Three ways of looking at touch coding

At SfN, a block of three posters by myself, Simon Peron and Daniel O’Connor will showcase three ways to approach the problem of touch coding.

My work on whisker force measurements, and single cell and silicon probe based cortical recordings during active objection localization :

Program#/Poster#:	677.18/KK18
Presentation Title:	Encoding whisking-related variables in the mouse barrel cortex during object localization
Location:	Hall F-J
Presentation time:	Tuesday, Oct 16, 2012, 2:00 PM – 3:00 PM
Authors:	*S. A. HIRES, D. O’CONNOR, D. GUTNISKY, K. SVOBODA; Janelia Farm Res. Campus, ASHBURN, VA

Simon Peron’s work on recording a complete representation of touch using in-vivo imaging with new G-CaMP variants during a similar behavior :

Program#/Poster#:	677.12/KK12
Presentation Title:	Towards imaging complete representations of whisker touch in the mouse barrel cortex
Location:	Hall F-J
Presentation time:	Tuesday, Oct 16, 2012, 4:00 PM – 5:00 PM
Authors:	*S. P. PERON1, V. IYER2, Z. GUO2, T.-W. CHEN2, D. KIM2, D. HUBER3, K. SVOBODA2;

Daniel O’Connor’s work on constructing synthetic perception of touch and object localization via cortical cell-type specific optogenetic stimulation during behavior :

Program#/Poster#:	677.06/KK6
Presentation Title:	Neural coding for object location revealed using synthetic touch
Location:	Hall F-J
Presentation time:	Tuesday, Oct 16, 2012, 2:00 PM – 3:00 PM
Authors:	*D. H. O’CONNOR1, S. A. HIRES1, Z. GUO1, Q.-Q. SUN2, D. HUBER1, K. SVOBODA1;

This is a must-see session for people interested in touch coding, the whisker system, in-vivo cortical imaging, or synthetic perception via optogenetics.

I hope to see you there.

Rapid warping of two-photon illumination wavefronts

A short paper in Optics Express looks interesting.  In A high speed wavefront determination method based on spatial frequency modulations for focusing light through random scattering media, Meng Cui presents a method for rapidly determining the optimal wavefront to ‘cancel out’ the scattering when 785nm light passes through turbid media.  In his example, a glass diffuser was used, but the clear goal for this work is to replace the glass with a brain.

To understand why this is so important for in vivo two-photon imaging, let’s review how 2-p imaging works. Light from a laser is focused to a point and swept across the field in a raster. The resulting fluorescence is of a different wavelength and can thus be filtered out from the excitation light. For each voxel, all the fluorescence that re-enters the objective is collected, regardless of its source.  The total amount of fluorescence collected for that timepoint in the sweep is assigned as the brightness of that voxel. Since the user knows where the laser was being aimed, scattering of fluorescence emission may reduce the brightness but will not blur the image.  However, scattering of the excitation light can dramatically reduce the excitation at the target voxel while increasing the off-target excitation of its neighbors. This causes a rapid increase in background fluorescence and blur at increasing brain depth.

The vasculature was labeled by injecting flourescein dextran into the circulatory stream. The light source was a regenerative amplifier. ‘‘0 mm’’ corresponds to the top of the brain. Left, XZ projection. Right, examples of XY projections. Note the increase in background fluo- rescence deeper than 600 mm in the brain due to out-of-focus 2PE. (Theer et al., 2003)

Previous reports work has shown that one can use adaptive optics to adjust the phase of the wavefront of the excitation light to correct for the scattering of the excitation.  However, determination of the optimal wavefront for a field of view took minutes, which could be problematic for imaging in an awake animal.  Any changes in the precise position of the brain might change the optimal wavefront.  Ideally, one would want a system that could optimize the wavefront every second, or even before every frame of acquisition (typically 4-8 Hz in a raster scan in vivo experiment)

Scattering in the brain warps two-photon excitation light, but adaptive optics can correct this.

