How Fluorescence Works - The Science

ORIGINAL: NurdRage

In this video we explore the colorful science of fluorescence.

A really cool way to play with fluorescence at home is get a blue or violet laser pointer and shine it into a dish or jar of water where you have added a drop of fluorescent highlighter fluid. You'll clearly see the beam as the solution fluoresces in its path.

Now the common definition of fluorescent is something that glows a visible color when exposed to ultraviolet light. Fluorescence is actually much broader than that and you don't need ultraviolet light in particular. For example in the yellow fluorescent dye "rubrene" both violet and green lasers will activate it and glow yellow. This proves you don't always need ultraviolet light. But a red laser will not activate a yellow dye. Why is that?

What's happening in fluorescence is that the incoming light raises the energy of the electrons in the molecule to an excited state. The electrons then lose a bit of energy due to vibrations of the molecules. And finally the electrons return to the ground state by releasing light. Now since energy cannot be created or destroyed and a bit of energy was already lost as heat in the vibrations of the molecules, the energy of light emitted must have lower energy than the light absorbed.

So since the yellow fluorescent dye emits yellow light, we need to use light of higher energy like violet and green for it to glow. Red is lower energy than yellow light so it can't excite the dye.

It also won't work if you use the same color as the dye like a green laser onto a green dye. This is because you almost always lose a bit of energy and therefore it has to emit a different color or none at all.

Synthetic circuits integrating logic and memory in living cells

ORIGINAL: NATURE BIOTECHNOLOGY | RESEARCH | LETTER

Piro Siuti, 

John Yazbek

& Timothy K Lu

Affiliations

Contributions

Corresponding author Nature Biotechnology (2013) doi:10.1038/nbt.2510Received 25 October 2012 Accepted 17 January 2013 Published online 10 February 2013

Logic and memory are essential functions of circuits that generate complex, state-dependent responses. Here we describe a strategy for efficiently assembling synthetic genetic circuits that use recombinases to implement Boolean logic functions with stable DNA-encoded memory of events. 

Application of this strategy allowed us to create all 16 two-input Boolean logic functions in living Escherichia coli cells without requiring cascades comprising multiple logic gates. We demonstrate long-term maintenance of memory for at least 90 cell generations and the ability to interrogate the states of these synthetic devices with fluorescent reporters and PCR. 

Using this approach we created two-bit digital-to-analog converters, which should be useful in biotechnology applications for encoding multiple stable gene expression outputs using transient inputs of inducers. We envision that this integrated logic and memory system will enable the implementation of complex cellular state machines, behaviors and pathways for therapeutic, diagnostic and basic science applications.

At a glance

A simple rule is used to translate desired computational functions into [promoter(s)]-[terminator(s)]-[output] designs, which can be constructed with straightforward Gibson assembly. In all of the E. coli cells used in this work, AHL (inpu… 

Figure 2: Recombinase-based logic gates can implement a complete set of two-input–one-output Boolean logic gates without needing to cascade multiple universal gates together.

(a) Percentage of cells maintaining GFP expression, assayed by flow cytometry after gating by forward and side scatter, in cells containing an AND gate induced to the ON state after day 0 and continuously diluted and grown without input si…

Cells were exposed to no inputs, AHL only, aTc only, or AHL and aTc simultaneously. (a–c) Various digital combinations of the input inducers result in multiple levels of analog gene expression outputs on the basis of the varying strengths… 

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Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Piro Siuti & 

Timothy K Lu

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Piro Siuti & 

Timothy K Lu

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

John Yazbek

Contributions

T.K.L. conceived of this study. P.S. and J.Y. implemented, constructed and performed all experiments. All authors analyzed the data, discussed results and wrote the manuscript.

Competing financial interests

P.S., J.Y. and T.K.L. have filed a provisional application with the US Patent and Trademark Office on this work.

Corresponding author

Correspondence to: 

Timothy K Lu

Supplementary information

Author information

Supplementary information

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Supplementary Text and Figures (6.5M)

Supplementary Figures 1–4, Supplementary Table 1 and Supplementary Data

Mice With Firefly Genes Glow in Response to Tumor Growth

ORIGINAL: MedGadget

by GENE OSTROVSKY

Jan 24, 2013

The progression of P16 increases in mice as they age visible from the younger mice (left) to the older mice (right).

Researchers at University of North Carolina have engineered laboratory mice which exhibit a firefly gene that could help scientists study cancer development. The p16INK4a (p16) gene is known to play a role in tumor suppression, so the team introduced the firefly gene so that it would be activated whenever the p16 gene is.

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Over time the mice with induced tumors were tracked and the glow was used to follow the activity of the p16 gene as it reacted to tumor progression. Some findings were that older mice glowed brighter, as expected, and the sites where cancer seemed to originate were particularly luminescent.

