Category Archives: 1 Fluorescent dyes & proteins

Quantum dots for smFISH

Single molecule FISH is currently the best method to get accurate measurements of mRNA levels at single molecule, single cell level in cell culture or tissue slices with a spatial resolution of ~200 nanometer (or less). One of the drawbacks of this method is the deterioration of the fluorescent signal (bleaching) of the organic dyes that are used to label the probes. Andrew Smith’s lab from University of Illinois now show how FISH can work with quantum dots instead of organic dyes. This provides better fluorophore stability and also the possibility to have more colors with less overlap of the emission spectra.

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Fluorescent protein databases

Two new awesome databases for fluorescent proteins are now available: FPbase and Fluorescent Biosensor database.

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Quantum dots for Immunofluorescence — Rapha-z-lab

An important post on quantum dots:

Guest post by Dave Mason In modern cell biology and light microscopy, immunofluorescence is a workhorse experiment. The same way antibodies can recognise foreign pathogens in an animal, so the specificity of antibodies can be used to label specific targets within the cell. When antibodies are bound to a fluorophore of your choice, and in […]

via Quantum dots for Immunofluorescence — Rapha-z-lab

Poking holes into membranes to label proteins for live imaging

There are two major way to label inner proteins, structures or organnelles for live cell imaging. The most common method is fusing the studied protein to a fluorescent protein. A second approach is the addition of labeling agents from outside the cells. However, many labels cannot penetrate through the cell membrane. This is true to some, but not all dyes, but more importantly, to larger agents, such as antibodies or DNA/RNA oligos. To allow these agents to enter cells, researchers can use microinjection, electroporation, bead-loading, or transfection (e.g. of short oligos).

In a paper just published in eLife, a new technique is described to form temporary holed in the cell membrane. These holes allow delivery of any labeling agent into cells. Continue reading

Roger Tsien – the scientist that colored our research

Roger Tsien died a few days ago, at the relatively young age of 64. He was a UCSD scientist, a Nobel laureate and he was one of the first to see the significance and usefulness of GFP.

I’ve never met him. But, I guess, this blogs owes him its existence.

I don’t want to discuss his body of work, his achievements, or awards he won (e.g. the Nobel award). Many wrote nice things about him, such as here, here or here and all over the internet, with nice pictures of fluorescent proteins used in research.

I thought it will be nice to look back at his first GFP paper.

title1

His goal in this paper was to investigate the formation of the fluorophore of GFP. Specifically, he asked:

“What is the mechanism of fluorophore formation? How does fluorescence relate to protein structure? Can its fluorescence properties be tailored and improved-in particular, to provide a second distinguishable color for comparison of independent proteins and gene expression events?”

Already here he looked to utilize GFP – to improve it, to change it, so it can be useful for fluorescent studies in biology.

He used random mutagenesis of the GFP cDNA to screen for mutants with altered brightness and emission. A simple yet powerful method, still used today, to find new FPs with exciting and useful properties.

Here is an ex/em spectral analysis of some of the mutants:

spectra

One mutant, I167T, proved to be almost twice as bright as the WT GFP protein.

But the most exciting was the finding of a blue FP (Y66H):

blue mutant

To sum up in his words:

“The availability of several forms of GFP with such different excitation and emission maxima [the most distinguishable pair being mutant P4 (Y66H) vs. mutant Pll (I167T)] should facilitate two-color assessment of differential gene expression, developmental fate, or protein trafficking. It may also be possible to use these GFP variants analogously to fluorescein and rhodamine to tag interacting proteins or subunits whose association could then be monitored dynamically in intact cells by fluorescence resonance energy transfer (19, 20). Such fluorescence labeling via gene fusion would be site-specific and would eliminate the present need to purify and label proteins in vitro and microinject them into cells.”

He saw the future, and it was bright green.

 
ResearchBlogging.orgHeim, R., Prasher, D., & Tsien, R. (1994). Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proceedings of the National Academy of Sciences, 91 (26), 12501-12504 DOI: 10.1073/pnas.91.26.12501

Design guidlines for tandem fluorescent timers

Almost 4 years ago, I wrote a post on tandem fluorescent timers (tFTs). The idea is to have two different fluorescent proteins fused together to the protein of interest. In the paper from 4 years ago, it was superfolder GFP (sfGFP) and mCherry. sfGFP matures very fast (within minutes) and mCherry  matures more slowly (t1/2 ~40min). The ratio beween green to red fluorescent signal indicates the percentage of new vs old proteins, thus acts as a “timer”.  This latests paper on tFTs from the same group of Michael Knop’s lab, found that analyzing tFTs might be more complicated due to some possible problems of this system.

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Counting exosome secretion

Last month I wrote a post about exosome internalization by recipient cells.  One of the topics I discussed was the lack of good quantitative data in the exosomal field, and what the current data tells us about the efficiency and capacity of exosome-mediate cell-to-cell communiation.

Today I came across an interesting paper in which the researchers try to get quantitative data of exosome secretion by the donor cells.

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