I’ll let Meng Cui explain the technique in his own words

Elastic scattering is the dominant factor limiting the optical imaging depth in tissues. Take gray matter as an example, at 800 nm the scattering coefficient is 77 /cm and the absorption coefficient is 0.2 / cm. If there is a way to suppress scattering, the optical imaging depth could be greatly improved. Despite the apparent randomness, scattering is a deterministic process. A properly engineered wave can propagate inside scattering media and form a focus, a well understood phenomenon in the time reversal and optical phase conjugation (OPC) studies…

For applications on biological tissues, acquisition time on the order of one millisecond (ms) per degree of freedom is desired. Deformable mirrors can provide a high modulation speed. However the degrees of freedom are rather limited. A phase-only SLM can provide about one million degrees of freedom at a much lower modulation speed. In this work, I present a novel method, capable of providing as many degrees of freedom as a SLM with a data acquisition time of one ms per degree of freedom. The method was employed to focus light through a random scattering medium with a 400 ms total data acquisition time, ~three orders of magnitude faster than the previous report [25].

The essence of a COAT system is to phase modulate different input spatial modes while detecting the output signal from the target. To greatly improve the operation speed, the experiment requires a device that can provide fast phase modulation and can access a large number of spatial modes very quickly. To meet these two requirements, a pair of scanning Galvanometer mirrors was used to quickly visit different modes in the spatial frequency domain or k space, and a frequency shifted reference beam was provided for a heterodyne detection. The wavefront profile was first determined in k space and then transformed to the spatial domain. The spatial phase profile was displayed on a SLM to focus light onto the target. In such a design, the number of degrees of freedom is limited by the number of pixels on the SLM and the experiment speed is determined by the scanning mirror speed…

Compared to existing techniques, the reported method can provide both a high operation speed and a large number of degrees of freedom. In the current design, the operation speed is limited by the scanning mirror speed and the maximum number of degrees of freedom is limited by the SLM pixel number. In this demonstration, 400 spatial modes in k space were visited and the determined phase profile was displayed on the SLM. Depending on the scattering property of the media, more (up to 1920 x 1080) or less number of degrees of freedom can be used to optimize the focus quality and the operation speed.

Using a stepwise position scanning, the method achieves an operation speed of one ms (400 μs transition time + 600 μs recording time) per spatial mode, ~three orders of magnitude faster than the previous report. Using a continuous position scanning and a faster position scanner such as resonant scanning mirrors, polygon mirror scanners, or acousto-optic deflectors, the operation speed can be potentially increased by at least one order of magnitude. It is anticipated that the reported technique will find a broad range of applications in biomedical deep tissue imaging.

Cameleon-Nanos : High Affinity GECIs

Takeharu Nagai’s lab has published in Nature Methods, Spontaneous network activity visualized by ultrasensitive Ca²⁺ indicators, yellow Cameleon-Nano, demonstrating a new set of calcium indicators based on yellow cameleon. Back when he was still Take-san, Take’s ability to churn out and manually screen hundreds of cameleon variants was impressive and inspiring. With high-throughput GECI pipelines now ramping up at Janelia, the idea of laboriously screening 200 variations on a theme (be it cameleons or GluSnFRs), seems a bit archaic. However, this paper is a good example of the progress that can still be made by understanding the needed sensor parameters and fiddling with the primary amino acid structure in a relatively low-throughput way. Take-sensei’s results are another example of the pramatic rule in protein design, “when in doubt, tinker with the linker.”