The researchers used these mice to make several unexpected discoveries. First, the group was able to track the accumulation of senescent cells in aging mice by assessing how brightly each mouse glowed. Surprisingly, the brightest animals were no more likely to die from spontaneous cancer than dimmer animals of the same age. That is, the number of senescent cells in the mouse did not predict its risk of dying.

Another surprise came from the disparities in p16 levels among the mice. The authors studied a large group of genetically identical animals that were all housed in the same way and fed the same diet. However, despite identical genetic and environmental conditions, the brightness of individual mice at any given age was highly variable, suggesting that factors beyond genetics and diet influence aging.

The glowing mice also provide a window into the formation of cancers. Expression of p16 is activated in the earliest stages of cancer formation to suppress cancer. Usually activation of p16 prevents cancer, but rarely this tumor suppressor mechanism fails and tumors develop, while still activating the p16 gene. As such, all tumors forming in these mice strongly glowed, allowing researchers to monitor early tumor formation in a wide variety of cancer types. In contrast to expectations, the researchers also found that p16 was activated not only in the tumor cells themselves, but also in normal, neighboring cells.

Biofilms

ORIGINAL: http://www.cell.com

Taking out the defender

by Maria Alhede and Thomas Bjarnsholt, University of Copenhagen

The in vivo interaction between a Pseudomonas aeruginosa biofilm, on a silicone implant, and the responding polymorphonuclear leukocytes.

Image: SEM imaging depicts the interaction at day 1 post insertion of the implant in the peritoneal cavity of a mouse. The leukocytes (yellow) are damaged with obvious cavities in the cell membrane and killed by the bacteria (cyan) following contact with the biofilm. The SEM image was pseudo colored in Photoshop CS5 using a Wacom Cintiq 24HD, by Michael Larsen.

Predictably beautiful

by Fernan Federici, PJ Steiner, Tim Rudge, and Jim Haseloff, University of Cambridge

As the bacteria grow within a biofilm, they organize themselves into reproducible patterns and shapes that can be predicted with mathematical models.

Image: Confocal microscopy of a bacterial biofilm composed of Bacillus subtilis expressing fluorescent proteins (TagRFP-T, sfGFP, TagBFP, mKate2 and mOrange2) to identify distinct lineages of bacteria in the biofilm. Images were taken at the Haseloff lab within the Department of Plant Sciences.

Predictably beautiful

by Fernan Federici, PJ Steiner, Tim Rudge, and Jim Haseloff, University of Cambridge

As the bacteria grow within a biofilm, they organize themselves into reproducible patterns and shapes that can be predicted with mathematical models.

Image: Confocal microscopy of a bacterial biofilm composed of Escherichia coli expressing the fluorescent proteins mCherry and sfGFP. The image was acquired on a Leica SP5 confocal microscope using a 2.5X dry objective. Images were taken at the Haseloff lab within the Department of Plant Sciences

Do we have a quorum?

by Tim Rudge, PJ Steiner, Fernan Federici, and Jim Haseloff, University of Cambridge

Under some conditions cells grow as "ropes," leading to interesting network-like structures.

Image: Confocal image of Bacillus subtilis cells in an early stage of biofilm formation. Images were taken at the Haseloff lab within the Department of Plant Sciences.

Keep your friends close

by Tim Rudge, PJ Steiner, Fernan Federici, and Jim Haseloff, University of Cambridge

Complex biofilm colonies can have eye-catching morphological features and spatial organization.

Image: A three-dimensional confocal stack of a Bacillus subtilis colony growing on agarose. Images were taken at the Haseloff lab within the Department of Plant Sciences.

Textured colony

by Valerie A. Ray and Karen L. Visick, Loyola University Chicago

Under certain conditions, Vibrio fischeri can form biofilms, as shown here by the formation of a "wrinkled" colony with substantial three-dimensional architecture.

Image: Vibrio fischeri was spotted onto and grown on solid agar and this image was captured using a Zeiss Stemi 2000-C dissecting scope.

What happens when you rinse?

by Shoji Takenaka, Betsey Pitts, Phil Stewart, Center for Biofilm Engineering, Montana State University

Have you ever wondered what your mouthwash is doing to the biofilms in your mouth?

Movie: Oral bacteria in a biofilm, shown in pink, red and yellow, lose fluorescence as a commercially available mouthwash penetrates the biofilm clusters. Time-lapse laser scanning confocal imaging, using a Leica TCS -SP5 II confocal microscope, 40X objective magnification.

Fungal biofilm in 3D

by Frederick Lanni, Haibing Teng, Jonathan Finkel, and Aaron Mitchell, Carnegie Mellon University

Candida albicans wild-type biofilm at 48 hr.

