated some very pretty movies. I’m showing one here, a time lapse acquired on a Spectral Applied Research Diskovery system, operated in spinning disk confocal mode. The first movie is the full field of view, as captured on an Andor Zyla 4.2. The first movie shows the full 2k x 2k field of view, downsampled. The second movie show a full resolution crop from the movie.

Lots of new fluorescent proteins at Addgene

Michael Davidson, at Florida State University, is contributing his entire collection of over 3000 fluorescent protein plasmids to Addgene. This is going to be a great resource for fluorescent imaging; currently there are 136 fluorescent protein vectors with no tags, and 139 fusions of different proteins to mEmerald alone.  Many more will be posted as they are curated.

Mike is one of the unsung heroes of fluorescent microscopy: in addition to maintaining the Molecular Expressions, MicroscopyU, Zeiss Campus, and a number of other websites, you will also see that he is an author on many fluorescent protein publications. He’s made a career out of testing the performance of fluorescent proteins by fusing them to as many as 40 different mammalian proteins and testing their localization and behavior. It’s the plasmids from these tests (among others) that are being deposited at Addgene.

Take a look at the plasmids at Addgene, and if they’re useful to you, thank Mike for all the hard work his group has put in to bring us better fluorescent proteins.

Photobleaching and Photoactivation

A few months ago, we purchased a Rapp Optoelectronics galvo-scanning system, along with 405 and 473 nm lasers from Vortran Laser Technology, to provide a photoactivation and photobleaching system for our high speed widefield system. This system is capable of photoactivating or photoconverting any protein that is switched by 405nm light (which is most of them) and photobleaching GFP, while simultaneously acquiring in the GFP, RFP, and Cy5 channels. The entire system is controlled through Micro-Manager.

Today, with help from Nico Stuurman, we took it out for a spin. Nico provided Drosophila S2 cells with either mEos2 or GFP-tubulin as well as help getting everything set up correctly. Here are two videos demonstrating what it can do. Both were acquired on a widefield microscope with a 100x / 1.4 NA objective.

Photoconversion of mEos2-labeled tubulin in the spindle of a Drosophila S2 cell. The video is sped up 20-fold from real time.
Photobleaching of GFP-labeled tubulin in a Drosophila S2 cell. First, a GFP aggregate is bleached, which rapidly recovers. Second, nearby microtubules are bleached, which do not recover over the same time scale. The acquisition doesn't stop during the bleaching, so you can see the bright flash as the bleaching laser is turned on. The video is sped up 1.5x from real time.

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Updates to Fluorescent Protein Visualization

I made some updates to the fluorescent protein visualization website that I’ve been meaning to do for some time: proteins that require an extrinsic chromophore (like the iRFPs) are now shown as squares, and the oligomerization state (dimer, etc.) is also shown on the graph.

Up next I intend to add a few new fluorescent proteins, including mCardinal, and then I want to allow the excitation and emission axes to be plotted in units of energy or wavenumber. As a reminder, the source code is available, and I encourage contributions.

Here’s what it currently looks like:

FPscreenshot

sptPALM

Some of you probably noticed that this blog was down for the last week. Unfortunately our server died right before Thanksgiving and I only now got it back up.  Please let me know if you encounter any problems with it.

To make up for the absence of the blog, I’ll share with you a cool movie we’ve just taken in the NIC. This is single particle tracking PALM or sptPALM [1] of the dopamine receptor labeled with mEos2.  Here, we’re imaging with pretty strong 561 nm illumination and very weak 405 nm illumination, so we’re continuously converting mEos2 molecules to the red state and imaging them until they bleach. Using this methodology we can track many different individual receptors over a long time and see how the population behavior changes when we stimulate the receptor.

References

  1. S. Manley, J.M. Gillette, G.H. Patterson, H. Shroff, H.F. Hess, E. Betzig, and J. Lippincott-Schwartz, "High-density mapping of single-molecule trajectories with photoactivated localization microscopy", Nature Methods, vol. 5, pp. 155-157, 2008. http://dx.doi.org/10.1038/nmeth.1176