The cameleon-nano family achieves greater apparent calcium affinity than YC2.60-4.60, reaching levels of up to 15nM.  They did this by increasing the flexibility of the linker by extending the standard Tsien/Miyawaki/Baird Gly-Gly-Ser linker with additional glycines.  In this case, the longer the linker between the CaM and M13 segments, the greater the apparent affinity. Interestingly, improvement by increasing linker flexibility is precisely the opposite the advice Atsushi and Take gave me for achieving high ratio changes with FRET reporters.  Back at RIKEN in 2002, they suggested I use short, stiff linkers to restrict the rotational freedom of the fluorescent pairs.  Then one could find orientations where relative rotation of dipole moment gave much greater FRET changes than would be expected from changes in FP distance alone. Take and Atsushi’s big YC2.60/3.60 paper strongly supported this idea!  However, as our understanding of the ideal parameters of calcium sensor’s for in vivo imaging has grown, development directions have adjusted.

Cameleon-Nanos achieve higher signal/noise for sparse action potentials at the expense of linearity.  Like Fluo-4, the signal saturates at relatively low AP frequencies.  I think the absolute affinities measured for this family (15, 30, 50 and 140nM) should be considered very rough estimates. They extrapolated these values from stopped-flow binding experiments, because

Although we would like to measure the koff of YC2.60 and its high affinity variants such as YC-Nano15, we could not do it because it was very difficult to precisely control free Ca2+ concentration at around few tens of nM as far as we used EGTA (Kd for Ca2+ = 151 nM in 0.1 M ionic strength, pH 7.2 at 25 oC). For this purpose, much stronger Ca2+ chelator with a smaller Kd value was required. However there is no such Ca2+ chelator available now.

I’m not sure why they didn’t just use the higher affinity, Mg++ insensitive, chelator BAPTA to make the Kd measurements the right way, with a linear regression of log-log fluorescence/concentration values.  Due to instrument dead time, and the high affinity, I didn’t like stopped-flow based Kd measurements in the early GCaMP papers, and I don’t like them now.  Also, the apparent calcium Kd will be highly dependent on solution ionic strength and [Mg++] which is unreported. Despite these quibbles, which are important only inasmuch as they give insight into the mechanism of improvement and the direction of future development, the cameleon-nano family looks promising for mammalian brain imaging.  I still wonder if, assuming the reported Kd values are relevant in vivo, YC2.60 would be the best of the bunch, since cortical neurons have a resting Kd of ~50nM, which implies that a single AP transient of say 200nM free [Ca++] increase would push the calcium levels right up into the sweet-spot of YC2.60’s sensitivity.

This is all the more interesting given the recent results in YC3.60 imaging from Maz Hasan’s group.  Previously, he had shown that transgenic YC animals were pretty bad for imaging.  However, AAV-mediated gene delivery of YC3.60 has significantly improved the responses of the YC family. I’m not sure if they are really up to GCaMP3 levels under identical in vivo conditions, but they might have better long-term protein stability (or that might depend on which viral serotype is used.) What about cameleon-nanos, what about YC2.60?

Journal Scan – Calcium Imaging in Auditory and Visual Cortex

A few papers on in vivo calcium imaging have just come out and are worth a careful read.

The first two examine the fine organization of layer 2/3 of the mouse auditory cortex.  The canonical view of auditory cortex organization is that neurons are arranged in a tonotopic pattern, with a smooth gradient in auditory frequency tuning across the surface of the cortex.  Using two-photon imaging in anesthetized mice, the groups saw that, while there was an overall gradient, the tuning of neighboring neurons was highly variable.  These are similar results to what Sato et al and Kerr et al found in the whisker barrel cortex back in 2007.  Moral of the story : mapping brain organization by microstimulation or sparse sampling (as in the classic papers) can be very misleading.

UPDATE : David Kleinfeld kindly directed me to the 40 year old work by Moshe Abeles and others that showed a similar spread in frequency tuning using microelectrodes…

Now, back to the more recent papers…

Functional organization and population dynamics in the mouse primary auditory cortex – Rothschild G, Nelken I, Mizrahi A. Nat Neurosci. 2010 Mar;13(3):353-60. Epub 2010 Jan 31.

