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?
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