The problem of optical scattering was long thought to fundamentally limit the depth at which light could be focused through turbid media such as fog or biological tissue. However, recent work in the field of wavefront shaping has demonstrated that by properly shaping the input light field, light can be noninvasively focused to desired locations deep inside scattering media. This has led to the development of several new techniques which have the potential to enhance the capabilities of existing optical tools in biomedicine. Unfortunately, extending these methods to living tissue has a number of challenges related to the requirements for noninvasive guidestar operation, speed, and focusing fidelity. Of existing wavefront shaping methods, time-reversed ultrasonically encoded (TRUE) focusing is well suited for applications in living tissue since it uses ultrasound as a guidestar which enables noninvasive operation and provides compatibility with optical phase conjugation for high-speed operation. In this paper, we will discuss the results of our recent work to apply TRUE focusing for optogenetic modulation, which enables enhanced optogenetic stimulation deep in tissue with a 4-fold spatial resolution improvement in 800-micron thick acute brain slices compared to conventional focusing, and summarize future directions to further extend the impact of wavefront shaping technologies in biomedicine.
Normal development of the visual system in infants relies on clear images being projected onto the retina, which can be disrupted by lens opacity caused by congenital cataract. This disruption, if uncorrected in early life, results in amblyopia (permanently decreased vision even after removal of the cataract). Doctors are able to prevent amblyopia by removing the cataract during the first several weeks of life, but this surgery risks a host of complications, which can be equally visually disabling. Here, we investigated the feasibility of focusing light noninvasively through highly scattering cataractous lenses to stimulate the retina, thereby preventing amblyopia. This approach would allow the cataractous lens removal surgery to be delayed and hence greatly reduce the risk of complications from early surgery. Employing a wavefront shaping technique named time-reversed ultrasonically encoded optical focusing in reflection mode, we focused 532-nm light through a highly scattering ex vivo adult human cataractous lens. This work demonstrates a potential clinical application of wavefront shaping techniques.
Optical scattering of biological tissue limits the working depth of conventional biomedical optics, which relies on the detection of ballistic photons. Recent developed optical phase conjugation (OPC) technique breaks through this depth limit by harnessing the scattered photons and shaping an optical wavefront that can “undo” the optical scattering. The OPC system measures the complex light field exiting the tissue and reconstructs a phase conjugated copy of the measured wavefront, which propagates in the reversed direction to the source of the light. To focus light inside a scattering medium, an embedded light source or “guidestar” is often required. Therefore, developing guidestar mechanisms plays an important role in advancing the OPC technique for deep tissue optical focusing and imaging. In addition to having strong optical modulation efficiency and compact size, a favorable guidestar for biomedical applications should also have good biocompatibility, fast response time, and be noninvasive or require only minimally invasive procedure. While a number of guidestar mechanisms have been developed and showed promising for various biomedical applications, they all have their own limitations. We have been developing new guidestars and tailoring them to meet the need for biomedical imaging and therapies. We are going to present our recent progress in novel guidestar development, compare them with established guidestar mechanisms, and discuss their potential in biomedical applications.
Optical scattering of biological tissue limits the penetration depth of conventional optical techniques, which rely on the detection of ballistic photons. Recent developed optical phase conjugation (OPC) technique breaks through this depth limit by shaping an optical wavefront that can “undo” the optical scattering. Assisted with an ultrasound focus, this technique enables optical focusing inside biological tissue in a freely addressable fashion. However, ultrasound modulation efficiency is low and the focusing resolution is limited by the ultrasound. Here we present a new technique, time-reversed ultrasound microbubble encoded (TRUME) optical focusing, which is able to provide high focusing efficiency and sub-ultrasound resolution. This technique achieves the wavefront solution by taking the difference of the optical fields captured outside the sample before and after ultrasound-driven microbubble destruction. A conjugated wavefront was then reconstructed and sent back to the sample to form a focus at the site of microbubble destruction. We experimentally demonstrate that a focus with ~2 um size was formed through a 2-mm thick biological tissue using this method. While the size the microbubble sets the resolution of an individual focus, the scale of the ultrasound focus limits the focusing addressability of this technique. Importantly, by utilizing the nonlinear destruction of microbubbles, the TRUME technique breaks the addressable focus resolution barrier imposed by the ultrasound focus. We experimentally demonstrate a 2-fold improvement in addressability using this effect. Since microbubbles are widely used as ultrasound contrast agents in human, this technique provides a promising solution for focusing light deep inside biological tissue.