Cortical processing of auditory stimuli involves large populations of neurons with distinct individual response profiles. However, the functional organization and dynamics of local populations in the auditory cortex have remained largely unknown. Using in vivo two-photon calcium imaging, we examined the response profiles and network dynamics of layer 2/3 neurons in the primary auditory cortex (A1) of mice in response to pure tones. We found that local populations in A1 were highly heterogeneous in the large-scale tonotopic organization. Despite the spatial heterogeneity, the tendency of neurons to respond together (measured as noise correlation) was high on average. This functional organization and high levels of noise correlations are consistent with the existence of partially overlapping cortical subnetworks. Our findings may account for apparent discrepancies between ordered large-scale organization and local heterogeneity.

In vivo two-photon calcium imaging from dozens of neurons simultaneously in A1.

Dichotomy of functional organization in the mouse auditory cortex – Bandyopadhyay S, Shamma SA, Kanold PO. Nat Neurosci. 2010 Mar;13(3):361-8. Epub 2010 Jan 31.

The sensory areas of the cerebral cortex possess multiple topographic representations of sensory dimensions. The gradient of frequency selectivity (tonotopy) is the dominant organizational feature in the primary auditory cortex, whereas other feature-based organizations are less well established. We probed the topographic organization of the mouse auditory cortex at the single-cell level using in vivo two-photon Ca(2+) imaging. Tonotopy was present on a large scale but was fractured on a fine scale. Intensity tuning, which is important in level-invariant representation, was observed in individual cells, but was not topographically organized. The presence or near absence of putative subthreshold responses revealed a dichotomy in topographic organization. Inclusion of subthreshold responses revealed a topographic clustering of neurons with similar response properties, whereas such clustering was absent in supra-threshold responses. This dichotomy indicates that groups of nearby neurons with locally shared inputs can perform independent parallel computations in the auditory cortex.

Tonotopy exists in A1 and AAF on a large scale, but not on small spatial scales.

The third paper uses a GECI (YC3.6) to do chronic imaging in visual cortex. Their results are noteworthy in that they look at visual responses to both a passive viewing and an ACTIVE discrimination task in an awake, head-fixed mouse.  The patterns of neural activity between anesthetized, awake but passively receiving sensory input, and awake while paying attention and using the sensory input are likely to be hugely different. Recording from neurons that are actively involved in a discrimination task is essential to understanding how the cortex is actually processing information.  Although this paper is more focused on simply presenting the technique rather than in depth analysis of the activity, we will be seeing more of this style of neuroscience in high-profile journals very soon…

Chronic cellular imaging of mouse visual cortex during operant behavior and passive viewing  –  Andermann ML, Kerlin AM and Reid RC, Front. Cell. Neurosci. 4:3.

Nearby neurons in mammalian neocortex demonstrate a great diversity of cell types and connectivity patterns. The importance of this diversity for computation is not understood. While extracellular recording studies in visual cortex have provided a particularly rich description of behavioral modulation of neural activity, new methods are needed to dissect the contribution of specific circuit elements in guiding visual perception. Here, we describe a method for three-dimensional cellular imaging of neural activity in the awake mouse visual cortex during active discrimination and passive viewing of visual stimuli. Head-fixed mice demonstrated robust discrimination for many hundred trials per day after initial task acquisition. To record from multiple neurons during operant behavior with single-trial resolution and minimal artifacts, we built a sensitive microscope for two-photon calcium imaging, capable of rapid tracking of neurons in three dimensions. We demonstrate stable recordings of cellular calcium activity during discrimination behavior across hours, days, and weeks, using both synthetic and genetically-encoded calcium indicators. When combined with molecular and genetic technologies in mice (e.g., cell-type specific transgenic labeling), this approach allows the identification of neuronal classes in vivo. Physiological measurements from distinct classes of neighboring neurons will enrich our understanding of the coordinated roles of diverse elements of cortical microcircuits in guiding sensory perception and perceptual learning. Further, our method provides a high-throughput, chronic in vivo assay of behavioral influences on cellular activity that is applicable to a wide range of mouse models of neurologic disease.

