Symposium : A Revolution in Fluorescence Imaging

11 02 2009


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


Styryl dyes may inhibit synaptic release

19 11 2008

A new report in PNAS, Probing synaptic vesicle fusion by altering mechanical properties of the neuronal surface membrane, from Chuck Stevens’s lab, raises a serious concern about using styryl dyes to study release probability of synapses.  Styryl dyes, such as FM 1-43, partition into cell membranes and have been commonly used to measure synaptic release of vesicles in culture and brain slice.  The protocol is simple, bathe the neurons in dye, electrically stimulate to cause massive synaptic release and then dye uptake via vesicular endocytosis, wash off the dye, then observe the rate of destaining of the synapses following electrical stimulation.  This rate is directly related to the number of vesicle fusions during the final stimulation period. There is just one problem, Zhu and Stevens report that the presence of the dye reduces the release probability of the synapse.


FM 4-64 reduces synaptophysin-pHluorin response

FM 4-64 reduces synaptophysin-pHluorin response

They observed this by taking the FM dye measurements in neurons that expressed synaptophysin-pHluorin.  The fluoresence increase from the pHluorins was reduced in a [dye] dependent manner. A 15uM concentration of dye (not atypical for published experiments) reduced the pHluorin signal by 40%.  The dye had no effect on the calcium levels in the presynaptic terminals, indicating it was potentially due to an increased energetic cost of forming a fusion pore. Chuck then weaves together some basic principles with this data to make an estimate of the fusion pore size.  

While this paper may seem to cover a minor technical point, FM dyes have been used to make numerous inferences about presynaptic release properties, modes of vesicle recycling, the locus of LTP expression, and other basics of synaptic physiology. The thing that’s bugging me is if this effect is as prominent as advertised, how did people not notice the change in release probability with electrophysiological techniques?

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.

Giving synapses a ‘born on’ label

30 06 2008

Memories are thought to be encoded by the patterns of synaptic connections in the brain. Learning can either delete or change the strength of existing synapses, or add new synapses. Following a learning process, how can we tell which synapses were added to encode this new memory?  

One strategy is to make a timelapse movie of the synapses.  In mice, this can be accomplished by installing a cortical window on the skull, and imaging the changes in structure of GFP labelled neurons. However, this is technically demanding, only works with sparsely labeled neurons, and accesses only a small subset of the neurons which may be involved in the learning process.  

Ideally, one could have a tag which can discriminate between synapses existing before learning takes place, and new ones generated after learning has occurred. Whole brain regions could then be examined at a single timepoint to see where new synapses were added. In a large step towards that goal, Michael Lin et. al, from the lab of Roger Tsien, report TimeSTAMP, a genetic label for newly synthesized protein.

The authors engineered the NS3 protease from the hepatitis C virus (HCV) to cleave itself at just the right pace. They then fuse tags (fluorescent proteins or epitopes) before and after the cleavage site. This fusion is then tagged to the end of a protein of interest. Shortly after synthesis, the protein cleaves off the C-terminal tag, but the N-terminal is left on. This cleavage is inhibitable by a variety of small molecule blockers. In the presence of the blocker, the C-terminal tag stays on. By controlling when drug is applied, they can selectively label a set of proteins of a particular age with the tags.

The choice of NS3 protease was very clever, as it is a favorite drug target of biotech and pharma companies.  Many inhibitors of this protein have been synthesized, exhaustively characterized in vitro and in clinical trials. This work is a great example of the standard research flow going in reverse; a basic-science project from an academic lab is actually benefitting from pharma company research. Stability, bioavailablity and toxicity have already been worked out.  One of the biggest impediments is actually getting ahold of these compounds. Companies with their survival hanging on the clinical success of a single small molecule inhibitor are understandably reluctant to hand out stocks for academic research. Note the roller coaster stock price of Vertex following results of its NS3 protease inhibitor (VX-950) trials. 

The authors use PSD-95 tagged to TimeSTAMP as a proxy marker of synaptic age. In neuronal culture, they show that newly synthesized synapses have a C-tag / N-tag ratio of about twice as large as old synapses.

They extend the technique to whole fruit fly brains, showing a very heterogeneous distribution of CaMKII synthesis across Kenyon cells in different areas of the mushroom body.

So far TimeSTAMP has not been shown to work in mice. Mice were not included in the paper due to the long generation time for transgenics. Given the good signal to noise and the large number of possible inhibitor molecules, I think this technique could be quite powerful in mammalian systems. It’s big advantage would be to label large populations of neurons or synapses in diverse brain regions, including those inaccessible to two-photon microscopy. TimeSTAMP’s success in labeling new synapses in the intact brain will be dependent on finding a protein to tag at the synapse with low turnover over the course of a learning experiment. Though PSD-95 appears to be a reasonable marker in culture, others have shown a higher rate of turnover in vivo, making in unsuitable for a synaptic marker. 

Three quick paper picks

18 10 2007

Here are three papers that are worth reading over. No time for full reviews.

New Single-FP GECIs
The Russian fluorescent protein team has come out with some new single fluorescent protein G-CaMP/pericam-like sensors. They fiddled with the linker sites at the 145 and 148AA insertion points and found a great deal of fluorescence sensitivity to the amino acid composition at those sites. They note two new sensor constructs Case12 and Case16 that have 12-16.5x maximal changes in fluorescence upon calcium binding, a significant improvement over G-CaMP2. The tradeoff appears to be that they are dimmer. They show calcium responses in HeLa, PC-12 and cortical neuron cells, but no direct head-to-head with other sensors in cells.

Multipoint multiphoton microscopy
In this technical paper, an MIT group led by Peter So examines some issues surrounding multipoint excitation multiphoton microscopy. In theory, multipoint excitation will dramatically increase image acquisition rate through parallelization. However, this comes at the expense of large increases in background scattered light, which reduces optical resolution and penetration depth. They present an imaging system using multianode photomultiplier tubes that lets them acquire an 8×8 grid of multiphoton excitiation points. This technique, plus post-hoc deconvolution allows them to approach the resolution and depth of single point multiphoton systems, with a parallel array.

Effects of linker length and stiffness on FRET
This paper from Harold Erickson’s group carefully examines the role of linker length and stiffness in determining the amount of FRET transfer between CFP and YFP derivatives. They show that intrinsically unstructured domains of identical amino acid length produce significant differences in FRET between tethered FPs. They propose a formula for estimating the stiffness of linkers from the degree of FRET, which corroborates a 2006 study by Evers et al. They also demonstrate that the reported enhancement of FRET between the CyPet and YPet pair is due to an enhanced tendency for the proteins to dimerize. This reinforces my thinking that the most needed and generalizable improvement to two-FP FRET systems is an enhanced photostability of the FRET acceptor. Somebody please screen for a bleach-resistant YFP!  Props to Michael Lin for pointing out the paper.