Symposium : A Revolution in Fluorescence Imaging

11 02 2009

header-jellyfish

This coming Tuesday and Wednesday (Feb 17th & 18th) at UCSD, there will be a symposium honoring Roger Tsien, featuring presentations from 32 former and current members of the Tsien Lab. The topics are quite diverse, concentrated in genetically-encoded indicators, but also featuring fluorescent cell penetrating peptides for cancer therapy, photophore ligases for imaging synaptic development, and even a radical new design for the internal combustion engine.

The quality of speakers and subjects looks to be outstanding.  Here is a complete schedule.  You may notice that at 11:15 AM on Tuesday in Price Center East Ballroom, I will be presenting recent progress we have made in the development of genetically-encoded calcium indicators and their application to in vivo imaging.  Don’t miss that one!  :)  Roger’s talk, which will assuredly be equal parts absorbing, humorous, and illuminating, is at 4pm Wednesday in the Price Center Theater.

If you live in Southern California and are interesting in imaging technology, there isn’t a better place to be than this symposium.  If you can’t make it, Brain Windows will have a full write-up following the event.

Here is the un-official schedule.

Tuesday February 17th – Price Center East Ballroom

9:00 -9:05 Varda  Levram -Ellisman Opening

9:05-9:15 Palmer Taylor

Designing the next generation of genetically encoded sensors

9:15-9:30 Roger Heim

FRET for compound screening at Aurora/Vertex

9:30-9:45 Amy Palmer

Designing and using genetically encoded sensors: Lessons I learned from Roger

9:45-10:00 Robert Campbell

Beyond brightness: colony screens for fluorescent protein photo stability and biosensor FRET changes

10:00-10:15 Colette Dooley

GFP sensors for reactive oxygen species: Tying up loose ends and looking forward.

10:15-10:30 Peter Wang

Fluorescent Proteins and FRET biosensors for visualizing cell motility and mechanotransduction

Fluorescent proteins in neuroscience

11:00-11:15 Brian Bacskai

Aberrant calcium homeostasis in the Alzheimer mouse brain

11:15-11:30 Andrew Hires

Watching a mouse think: Novel fluorescent genetically-encoded calcium indicators applied to in vivo brain imaging

11:30-11:45 Alice Ting

Imaging synapse development with engineered photophore ligases

11:45-12:00 Rex Kerr

3D calcium imaging in C. elegans

Clinical applications

12:00-12:15 Todd Aguilera

Activatable Cell Penetrating Peptides for use in clinical contrast agent and therapeutic development

12:15-12:30 Quyen Nguyen

Surgery with Molecular Fluorescence Imaging Guidance

Fluorescent probes (Chemistry)

1:30-1:45 Tito Gonzalez

Voltage-Sensitive FRET Probes & Applications

1:45-2:00 Paul Negulescu

From watching ions to moving them

2:00-2:15 Timothy Dore

Roger-Inspired Photochemistry: Releasing Biological Effectors with 2PE

2:00-2:15 Joe Kao

Electron Paramagnetic Resonance Imaging in Living Animals

2:15-2:30 Brent Martin

Chemical probes for studying protein acylation

2:30-2:45 Jianghong Rao

Non-GFP based probes for imaging of the hydrolytic enzyme activity

Cellular research with and without Fluorescent probes

3:15-3:30 Carsten Schultz

Cell membrane repair visualized by GFP fusion proteins

3:30-3:45 David Green

Transcriptomes and Systems Biology: application to early mammalian embryogenesis

3:45-4:00 Clotilde Randriamampita

Paradoxical aspects of T cell activation revealed with fluorescent proteins

4:15-4:30 Wen-Hong Li

Studying dynamic cell-cell communication in vivo by Trojan-LAMP

4:30-4:45 Martin Poenie

Aim and Shoot: Two roles for dynein in T cell effector function

4:45-5:00 Gregor Zlokarnik

From bla to blah, blah in 20 years

5:00-5:15                        James Sharp

President, Zeiss MicroImaging Gmbh

February 18 2009 – Leichtag 107

Cellular research with and without fluorescent proteins

9:00-9:15 David Zacharias

Fluorescent Proteins, Palmitoylation and Cancer: two out of three ain’t bad

9:15-9:30 Jin Zhang

Visualization of Cell Signaling Dynamics: A Tale of MAPK

9:30-9:45 Paul Sammak

Nuclear organization and movement in pluripotent stem cells measured by Histone GFP H2B