Mapping visual responses in identified excitatory and inhibitory neurons in awake mice

BrainStorm 1 : The Calcium Memory Sensor

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.

Some interesting posters @ SfN

Here’s a few posters that caught my eye at SfN.  Click the meeting planner for the full abstract

Optimizing two-photon activation of channelrhodopsin-2 for stimulation at cellular resolution

J. P. RICKGAUER^1,2, D. W. TANK^1,2; 

Spiral pattern of 2-photon excitation can drive neurons to spike.  A low NA objective helps. Need to do piezo-based Z-scanning if you use high NA, don’t with low NA.

In vivo two-photon imaging 1 mm deep into cortical brain tissue with novel microprism probe 

*T. H. CHIA, M. J. LEVENE; 

A cute method to image 1mm into cortex with 2-photon imaging. They used 2-6 month old mice. The just took a triangular prism whose hypotenuse was silvered and stuck it in the cortex. Then they internally reflected the beam off the prism and fired it sideways into cortex. Got good SNR to 300um lateral distance.  Some clippling of beam at edges of the prism gave somewhat inconsistent spatial resolution.

Self-complementary adeno-associated viral vectors for fast, efficient labeling of neurons and astrocytes in visual cortex in vivo

R. L. LOWERY¹, Y. ZHANG², C. LAMANTIA¹, B. K. HARVEY³, A. K. MAJEWSKA¹; 

AAV is the way to go for expression of GECIs and ChR2 in vivo, but it takes a long time to express at high levels (2 weeks). They show that using a double stranded DNA version of AAV rather than single stranded gets protein expression up high much faster. Very high expression after one week. This is because the virus doesn’t need to take the time to make the second strand before expressing the protein.  See Xiao, X J. Virol 1998

Detection of single action potentials in vitro and in vivo with genetically-encoded Ca²⁺ sensors

S. MEYER ZUM ALTEN BORGLOH¹, D. J. WALLACE², S. ASTORI³, Y. YANG³, M. BAUSEN³, S. KUGLER⁴, M. MANK⁵, O. GRIESBECK⁵, J. NAKAI⁶, A. MIYAWAKI⁶, A. E. PALMER⁷, R. Y. TSIEN⁷, R. SPRENGEL³, J. N. D. KERR², W. DENK³, M. T. HASAN³; 

Everything in the poster was in the Nature Methods paper.  Conversation reveled that YC3.60 works as well or better than D3cpv. Only have done up to whisker evoked stimulation, no imaging of spontaneous YC3.60 signals yet.

Characterization of improved probes for the hybrid voltage sensor method of voltage imaging

D. WANG¹, Z. ZHANG², B. CHANDA¹, M. B. JACKSON¹; 

A nice little sensor optimization poster.  They took the hVOS hybrid voltage sensor of dipicrylamine with membrane tethered GFP and improved it by changing the chromophore to Cerulean, and by using the “membrane-staple” strategy. Having membrane anchors on both the N and C-termini gave better quenching. Fast response, ~0.5ms, and 20% dF/F.

Crystal structure of the genetically encoded calcium indicator gcamp2

*J. AKERBOOM¹, L. TIAN¹, S. VISWANATHAN¹, S. A. HIRES¹, J. S. MARVIN¹, E. R. SCHREITER², L. L. LOOGER¹; 

Jasper made crystal structures of G-CaMP2 in the apo and bound states.  Bound states crystalized as a heterodimer, but he was able to also crystalize the monomer. The structures show a pore to the chromophore in the apo state that is plugged in the Ca-bound state. Thus, the quenched apo state is due to solvent access to the chromophore.  This structural data should help rational design of better G-CaMP sensors.

Deep & local Channelrhodopsin-2 two-photon activation

An interesting paper on two-photon activation of channelrhodopsin-2 is out in Biophysical Journal. In In-depth activation of ChR2 sensitized excitable cells with high spatial resolution using two-photon excitation with near-IR laser microbeam, Mohanty et. al show cellular activation with a fast-scanning two-photon laser.