Detection of ultrasound (US)-modulated fluorescence in turbid media is a challenge because of the low level of fluorescent light and the weak modulation of incoherent light. A very limited number of theoretical and experimental investigations have been performed, and this is, to our knowledge, the first demonstration of pulsed US-modulated fluorescence tomography. Experimental results show that the detected signal depends on the acoustic frequency and the fluorescent target's size along the ultrasonic propagation axis. The modulation depth of the detected signal is greatest when the length of the object along the acoustic axis is an odd number of half wavelengths and is weakest when the object is an integer multiple of an acoustic wavelength. Images of a fluorescent tube embedded within a 22- by 13- by 30 mm scattering gel phantom (μs ∼ 15 cm−1, g = 0.93) with 1-, 1.5-, and 2 MHz frequency US are presented. The modulation depth of the detected signal changes by a factor of 5 depending on the relative size of the object and the frequency. The approach is also verified by some simple experiments in a nonscattering gel and using a theoretical model.
Ultrasound imaging has benefited from non-linear approaches to improve image resolution and reduce the effects of
side-lobes. A system for performing second harmonic ultrasound modulated optical tomography is demonstrated which
incorporates both pulsed optical illumination and acoustic excitation. A pulse acoustic inversion scheme is employed
which allows the second harmonic ultrasound modulated optical signal to be obtained while still maintaining a short
pulse length of the acoustic excitation. For the experiments carried out the method demonstrates a reduction in the
effective line spread function from 4mm for the fundamental to 2.4mm for the second harmonic. The first use of pulsed
ultrasound modulated optical tomography in imaging fluorescent targets is also discussed. Simple experiments show that
by changing the length of the acoustic pulse the image contrast can be optimized. The modulation depth of the detected
signal is greatest when the length of the object along the acoustic axis is an odd number of half wavelengths and is
weakest when the object is an integer multiple of an acoustic wavelength. Preliminary ultrasound modulated imaging
results are also presented where the target generates light within the medium without the use of an external light source.
Although signal to noise ratio is likely to be a major challenge, this result highlights a potentially useful application of
ultrasound modulated optical tomography in bio- or chemi-luminescence imaging.
Tissue scaffolds are an integral part of the tissue engineering process, assisting in the culturing of cells in three
dimensions. It is important to understand both the properties of the scaffold and the growth of cells within the scaffold.
This paper describes a system to characterise scaffolds using acoustic techniques alone and the development of an
ultrasound modulated optical tomography system to study the growth of cells within the scaffolds.
Our interest is in characterising the properties of gel-based and polymer foam-based scaffolds. Results from a purely
acoustic system have been used to investigate the properties of foam scaffolds manufactured from synthetic polyesters
poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) via a supercritical fluid process. As these are porous
materials, they are particularly challenging acoustically as the pores scatter sound significantly. However, it is
demonstrated that acoustic signals are detectable through a 6mm thick scaffold.
Although acoustics alone can be used to characterize many properties of the scaffolds, useful information can also be
obtained from optical techniques e.g. monitoring the growth of cells within the scaffold via optical absorption or
fluorescence techniques. Light scattering is of course a significant problem for relatively thick engineered tissue
(~5mm). The acoustic approach has been extended to include laser illumination and detection of the ultrasound
modulated optical pulse. Images of optically-absorbing materials embedded in gel-based tissue phantoms will be
presented demonstrating that a lateral resolution of 250μm and an axial resolution of ~90μm can be achieved in