Fluorescence guided surgery has high potential for improved patient outcomes but often lacks quantification of fluorophore depth which is needed to determine surgical margins of solid tumors. To address this need, a dual wavelength excitation approach was applied that capitalizes on the wavelength-dependent attenuation of light in tissue to provide depth information independent of fluorophore concentration. A portable near infrared dual wavelength excitation fluorescence imaging system was built and tested using tissue mimicking phantoms and is currently being tested to determine breast tumor margin status in a first-in-human clinical trial investigating LS301, a novel near infrared tumor-targeted contrast agent.
Photoacoustic microscopy (PAM) has been extensively applied in biomedical study because of its ability to visualize tissue morphology and physiology in vivo in three dimensions (3D). However, conventional PAM suffers from a rapidly decreasing resolution away from the focal plane because of the limited depth of focus of an objective lens, which deteriorates the volumetric imaging quality inevitably. Here, we propose a novel method to synthesize an ultra-long light needle to extend a microscope’s depth of focus beyond its physical limitations with wavefront engineering method. Furthermore, it enables an improved lateral resolution that exceeds the diffraction limit of the objective lens. The virtual light needle can be flexibly synthesized anywhere throughout the imaging volume without mechanical scanning. Benefiting from these advantages, we developed a synthetic light needle photoacoustic microscopy (SLN-PAM) to achieve an extended depth of field (DOF), sub-diffraction and motionless volumetric imaging. The DOF of our SLN-PAM system is up to 1800 µm, more than 30-fold improvement over that gained by conventional PAM. Our system also achieves the lateral resolution of 1.8 µm (characterized at 532 nm and 0.1 NA objective), about 50% higher than the Rayleigh diffraction limit. Its superior imaging performance was demonstrated by 3D imaging of both non-biological and biological samples. This extended DOF, sub-diffraction and motionless 3D PAM will open up new opportunities for potential biomedical applications.
Focusing light deep inside scattering media plays a key role in such biomedical applications as high resolution optical imaging, control, and therapy. In recent years, wavefront shaping technologies have come a long way in controlling light propagation in complex media. A prominent example is time-reversed ultrasonically encoded (TRUE) focusing, which allows noninvasive introduction of “guide stars” inside biological tissue to guide light focusing. By measuring the optical wavefront emanating from an ultrasound focus created at the target location, TRUE determines the desired wavefront non-iteratively, and achieves focusing at the target position via a subsequent optical time reversal. Compared to digital counterparts that employ slow electronic spatial light modulators and cameras, analog TRUE focusing relies on nonlinear photorefractive crystals that inherently accommodate more spatial modes and eliminate the troublesome alignment and data transfer required by digital approaches. However, analog TRUE focusing suffers from its small gain, defined as the energy or power ratio between the focusing and probing beams in the focal volume. Here, by implementing a modified analog TRUE focusing scheme that squeezes the duration of the time-reversed photon packet below the carrier-recombination-limited hologram decay time of the crystal, we demonstrated a photon flux amplification much greater than unity at a preset focal voxel in between two scattering layers. Although the energy gain was still below unity, the unprecedented power gain will nevertheless benefit new biomedical applications.
Intralipid is widely used as an optical scattering agent in tissue-mimicking phantoms. Accurate control when using Intralipid is critical to match the optical diffusivity of phantoms to the prescribed value. Currently, most protocols of Intralipid-based hydrogel phantom fabrication focus on factors such as Intralipid brand and concentration. In this note, for the first time to our knowledge, we explore the dependence of the optical reduced scattering coefficient (at 532 nm optical wavelength) on the temperature and the time of mixing Intralipid with gelatin-water solution. The studied samples contained 1% Intralipid and were measured with oblique-incidence reflectometry. It was found that the reduced scattering coefficient increased when the Intralipid-gelatin-water mixture began to solidify at room temperature. For phantoms that had already solidified completely, the diffusivity was shown to be significantly influenced by the temperature and the duration of the mixing course. The dependence of the measured diffusivity on the mixing conditions was confirmed by experimental observations. Moreover, the mechanism behind the dependence behavior is discussed.
To focus light beyond one transport mean free path, time-reversed ultrasonically encoded (TRUE) optical focusing has previously been implemented by both analog and digital devices. By allowing wavefront recording with finer resolution and larger aperture, the analog scheme, which uses photorefractive materials as the phase-conjugate mirror, generates a more complete set of time-reversed optical modes than the digital scheme. Here, we report the direct visualization of localized fluorescence excitation inside a turbid medium by photorefractive time reversal. Further, we imaged fluorescent targets embedded in a turbid phantom whose thickness was four transport mean free paths.