Action potential generation from Channelrhodopsin-2 with a two-photon beam has been difficult to achieve, presumably due to the small activation volume of the 2p spot. They show similar calcium transients in response to 2p stimulation as with one-photon stimulation. As depth increases, the one-photon response attenuates faster than the two-photon. Unfortunately, the supplemental info with  electrophysiology traces are not yet online.  Presumably, they are generating action potentials, but I’d like to see the raw data.  Interestingly, they also show calcium increases when the laser stays in once place.  This would imply that local depolarization causes local voltage-gated calcium channels to open, or that calcium is getting through the ChR2. I was under the impression that ChR2 has a low conductance for calcium, though this study by Caldwell et. al, in press for JBC, uses ChR2 specifically for its calcium permeability.

I’m not sure what to make of the first paper. Are they really able to fire action potentials with two-photon stimulation, at depth?  Or are the calcium traces they are seeing simply the result of localized calcium flux.  I’ll followup once the Supplemental Data becomes available.  Still worth a look if this is the sort of thing you are interested in.

Pulse shaping for 2-photon signal enhancement

Gains in signal to noise ratios of organic dyes and genetically encoded indicators often come in modest steps following screening of large numbers of compounds or clones. Improvements are usually specific to individual chromophores, leading to the pigeonholing of development efforts on a small handful of indicators that have already undergone systemic optimization (i.e. cameleons, G-CaMP and troponin-based GECIs). Indicator photobleaching imposes strict limits on the amount of information which can be extracted by optical indicators. Improvement of specific indicators and their constituents is a worthy and necessary goal, but more generalizable improvements can be made by changing the nature of the illumination source. A series of papers from a variety of groups has shown that careful manipulation of the structure of pulse laser illumination can produce dramatic improvements in signal/noise and photobleaching during non-linear (two-photon) imaging. This is generalizable to numerous optical indicators. Reduction of photobleaching and photoinduced tissue damage will be essential for continuous optical monitoring of sparse neural activity.

 My first encounter with these techniques came in 2003 during a lab presentation by Atsushi Miyawaki, who showed intriguing results with two-photon illumination of GFP. Kawano et al. shined ultra-short (28 femtosecond) pulses from a Ti-Sapphire laser on a plate of immobilized GFP. Due to the uncertainty principal, these ultra-short pulse durations cause a broad spread (~100nm) in the frequency of the laser pulse. They then actively modulated the phase of different frequency bands of the pulse. The interesting part is that they coupled this modulation to a feedback genetic algorithm that sought to increase the ratio of the GFP fluorescence to the intensity of the laser input. Over several hundred iterations of modulation, the system learned how to dramatically increase the output fluorescence over input power by tuning the phase of the frequency components. Using these optimally shaped pulses, they reduced the photobleaching rate by a factor of four! This was an impressive result, but it is unclear how useful this technique would be to live samples, in their heterogeneous aqueous environments. The tuning parameters might be less stable across a non-uniform sample.

The above paper raises intriguing questions on the nature of two-photon excited states and photobleaching. GFP and other fluorescent proteins have multiple bleaching modes, some permanent, some dark or UV-reversible. A more clear understanding of the photochemistry of bleaching could lead to improved illumination pulse design that could keep the chromophore away from these undesired states.

