Virtual Cell Staining

Invitrogen has a new online tool that lets you explore cell imaging from your computer. You can choose different combinations of cellular structures and fluorophores to create your version of the perfectly labeled fluorescent cell. Use the tool to find various products to label your cell, create a cell image, then print and share your image. I created the cell below. You can try it out here.

Multicolor two-photon microscopy with a new red fluorescent protein

Two-photon microscopy can be used with multiple fluorescent markers to simultaneously observe multiple processes in vivo. Red fluorescent proteins (RFPs) are a popular choice because cellular autofluorescence is reduced at its emission region and it allows greater imaging depth thanks to less light scattering. In addition, longer wavelengths are less damaging to proteins and DNA.

However, using red fluorescent proteins with other colors such as GFP or cyan fluorescent protein (CFP) comes with some challenges. The Ti-Sapphire lasers used for two-photon microscopy are expensive, so most systems have only one laser. This makes it hard to efficiently excite RFPs at the same time as GFPs or CFPs. Also, Ti:Sapphire lasers have low power output in the excitation wavelengths for RFPs.

One way to overcome these problems is to shift the excitation spectrum of RFPs by making RFPs with a large Stokes shifts. One such RFP exists already. Called, mKeima, it can be used for dual-color two-photon microscopy, but its biochemical and photochemical properties leave room for improvement.

Researchers at the Albert Einstein College of Medicine in New York applied random and rational mutagenesis to the monomeric far-red mKate fluorescent protein and produced two RFPs with large Stokes shifts. The RFP variants have six amino acid mutations compared to mKate. Compare to mKeima, the new variants have higher pH stability, better photostability and faster chromophore maturation and monomeric behavior.

They used the new RFPs, named LSS-mKate1 and LSS-mKate2, for two-photon microscopy simultaneously with ECFP and EGFP. Their multicolor intravital imaging study of the motility and Golgi-nucleus alignment of tumor cells as a function of distance from blood vessels in a live mouse xenograft model of breast cancer revealed that breast cancer cells showed polarization towards vessels at a distance of 40 microns.

Research paper:
Monomeric red fluorescent proteins with a large Stokes shift
Kiryl D. Piatkevich, James Hulit, Oksana M. Subach, Bin Wu, Arian Abdulla, Jeffrey E. Segall, and Vladislav V. Verkhusha
PNAS doi:10.1073/pnas.0914365107

See more posts on:

1) two-photon microscopy 2) fluorescent proteins

Multiphoton GFP photoconversion reveals cilia diffusion dynamics

Researchers measured protein diffusion through a sensory cilium. Courtesy of Peter D. Calvert.

Cilia are present on most cells and play important functions in vision, hearing, and smell. Scientist want to know more about the unknown functions of many cilia because mutations in genes that encode cilia proteins can cause serious diseases such as Bardet-Biedl syndrome, Usher syndrome, poly-cystic kidney disease, and retinal degeneration.

Cilia don’t synthesize proteins or membrane, and research is focused on how proteins are delivered to and removed from these organelles. Researchers believe that cilia impose selective barriers to the movement of proteins but haven’t been able to show this experimentally because some of the cilia’s structures have diameters near or below the resolution of light microscopy.

But now, a team of researchers led by Peter Calvert at SUNY Upstate Medical University along with researchers from Lehigh University and the University of California, Davis have measured the mobility of photoactivatable GFP in the connecting cilium of live Xenopus retinal rod photoreceptors and in subcellular compartments bridged by the connecting cilium. In addition, the team measured the overall time for the protein concentration to equilibrate within and between compartments.

The researchers photoconverted GFP at specified coordinates using multiphoton excitation from a Ti:sapphire laser tuned to 820 nm and focused to the diffraction limit. The multiphoton excitation produced spatially well-defined fields of photoconverted molecules and minimized the tissue’s exposure to visible light. They then used confocal excitation at 488 nm to monitor the equilibration of the photoconverted GFP with serial x–y scans that intersected the area of photoconversion.

The results showed that in the connecting cilium diffusion was somewhat slower than expected in an aqueous cell compartment, but that this structure was not a major barrier to protein diffusion. However, the diffusion in the inner and outer segments bridged by the connection cilium was found to be substantially slower than in other cells studied. The authors offer various explanations of what might cause this slower diffusion.

Research Paper:

The Journal of General Physiology, Vol. 135, No. 3, 173-196 Diffusion of a soluble protein, photoactivatable GFP, through a sensory cilium

Peter D. Calvert, William E. Schiesser, and Edward N. Pugh, Jr.

