Dr. Cui will present “Iterative Multiphoton Adaptive Compensation Technique for Deep Tissue Microscopy.” Fluorescence microscopy has become an indispensable tool for biomedical research. For deep tissue fluorescence imaging applications, two-photon microscopy is perhaps the most widely used technique. Optical wavefront distortions in tissue fundamentally limit the achievable imaging depth. To further improve the imaging depth, wavefront distortions need to be compensated. Here Dr. Cui presents the iterative multiphoton adaptive compensation technique (IMPACT), which utilizes iterative feedback and the nonlinearity of the two-photon signals to measure and compensate wavefront distortion. He will discuss the imaging results on a variety of biological tissue and compare his team's technique with conventional adaptive optics methods.
Questions & Answers from the Webinar:
1. I've shown that detection of breast tumors of 2-4mm diameter should produce >99% cure when they are ablated. Could you get this resolution noninvasively, for screening? (Dick Gordon DickGordonCan@gmail.com)
[LW:> ] Our breast imaging PAT system does have that resolution. However, we need to image human subjects to validate it.
2. Prof Wang, to use NIR wavelength for PA, what would be the more economical substitution for OPO or dye laser?
[LW:> ] Ti:sapphire might be another option. We hope laser companies will be motivated to produce more economical lasers for PAT.
3. Why does the imaging plane stay fixed during the optimization?
[MC:>] The wavefront optimization basically is an in-line holography. For that, we need signal field and reference field. In our application, we modulate half pixels (signal field) and keep the other half stationary (reference field). After one half is done, we keep them fixed (serve as the reference) and modulate the other half. For the microscopy work, we typically run three iterations.
4. What is the requirement on the signal-to-noise for IMPACT to work?
[MC:>] Because the wavefront measurement is done through interferometry, the SNR is close to the shot noise limited value. For shallow imaging depth, the inherent wavefront distortion is small and therefore the wavefront correction need to have lambda/10 accuracy. For deep imaging depth, the inherent wavefront distortion is large. Wavefront correction with less accuracy can already provide a huge improvement in both resolution and signal level, and the requirement on SNR is therefore lower.
5. Does this method work equally well for single photon microscopy?
[MC:>] Nonlinearity in two-photon microscopy can help and accelerate the convergence of the wavefront measurement. We do have a version that is based on detecting the back scattered light via coherence gating for one-photon applications (http://dx.doi.org/10.1364/OE.20.016532).
6. Is there a temperature dependence?
[MC:>] From our experiments on animal imaging, the animal is in anesthesia and its body temperature is maintained. We did not observe any temperature dependence.
7. Since the interfering beams travel separate paths, what is the error induced by environmental perturbation?
[MC:>] For the IMPACT work, the interference happens as in an in-line holography that has passive phase stability (> 30 seconds). Given the fast MEMS mirror to accelerate the measurement, the environmental perturbation is not an issue. For the spatial frequency domain work, the two beams (xy beam and SLM beam) travel through different paths and therefore do not have passive phase stability. However, in the spatial frequency domain work, the system is a lot faster. The overall measurement time in our first demonstration is 0.4 second, which is much faster than the environment induced phase drift. Our ongoing development of the spatial frequency domain method will be even faster.
8. How does the wavefront correction effect detection of tumors, for example? Is it possible that the Phase Conjugation might mask tumor detection?
[MC:>] So far, we have only tested mouse brain and mouse lymph node. For these studies, we need high spatial resolution. The imaging depth is still less than the diffusive limit. Perhaps this is still not deep enough for tumor detection.
9. What SLM's do you use for fast data acquisition?
[MC:>] The high speed MEMS is from Boston Micromachine corporation.