Symposium : A Revolution in Fluorescence Imaging

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

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Raw Data : Vesicular Release from Astrocytes, SynaptopHluorange

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?

Optical imaging of neuronal glutamate release and spillover with GluSnFR

This post is difficult to craft. I’ve been struggling with whether to write an epic post describing the history of glutamate imaging, the major advances and players in the field and where I fit into it, or a simple post focused on my new paper. Since glutamate imaging is my field, I’ve got tons to say about it, but also there is probably no way to avoid significant personal bias in my account. So, I’ll go with the short form. For those interested in further reading, please check out these earlier reports, including our brief mention of neuronal glutamate measurements with GluSnFR prototypes, neuronal glutamate measurement with FLIPE and the optimization of FLIPE constructs from Wolf Frommer’s group, and the use of FLIPE’s in brain slice to look at broad patterns of glutamate release from the Huguenard group.

In this PNAS paper, Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters, by Hires et al. from the group of Roger Tsien, the authors report on the optimization of GluSnFR, a genetically-encoded Glutamate Sensitive Fluorescent Reporter, and its application to the study of glutamate spillover. A cyan and yellow fluorescent protein bracket a glutamate periplasmic binding protein. Glutamate binding to the PBP causes a conformational change and a reduction in the amount of FRET between the fluorescent proteins. Glutamate concentration can be quantitatively determined by observing the ratio of the blue to yellow fluorescence. When fused to an extra-cellular membrane targeting motif and expressed in neurons, optical responses to synaptic glutamate release were detected.

FRET constructs are very fickle, with their response being very sensitive to how the sensor components are fused together. This paper clearly demonstrates this, as 176 linker combinations were screened for maximal ratio change and only one was far superior to all others. The optimized SuperGluSnFR showed a 44% ratio change between zero and saturating glutamate levels in Ringer’s solution, a 6.2-fold improvement over the original prototype. Importantly, the screen took place in a system, HEK cells with surface displayed GluSnFRs, that was physiologically similar to the neuronal system where the sensor was ultimately used. This ensured that the screen discovered useful improvements, rather than ones that worked great in the screening system, but did not express or respond well when expressed in neurons. Previous glutamate sensor optimization in bacteria lead to large responses in vitro that did not translate well when ported to surface-displayed plasmids. Note though that this optimized sensor, FLI⁸¹PE, has found use when bath applied to brain slice.

SuperGluSnFR was used to address questions about glutamate spillover. Under what conditions might glutamate spill beyond the synaptic cleft? How long does this spillover last, and what effects might it have? The paper makes the first direct, quantitative measurements of the timecourse of glutamate spillover. It shows that, at least in cell culture, spillover following burst stimulation can cause a significant glutamate transient along the entire dendritic surface, not just at the synaptic active zone. After a single action potential, spillover is insufficient to activate any extrasynaptic glutamate receptors. But, after a burst of stimulation, sub-micromolar glutamate levels persist long enough to activate extrasynaptic NMDA receptors. This could have a tremendous impact on dendritic computation, synaptic independence and heterosynaptic long term potentiation or depression.

There are two significant limitations to the conclusions of this paper. First is that the experiments were done in dissociated hippocampal culture at room temperature. Glutamate transporters are faster at physiological temperatures, and the geometry of the neuropil in vivo might reduce the impact of spillover. Secondly, there is no electrophysiology to directly support the NMDAR activation assertion. Hopefully, some other group will pick up this thread and do more rigorous testing. GluSnFR imaging in acute brain slice should be easy enough using in utero electroporation techniques, and many, many labs have the electrophysiology experience needed. I’m tempted to do the experiments myself, but there is simply no time!

If anyone would like to try SuperGluSnFR for their own work, send me an email and I’ll be happy to send out an aliquot!