Fluorescent reporter allows imaging of single cells infected with hepatitis C

Hepatitis C virus (HCV) infects 2 to 3 percent of the world population. The recent development of methods to culture the virus may lead to antiviral therapies, but better imaging methods are needed to understand the infection process. In the February issue of Nature Biotechnology researchers at Rockefeller University, MIT and other institutions report on a cell-based fluorescent reporter that allows imaging of live or fixed individual HCV-infected cells.

They constructed the reporter by fusing a fluorescent protein--either EGFP, mCherry, or Tag RFP--to the C-terminal region of a known substrate of the HCV NS3-4A protease. They were able to use this reporter to distinguish between infected and uninfected cells and to image previously unseen events such as viral propagation and host response. The researchers used a Nikon Eclipse TE300 for wide-field fluorescence imaging, a Zeiss inverted Axiovert 200 laser scanning confocal microscope for imaging fixed samples, the Axiovert 200 with a Perkin-Elmer spinning-disk confocal head for imaging live samples, and a Olympus IX71 inverted microscope for time-lapse imaging.

The reporter system doesn’t require genetic modification of the viral genome and worked for all the HCV genomes they tested, which gives it potential for use in identifying new infectious isolates directly from patient samples, according to the researchers. They were also able to combine the HCV reporter with a fluorescent marker of cellular stress to correlate viral and host events.

Research Paper:
Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system, Nature Biotechnology 28, 167 - 171 (2010) 
doi:10.1038/nbt.1604

White light laser allows orange fluorescent protein to be useful FRET acceptor

Ammasi Periasamy of the University of Virginia as well as colleagues from Florida State and Indiana Universities recently studied the performance of an orange fluorescent protein’s performance as an acceptor in a FRET pairs. Orange fluorescent proteins bridge the gap between green and red/far-red fluorescence proteins but are hard to excite with common fixed wavelength lasers. However, the researchers wanted to see if using a supercontinuum light source--also called a white light laser (WWL)--could help. The researchers studied monomeric Kusabira orange 2 (mKO2) as an FRET acceptor partnered with a monomeric teal (mTFP) donor on a standard FRET construct in live cells. They used a supercontinuum laser with continuous emission from 470 to 670 nm that allowed selection of individual wavelengths in 1-nm increments. The researchers found that that mKO2 and mTFP are good FRET partners with the right imaging setup, specifically exciting the mTFP with an Argon 458 nm laser line and using 540-nm excitation from the supercontinuum laser for mKO2.

See other FRET-related posts here.

Research paper:

http://dx.doi.org/10.1117/1.3227036
Yuansheng Sun, Cynthia F. Booker, Sangeeta Kumari, Richard N. Day, Mike Davidson, Ammasi Periasamy, Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser. J. Biomed. Opt. Vol. 14, 054009.

LED flashlight used as fluorescence microscopy light source

Mercury gas discharge lamps, the traditional light sources for

fluorescence microscopy, come with several disadvantages. They have a limited lifetime, a fluctuating intensity, give off heat, and can sometimes explode. LED illumination overcomes these limitations, but most commercial LED illumination systems are expensive because they are made to excite at many different wavelength ranges.

Researchers from Vanderbilt University wanted to find a cheaper way, so they tried an off-the-shelf LED flashlight as an illumination source for fluorescence microscopy. They picked out the brightest flashlight available at a camping store, which was a $50 Inova Bolt 4.6 W/6 V flashlight. It had a strong emission in the 440 to 480 nm range, which is useful for excitation of several common fluorescence probes including enhanced GFP. They hooked up the flashlight to a power supply so that it could be turned off and on from outside the light-tight box they were using and also created an adaptor to fit the flashlight to the microscope’s fluorescence lamp port.

Although the flashlight was less intense than a mercury light source, they did achieve images with the LEDs that were comparable to those from a mercury light source by increasing the CCD camera exposure to four to five times that used for a 100 W mercury lamp. The researchers say that the power supply and flashlight together costs around $90, less than a single replacement mercury lamp.

Read more about this work in the paper:

J.B. Robertson, Y. Zhang & C.H. Johnson. Light-emitting diode flashlights as effective and inexpensive light sources for fluorescence microscopy, Journal ofMicroscopy, Vol. 236, Pt 1 2009, pp. 1–4, doi: 10.1111/j.1365-2818.2009.03208.x

Microscopy camera for bioimaging

QImaging has released the EXi Aqua Bioimaging Microscopy Camera, which replaces the company’s Retiga EXi. The 20 MHz, 14-bit EXi Aqua camera has an IEEE 1394b Firewire interface and achieves mega pixel resolution with a Sony ICX285 interline sensor.