Movie: Image stacks were collected by serial focus imaging from the apical to basal region using Zeiss LSM510 Meta confocal imaging. 3D projections were compiled using Volocity 3D image analysis software, PerkinElmer.

Treating fungal infections

by Nicole Robbins (University of Toronto), Ranjith Rajendran (University of Glasgow ), Gordon Ramage (University of Glasgow ), and Leah E. Cowen (University of Toronto)

Some biofilms can be deadly, especially in immunocompromised individuals. Serious fungal infections such as invasive aspergillosis require aggressive treatment.

Image: Aspergillus fumigatus biofilm grown in vitro and subjected to voriconazole treatment. Image was obtained with a scanning electron microscope (Leo 435 VP) in the high-vacuum mode at 15 kV (Magnification 500X).

What happens when the chaperone is not around?

by Nicole Robbins (University of Toronto), Ranjith Rajendran (University of Glasgow ), Gordon Ramage (University of Glasgow ), and Leah E. Cowen (University of Toronto)

Treatment of an Aspergillus fumigatus biofilm with caspofungin and the Hsp90 inhibitor geldanamycin induces cellular damage.

Image: Image was obtained with a scanning electron microscope (Leo 435 VP) in the high-vacuum mode at 15 kV. (Magnification 500X).

DNA used as rewritable data storage in cells

ORIGINAL: ScienceNews

By Alexandra Witze

Monday, May 21st, 2012

Genetically encoded memory could track cell division inside the body

They aren’t yet competition for Intel, but bioengineers have created a one-bit “memory” made of DNA that can record, erase and rewrite data within living cells.

One day, doctors might be able to insert such devices into a cancer patient to tally how many times a cell divides and flag when to shut the cancer down. Or researchers might track exactly what happens inside cells as they age.

The work is a step forward in synthetic biology, a new field in which scientists create tools to control life’s basics from the cell on up.

“We can write and erase DNA in a living cell,” says Jerome Bonnet, a bioengineer at Stanford University. “Now we can bring logic and computation inside a cell itself.”

Bonnet and his colleagues, led by Stanford’s Drew Endy, describe the feat in a paper published online May 21 in the Proceedings of the National Academy of Sciences.

Figure S2: Alternate architecture for a reset circuit. A, Schematic diagram of the decoupled reset circuit where integrase is expressed from a low-copy plasmid while excisionase is expressed from a medium-copy plasmid. B, Cells bearing the chromosomal LR DNA register were transformed with both plasmids encoding integrase and excisionase, pulsed with arabinose and analyzed by flow cytometry. Cells relaxed to the BP state after induction with approximatively 85% efficiency.

Scientists have long dreamed of putting tiny computers inside the body to monitor and perhaps even control what’s going on. But nobody has yet made a silicon-based computer chip small enough to embark on a fantastic computing voyage inside a cell.

So researchers are turning instead to biological tools, such as enzymes and DNA. Some biologists have devised DNA switches that can be turned on and off within a cell. And in 2009, bioengineers reported making a genetic “counter” that could tally the number of times a particular event, like a cell dividing, took place (SN: 6/20/09, p. 5).

But these previous efforts made systems that could write a piece of information only once. Truly useful digital data storage allows the information to be erased and rewritten over and over again, like burning new information onto a CD with each pass. “What we didn’t have is some kind of logic that also has memory,” says Pakpoom Subsoontorn, a graduate student on the team.

The researchers chose DNA as the stuff of memory and used enzymes called recombinases as the tools to flip it on and off. Those enzymes came from bacteriophages, which are viruses that infect bacteria. These viruses use one enzyme to integrate into the genome of the bacterium they’re infecting.

In the experiment, the enzyme traveled to a particular place on the sequence of DNA that contains genetic information and flipped a small section so that it read backward. Sending a second signal then flipped the sequence back to its original state. The flipped and unflipped versions thus represent the “0” and “1” states of a computer bit, says Bonnet.

Working in the bacterium Escherichia coli, the team also tweaked the DNA so that it would fluoresce in different colors depending on the orientation of the strand in question. By watching the cells’ glow change between red and green and then back again, the scientists could tell when the DNA strand had been flipped.

So far, Endy’s team has just one bit of memory. Next they hope to scale up to eight bits, or a byte — a goal that could take many more years, Bonnet says. The scientists are also working on speeding up the flips; it currently takes about an hour to invert a DNA segment.

But the team has gotten the flips to hold for more than 100 generations within a living cell, a laboratory first. “This was an important proof of concept that it was doable,” says Bonnet. “Now we want to build a more complex system, something other people can use.”

Though interesting, it’s not yet clear whether DNA-based memory will ever replace silicon-based memory for certain applications, says Roger Brent, a researcher at the Fred Hutchinson Cancer Research Center and director of the Center for Biological Futures in Seattle. “It will need to prove itself in the marketplace of ideas.”