Early last year, Stenfan Hell’s group demonstrated a dramatic reduction in one and two-photon photobleaching by avoiding recurrent excitation of the GFP chromophore when it was in a dark absorbing state.Standard two-photon imaging procedure is to illuminate with a Ti-Sapphire laser pulsed at 80MHz, with a interpulse gap of 12.5ns. This gap is five times longer then the 2.4ns fluorescent lifetime of EGFP, giving the chromophore plenty of time to emit a photon and decay from the excited singlet S1 state. But is the singlet state the precursor to most photobleaching? Donnert et al. varied the pulse rate from 40 to 0.5MHz and discovered that photobleaching was dramatically reduced at the lower pulse rates, especially below 1MHz (1us interpulse interval). Under one photon illumination, total photons extracted from GFP before bleaching was increased 20-fold, while the rhodamine dye Atto532 increased by 8-fold. This suggests that the primary precursor to photobleaching is not the S1 state but is due to photon absorbtion during a dark triplet state T1, which has a relatively long lifetime of ~1us. Don’t illuminate during this state, and prevent most photobleaching! Under two photon illumination (800nm), even greater reductions in photobleaching of 25 and 20-fold respectively took place. This is particularly important because the much higher illumination power used in 2p excitation normally causes a dramatic, non-linear increase in the rate of photobleaching over 1p imaging in the region of focus.

What is special about the T1 triplet state that makes it more prone to causing photobleaching? Is it simply the longer lifetime gives a greater opportunity for an additional photon to hit, jumping to T2 and inducing a photochemical breakdown of the chromophore or the surrounding residues? Does T1 have a broader range of vibrational energies that can more easily engage the variety of photobleaching reactions than S1? How does the photobleaching rate of S2 compare to T2?

Despite the impressive reduction in photobleaching, and hence the greater S/N for a given bleach rate, Hell’s approach has a major drawback. Slowing the pulse train down ~100-fold also slows acquisition time down 100-fold. Therefore this technique is most useful for fixed specimens. Real-time, high resolution imaging of dynamic processes would be seriously degraded. There are work-arounds, such as wide-field pulsed illumination, rapid laser sweeping or multipoint parallel illumination, but these require additional technical development to make them feasible for the average, or even well-above average investigator.

Is there any related solution that can be easily applied to imaging live, dynamic cells? In this month’s Nature Methods, Na et al. from Eric Betzig’s group present an ‘exciting’ approach. They note that with conventional two photon illumination, most of the available laser power is wasted, intentionally blocked to reduce photodamage. As alluded to above, photodamage increases non-linearly with illumination intensity, making two-photon illumination methods particularly harmful. The authors demonstrate that this damage increases proportional to intensity to the ~2.4 power. To try to attenuate this effect, they used a series of mirrors to split the single ultra-short intense pulse (140 femtosecond) in half, and half again and again and again… They end up with 128 pulses of 1/128 the full intensity nearly evenly spaced every 37 picoseconds. This entire pulse group has a duration of 12ns, and with a 80MHz pulse cycle (12.5ns inter pulse interval) illuminates the chromophore with a steady stream of relatively dim pulsed light.

This split pulse illumination dramatically enhances acquisition speed and signal/noise. Images acquired at a rate of 0.4us/pixel with splitting look far more clear than those acquired at 25.6us/pixel without splitting. Photobleaching is reduced by a factor of four and acute photodamage is also reduced. Additional splitting may be possible and further improve the photobleaching attenuation. Importantly, they demonstrate this technique with GFP in fixed brain slices and in live worms and imagine dynamic responses with a calcium dye in living hippocampal slices. This technique appears to let you eat your cake and have it too. The implementation of the pulse splitting is modular, appears relatively simple to those with customizable two-photon instruments and works with existing Ti-Sapphire lasers. I anticipate rapid adoption by serious imaging labs.

Each of the above advances attacks the problem of indicator photobleaching by a different approach, and each focuses on a different aspect of the photochemistry. Theoretically, one could even combine all three for maximum photon collection efficiency before photobleaching, though this would also require combining the drawbacks of each. Photobleaching will continue to be a major concern in the imaging of dynamic processes, particularly when the signal is not synchronized with the onset of image acquisition. These techniques show substantial progress towards alleviating this concern, and I’m heartened to see a number of excellent labs are focusing so much energy on it.

A final question : How will each of these techniques affect acceptor photobleaching (I’m looking at you Citrine and Venus) in FRET imaging experiments? Do the same processes apply when the excitation is coming from a FRET donor?

Three quick paper picks

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.