The camera is monochrome but can acquire color with an optional RGB filter module or Bayer mask color CCD option. The camera can read out at up to 10.9 full-resolution frames per second, has flexible binning, and exposure times that are adjustable from 10 microseconds to 17.9 minutes. It is cooled to 0 ºC and exhibits ~20% less read noise than the Retiga EXi. The camera’s QCam driver integrates with a variety of popular third-party software packages.

The camera is useful for many applications, including immunofluorescence imaging, fluorescent protein imaging, time-lapse fluorescence imaging, calcium imaging, and FRET as well as phase-contrast, DIC, and bright field microscopy. More information here.

GFP images of bioengineered tooth

Researchers at Tokyo University of Science, Organ Technologies Inc, Tohoku University, and Tokyo Medical and Dental University, all in Japan, have successfully transplanted bioengineered tooth germ into a mouse, achieving a fully functioning tooth replacement. The work is reported in an open access PNAS  paper. The bioengineered tooth had the correct structure and hardness and also responded to mechanical stress and pain. The researchers think that this is a substantial advance in bioengineering, and that the technology might be useful for use with replacement of other organs.

Open access paper:
Fully functional bioengineered tooth replacement as an organ replacement therapy.Etsuko Ikedaa et al. PNAS, doi: 10.1073/pnas.0902944106.

The image above shows a merged bright field and dark field image of the teeth. 

These images show the nerve fibers in the pulp (top) and the periodontal ligament (bottom) of the bioengineered tooth. 

Images copyright Tokyo University of Science. Courtesy of Takashi Tsuji. 

Printed FRET pairs

After reading the headline you may be asking why someone would want to print FRET pairs. After all, FRET is typically used to study dynamic protein interactions. Biological applications are indeed what FRET is known for, however scientists are beginning to see that fluorescent proteins might be useful in fabricated devices such as those used for high-density optical memory and switches. If so, FRET could be a useful way to add functionality to devices making use of fluorescent proteins.

To study this possibility, Vinod Subramaniam and colleagues at the University of Twente in The Netherlands printed 2-D and 3-D patterns of fluorescent protein FRET donor/acceptor pairs. The work is detailed in a recent Langmuir research paper.

By examining the spectra and lifetime of the fluorescent proteins using wide-field fluorescence and confocal microscopy the researchers found that FRET occurred at the interfaces between the donor and acceptor molecules. The researchers point out that fluorescent lifetime imaging microscopy (FLIM) is useful to in making sure the function of the patterned fluorescent proteins is not compromised.

In the future, patterning of FRET pairs could be combined with nanopatterning technology to produce nanoscale devices.

Research paper: FRET Pair Printing of Fluorescent Proteins, Maryana Escalante, Christian Blum, Yanina Cesa, Cees Otto, Vinod Subramaniam, Langmuir Article ASAP

Fluorescent proteins for mouse embryo imaging

Fluorescent proteins have led to numerous discoveries in biology and have become so important that the work on the original green fluorescent protein won a Nobel prize last year. In the last decade or so, researchers have added many new colors to the original green fluorescent protein, have fine tuned these proteins to make them brighter and easier to use, and have introduced new techniques using fluorescent protein characteristics that were modified or even discovered.  

With so many new developments I was glad to see that researchers from the Sloan-Kettering Institute in New York have published an excellent review paper in Biotechnology Trends that puts the most important information tied to fluorescent proteins in one place.  The review somewhat focuses on mouse embryo imaging, but doesn't skip over the history or the basics.

The writers include a helpful table detailing the excitation/emission, oliogmeric state, commercial availability and reference for almost 30 of most common ones. I have read the original publications on many of these proteins but did'nt realize just how many "common" ones there were until seeing this list. I found myself clicking on several of the references to remind me which research groups were involved in development or discover of the various fluorescent proteins. 

The authors believe that combining fluorescent proteins with mouse genetics is essential for unraveling mechanisms involved in regulating embryonic development. Thus, they detail studies several applications related to mouse imaging and point out that development of mouse strains for live imaging has been a limiting step in many applications.

The authors do include discussion of some alternative technologies such as quantum dots and small molecule probes, but seem to think that fluorescent proteins are the way of the future, at least for mouse embryo imaging. I think that quantum dots are quite promising for imaging and are often easier to use than fluorescent proteins. 

Do you think quantum dots will eventually replace fluorescent proteins?

Trends in Biotechnology paper: Live-imaging fluorescent proteins in mouse embryos: multi-dimensional, multi-spectral perspectives