Branching out

9:45-10:00 Yong Yao

NIH Toolbox Program

10:00-10:15 Oded Tour

The Tour Engine – A novel Internal Combustion Engine with the potential to boost efficiency and cut emissions

Into the future

10:45-11:00 Xiaokun Shu

Visibly and infrared fluorescent proteins: photophysics and engineering

11:00-11:15 Michael Lin

Engineering fluorescent proteins for visualizing newly synthesized proteins and improving FRET-based biosensors

11:15-11:30 Jeremy Babendure

Training our next generation of Fluorescent Protein Enthusiasts

Main Event – Price Center Theater

4:00-5:00 Roger Tsien

Chancellor invitational lecture 2008 Nobel Prize in Chemistry






BrainStorm 1 : The Calcium Memory Sensor

9 01 2009

As mentioned in the previous post, this is the first installment of BrainStorm, a section of ideas I have under development, but don’t have the time to physically work on.  This section will contain organically developed ideas, organized by project.  Reader feedback is encouraged.

How can we identify the group of neurons that encode a particular thought?  

I don’t want to simply see correlations of in activity of a few scattered neurons with a given thought, but identify the entire neuronal ensemble.  Which neurons are active at a precise moment in a task?  How are they wired together? Which are the drivers of activity?

Existing technology is inadequate to identify the entire neural ensemble that encodes a thought. Immediate early gene expression  patterns have not been shown to be precisely correlated with brain activity, and have a temporal resolution on the order of minutes. Genetically encoded calcium sensors (GECIs) have the necessary temporal and spatial resolution, but their response is nearly as fleeting as a thought, making it impossible to simultaneously record from networks of thousands of possible participants with current microscopy techniques.

In BrainStorm 1, I will outline a technology, photoswitchable genetically-encoded calcium memory sensors, that can identify all the neurons in a large network that are active during user-specified, aribitrarly brief or long time periods.  I will propose four potential strategies for construction of these sensors, and detail practical considerations for sensor design, screening and application.





Fluorescent Proteins in Scholarpedia

12 12 2008

I just discovered that the scholarpedia article on fluorescent proteins was published back in July.  This article is an excellent review of all fluorescent proteins that contains both classic and very current references.  I recommend it as the first place to go to learn about fluorescent proteins in detail. It makes my contributions to the GFP page of wikipedia look quite primitive.

The author, Rob Campbell, is relatively famous in the field of fluorescent proteins for his monomerization of dsRed, the brilliant red fluorescent protein from coral. Many of the fluorescent proteins in Brain Windows title bar were derived from mutations of this monomeric red fluorescent protein. Less well known is that he taught me how change restriction sites on plasmid DNA via overlap extension PCR during my first year of grad school. Great work Rob!

Red Discosoma Coral

Red Discosoma Coral





Raw Data : Vesicular Release from Astrocytes, SynaptopHluorange

15 11 2008

When I was working on my Ph.D. thesis, I was trying to find some biological question to definitively answer with GluSnFR, my glutamate sensitive fluorescent reporter. One possibility was the study of glutamate release from astrocytes. Around that time, 2003/2004, there was increasing evidence that glutamate was not just scavenged by astrocytes, but was also released from astrocytic vesicles. It released in response to calcium elevations within the cell. Existing methods for measuring this release were somewhat crude, so it seemed a great test system for GluSnFR.

Unfortunately, since there seemed to be no specialized areas on the astrocyte where the vesicles fused, and the release rate was relatively slow, we were unable to detect glutamate release with GluSnFR. I thought this might be a problem of not knowing when and where to look. So my collaborator, Yongling Zhu, and I expressed pHluorins fused to VAMP or to synaptophysin in astrocyte cultures. When we looked at them under the microscope, they just looked green, no action…

But then we left the excitation light on for a few minutes. I happened to look back into the scope after they had been bathing in bright blue light and was astonished. I could directly see, by eye, spontaneous bursts of fluorescence across the cells. It was absolutely magnificent. The long application of light had bleached all of the surface expressed, bright pHluorins. But the pH-quenched pHluorins in the vesicles were resistant to bleaching. On this dimmer background, the fusion events were plain as day.