Focusing light inside highly scattering media beyond the ballistic regime is a challenging task in biomedical optical imaging, manipulation, and therapy. This challenge can be overcome by time reversing ultrasonically encoded (TRUE) diffuse light to the ultrasonic focus inside a turbid medium. In TRUE optical focusing, a photorefractive crystal or polymer is used as the phase conjugate mirror for optical time reversal. Accordingly, a relatively long ultrasound burst, whose duration matches the response time of the photorefractive material, is used to encode the diffuse light. With this long ultrasound burst, the resolution of the TRUE focus along the acoustic axis is poor. In this work, we used two transducers, emitting two intersecting ultrasound beams at 3.4 MHz and 3.6 MHz respectively, to modulate the diffuse light within their intersection volume at the beat frequency. We show that light encoded at the beat frequency can be time-reversed and converge to the intersection volume. Experimentally, TRUE focusing with an acoustic axial resolution of ~1.1 mm was demonstrated inside turbid media, agreeing with the theoretical estimation.
Time-reversed ultrasonically encoded (TRUE) optical focusing is an emerging technique that focuses light deep into scattering media by phase-conjugating ultrasonically encoded diffuse light. In previous work, the speed of TRUE focusing was limited to no faster than 1 Hz by the response time of the photorefractive phase conjugate mirror, or the data acquisition and streaming speed of the digital camera; photorefractive-crystal-based TRUE focusing was also limited to the visible spectral range. These time-consuming schemes prevent this technique from being applied in vivo, since living biological tissue has a speckle decorrelation time on the order of a millisecond. In this work, using a Tedoped Sn2P2S6 photorefractive crystal at a near-infrared wavelength of 793 nm, we achieved TRUE focusing inside dynamic scattering media having a speckle decorrelation time as short as 7.7 ms. As the achieved speed approaches the tissue decorrelation rate, this work is an important step forward toward in vivo applications of TRUE focusing in deep tissue imaging, photodynamic therapy, and optical manipulation.
Focusing light inside highly scattering media is a challenging task in biomedical optical imaging, manipulation, and therapy. A recent invention has overcome this challenge by time reversing ultrasonically encoded diffuse light to an ultrasound-modulated volume inside a turbid medium. In this technique, a photorefractive (PR) crystal or polymer can be used as the phase conjugate mirror for optical time reversal. Accordingly, a relatively long ultrasound burst, whose duration matches the PR response time of the PR material, is usually used to encode the diffuse light. This long burst results in poor focusing resolution along the acoustic axis. In this work, we propose to use two intersecting ultrasound beams, emitted from two ultrasonic transducers at different frequencies, to modulate the diffuse light at the beat frequency within the intersection volume. We show that the time reversal of the light encoded at the beat frequency can converge back to the intersection volume. Experimentally, an acoustic axial resolution of ∼1.1 mm was demonstrated inside turbid media, agreeing with theoretical estimation.
For years, ultrasound-modulated optical tomography (UOT) has been proposed to image optical contrasts deep inside
turbid media (such as biological tissue) at an ultrasonic spatial resolution. The reported imaging depth so far, however,
has been limited, preventing this technique from finding broader applications. In this work, we present our latest
experimental explorations that push UOT to clinically useful imaging depths, achieved through optimizing from different
aspects. One improvement is the use of a large aperture fiber bundle, which more effectively collects the diffused light,
including both ultrasound-modulated and unmodulated portions, from the turbid sample and then sends it to the
photorefractive material. Another endeavor is employment of a large aperture photorefractive polymer film for
demodulating the ultrasound-induced phase modulation. Compared with most UOT detection schemes, the polymer film
based setup provides a much higher etendue as well as photorefractive two-beam-coupling gain. Experimentally, we have
demonstrated enhanced sensitivity and have imaged through tissue-mimicking samples up to 9.4 cm thick at the
ultrasonically-determined spatial resolutions.
Time-reversed ultrasonically encoded (TRUE) optical focusing focuses light beyond one transport mean free
path by phase-conjugating the ultrasonically tagged light. However, in previous works, only a small portion of the tagged
light was phase-conjugated by using a photorefractive Bi12SiO20 crystal, due to its small active area (1x1 cm2). In this work, we report high-efficiency TRUE focusing using a large-area photorefractive polymer (5x5 cm2), which
demonstrated ~40 times increase in focused energy. Further, we imaged absorbers embedded in a turbid sample of
thickness of ~12 transport mean free paths.
Time-reversed ultrasonically encoded (TRUE) optical focusing achieves light focusing into scattering media beyond one transport mean free path, which is desirable in biomedical optics. However, the focused optical energy needs to be increased for broad applications. Here, we report the use of a photorefractive polymer (PRP) as the phase conjugate mirror in TRUE optical focusing. The PRP boosted the focused optical energy by ∼ 40 times in comparison to the previously used photorefractive Bi12SiO20 crystal. As a result, we successfully imaged absorbing objects embedded in the middle plane of a tissue-mimicking phantom having an optical thickness of 120 scattering mean free paths.