Unfortunately, the green color overlapped with the emission of GluSnFR, so we couldn’t use it for a spatiotemporal marker of when and where to look for glutamate release. We tried using some ph-sensitive precursors to mOrange and mOrange2, developed by Nathan Shaner, but these seemed to block the release events. Since then, others have shown the functional relevance of glutamate release from astrocytes, and I turned the focus of GluSnFR measurements to synaptic spillover. This was one of the projects that was tantilizingly close, but got away. This movie of VAMP-pHluorin is almost five years old now, but it still looks cool… Enjoy!

If you are curious, this is what the Synaptophysin-mOrange looked like when we expressed it in hippocampal neuron cultures. Ammonium Chloride caused a massive fluorescence increase, by alkalizing the synaptic vesicles. Unfortunately, we never were able to see release via electrical stimulation. Details are in my thesis. Maybe someone else wants to give it a shot?





2008 Nobel Prize in Chemistry to GFP

8 10 2008

This morning, the Nobel committee recognized the work of Osamu Shimomura, Martin Chalfie and Roger Tsien “for the discovery and development of the green fluorescent protein, GFP” by awarding them the Nobel Prize in Chemistry for 2008.  A video of a great lecture on fluorescent proteins by Roger Tsien is available here.

Green Fluorescent Protein

Green Fluorescent Protein

Shimomura first discovered GFP during the study of the bioluminescent protein aequorin, the mechanism by which certain jellyfish glow.  In the footnote to his seminal paper on aequorin purification, he noted the additional presence of “a protein giving solutions that look slightly greenish in sunlight through only yellowish under tungsten lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of a Mineralite, has also been isolated from squeezates.” 

Aequorea Victoria

Aequorea Victoria

Chalfie took the cDNA of GFP and first expressed it bacteria and worms.  He demonstrated GFP could be used as a molecular tag. Surprisingly, the protein folded and functioned without the use of co-factors specific to the jellyfish.

Tsien developed GFP into the many useful variants we use today.  He reported the S65T point mutation that greatly improved its fluorescent characteristics. His lab also evolved GFP into many other color variants, and demonstrated that these variants could be used as genetically-encoded intracellular sensors for calcium, enzyme action, and glutamate.

Chromophore of the S65T mutant of GFP

The odd man out in this triumvirate is Douglas Prasher.  With a tiny lab and budget, Prasher discovered the primary sequence of GFP and cloned the cDNA of GFP. Unfortunately, around the time of his work’s publication, his grant ran out. Prasher sent out DNA samples to Chalfie, Tsien and others, shut down his lab and left science. Prasher’s contribution was the essential foundation for the explosion of developments in the field.

Some argue that Tsien would have already won the Nobel prize for calcium signaling if not for his contribution to GFP. As a graduate student, he invented the high affinity calcium chelator BAPTA. Using BAPTA as a foundation, he created a large family of fast, bright calcium dyes, including fura-2.  Nearly every fluorescent dye for calcium was either his invention or a close variant of one of these. The importance of these tools for understanding intracellular communication cannot be overstated.

Transgenic GFP mouse





The great GECI shootout

21 07 2008

Dierk Reiff’s lab has done another head-to-head in vivo showdown between various GECIs and a synthetic dye. Their paper, Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in vitro, is very interesting and deserves a full write-up. I will present a detailed analysis of the paper in a future update.  For now, check the abstract.

Recent advance in the design of genetically encoded calcium indicators (GECIs) has further increased their potential fordirect measurements of activity in intact neural circuits. However, a quantitative analysis of their fluorescence changes ({Delta}Fin vivo and the relationship to the underlying neural activity and changes in intracellular calcium concentration ({Delta}[Ca2+]i) has not been given. We used two-photon microscopy, microinjection of synthetic Ca2+ dyes and in vivocalibration of Oregon-Green-BAPTA-1 (OGB-1) to estimate [Ca2+]i at rest and {Delta}[Ca2+]i at different action potential frequencies in presynaptic motoneuron boutons of transgenic Drosophila larvae. We calibrated {Delta}F of eight different GECIs in vivo to neural activity, {Delta}[Ca2+]i, and {Delta}F of purified GECI protein at similar {Delta}[Ca2+in vitro. Yellow Cameleon 3.60 (YC3.60), YC2.60, D3cpv, and TN-XL exhibited twofold higher maximum {Delta}F compared with YC3.3 and TN-L15 in vivo. Maximum {Delta}F of GCaMP2 and GCaMP1.6 were almost identical. Small {Delta}[Ca2+]i were reported best by YC3.60, D3cpv, and YC2.60. The kinetics of {Delta}[Ca2+]i was massively distorted by all GECIs, with YC2.60 showing the slowest kinetics, whereas TN-XL exhibited the fastest decay. Single spikes were only reported by OGB-1; all GECIswere blind for {Delta}[Ca2+]i associated with single action potentials. YC3.60 and D3cpv tentatively reported spike doublets. In vivo, the KD(dissociation constant) of all GECIs was shifted toward lower values, the Hill coefficient was changed, and the maximum {Delta}F was reduced. The latter could be attributed to resting [Ca2+]i and the optical filters of the equipment. These results suggest increased sensitivity of new GECIs but still slow on rates for calcium binding.





Sensing salty currents with Mermaids

16 07 2008

A new genetically-encoded voltage sensor paper is out from a friend and former mentor of mine, Atsushi Miyawaki. One memorable moment when working in his lab during the RIKEN summer program of 2002 was when Atsushi took me into his office and whipped out a custom green laser pointer. These had been banned in Japan, as fans would shine their powerful light into the eyes of pitchers and batters at baseball games. Atsushi was really proud of his. He smiled and then started sweeping the light point over the rocks in his fishtank. Each ‘rock’ was actually coral his lab had collected from fluorescent protein hunting trips, and each glowed a different color when the green light hit it. He has been putting these novel discoveries to good use.

In Improving membrane voltage measurements using FRET with new fluorescent proteins, Tsutsui et. al take two fluorescent proteins discovered and engineered by the Miyawaki lab, mUKG and mKOk, and graft them onto the Ci-VSP scaffold used in VSFP2.1 (also developed at RIKEN).  The green and orange fluorescent proteins undergo significant FRET transfer which is voltage dependent.  They get 40% dR/R per 100mV with a 2 component association rate of around 10 and 200ms. Unsurprisingly, the kinetics speed up at physiological temperatures to 5-20ms on and off.  They are able to pick up single pseudo-action potentials in Neuro2A cells, though the response is highly filtered. They are also able to see very clear spontaneous waves of potential change in cardiomyocytes (23% dR/R) and single spikes in cultured neurons (2% dR/R for 1AP). They dub this voltage sensor “Mermaid”.

The authors state that they used the new FPs due to their improved photostability and especially pH resistance. 

Additionally, because Aequorea GFP variants are pH-sensitive, and neuronal activity causes considerable acidification, the responses of sensors to depolarization in intact neurons may be overwhelmed by sustained changes resulting from acidification.

Granted that mOrange2 is pretty pH-sensitive, but I’m not sure this is a real issue, or a potential issue to justify using their new FPs.  From the spectra of mUKG vs. EGFP, it would seem that EGFP’s 10nm further redshifted emission would be a superior FRET pair for mKOk.  It smells like there may be a bit of bundling of various independent projects into this paper.  However, they do make a good point that this pair will have a different preferred dipole orientation than existing FRET pairs, which could lead to improved performance in some constructs.  

Things I’m still wondering :

  • Have they tried using the improved VSFP3.1 scaffold? This was shown to be much faster than 2.1.  I suspect the mUKG is not as tolerant to C-terminal truncation than CFP and GFP.  
  • What about using EGFP as the donor?  Could you then use the VSFP3.1 scaffold?
  • Is there a rapid non-FRET quenching of the donor upon depolarization as seen in VSFP3.1?
  • Why is the single wavelength fluorescence increasing in both channels in figure 2d?  Is there some photoactivation going on?
  • I’d love to see a head to head comparison of VSFP3.1 and Mermaid under identical conditions. Also responses in brain slice at physiological temperatures.







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