Ultrasound-modulated optical tomography (UOT) has the potential to reveal optical contrast deep inside soft biological tissues at an ultrasonically determined spatial resolution. The optical imaging depth reported so far has, however, been limited, which prevents this technique from broader applications. Our latest experimental exploration has pushed UOT to an unprecedented imaging depth. We developed and optimized a UOT system employing a photorefractive crystal-based interferometer. A large aperture optical fiber bundle was used to enhance the efficiencies for diffuse light collection and photorefractive two-wave-mixing. Within the safety limits for both laser illumination and ultrasound modulation, the system has attained the ability to image through a tissue-mimicking phantom of 9.4 cm in thickness, which has never been reached previously by UOT.
We report an experimental investigation of time-reversed ultrasonically encoded optical focusing in biological tissue. This technology combines the concepts of optical phase conjugation and ultrasound modulation of diffused coherent light. The ultrasonically encoded (or tagged) diffused light from a tissue sample is collected in reflection mode and interferes with a reference light in a photorefractive crystal (used as a phase conjugation mirror) to form a hologram. Then a time-reversed copy of the tagged light is generated and traces back the original trajectories to the ultrasonic focus inside the tissue sample. With our current setup, we can achieve a maximum penetration depth of 5 mm in a chicken breast sample and image optical contrasts within a tissue sample with a spatial resolution approximately equaling 1/2 of the ultrasound focal diameter.
In optical scattering media such as biological tissue, light propagation is randomized by multiple scattering. Beyond one
transport mean free path, where photon propagation is in the diffusive regime, direct light focusing becomes infeasible.
The resulting loss of light localization poses serious challenge to optical imaging in thick scattering media. Ultrasound
modulated optical tomography (UOT) combines high optical contrast and good ultrasonic resolution, and is therefore an
ideal imaging modality for soft biological tissue. A variety of detection techniques have been developed in UOT in an
effort to discriminate the ultrasonically encoded diffused light as the imaging signal. We developed a photorefractive
crystal based detection system, which has the ability to image both the optical and acoustic properties of biological
tissues. With the improved photorefractive crystal based detection, tissue-mimicking phantom samples as thick as 9.4 cm
can be imaged. We further exploit the virtual source concept in UOT and combine it with optical time reversal to achieve
diffusive light focusing into scattering media. Experimental implementation of this new technology is presented.
The problem of how to effectively deliver light dynamically to a small volume inside turbid media has been intensively
investigated for imaging and therapeutic purposes. Most recently, a new modality termed Time-Reversed Ultrasonically
Encoded (TRUE) optical focusing was proposed by integrating the concepts of ultrasound modulation of diffused light
with optical phase conjugation. In this work, the diffused photons that travel through the ultrasound focal region are
"tagged" with a frequency shift due to the ultrasound modulation. Part of the tagged light is collected in reflection mode
and transmitted to a photorefractive crystal, forming there a stationary hologram through interference with a coherent
reference optical beam. The hologram is later read by a conjugated optical beam, generating a phase conjugated wavefront
of the tagged light. It is conveyed back to the turbid medium in reflection mode, and eventually converges to the ultrasound
focal zone. Optical focusing effects from this system are demonstrated experimentally in tissue-mimicking phantoms and
ex vivo chicken breast tissue, achieving effective round-trip optical penetration pathlength (extinction coefficient
multiplied by round-trip focusing depth) exceeding 160 and 100, respectively. Examples of imaging optical inclusions with
this system are also reported.
In turbid media such as biological tissue, multiple scattering hinders direct light focusing at depths beyond one transport mean free path. As a solution to this problem, time-reversed ultrasonically encoded (TRUE) optical focusing is proposed based on ultrasonic encoding of diffused laser light and optical time reversal. In TRUE focusing, a laser beam of long coherence length illuminates a turbid medium, where the incident light undergoes multiple scattering and part of it gets ultrasonically encoded within the ultrasonic focal zone. A conjugated wavefront of the ultrasonically encoded light is then generated by a phase conjugate mirror outside the medium, which traces back the trajectories of the ultrasonically encoded diffused light and converges light to the ultrasonic focal zone. Here, we report the latest experimental improvement in TRUE optical focusing that increases its penetration in tissue-mimicking media from a thickness of 3.75 to 7.00 mm. We also demonstrate that the TRUE focus depends on the focal diameter of the ultrasonic transducer.
Time-reversed ultrasonically encoded (TRUE) optical focusing was recently proposed to deliver light dynamically to a tight region inside a scattering medium. In this letter, we report the first development of a reflection-mode TRUE optical focusing system. A high numerical aperture light guide is used to transmit the diffusely reflected light from a turbid medium to a phase-conjugate mirror (PCM), which is sensitive only to the ultrasound-tagged light. From the PCM, a phase conjugated wavefront of the tagged light is generated and conveyed by the same light guide back to the turbid medium, subsequently converging to the ultrasonic focal zone. We present experimental results from this system, which has the ability to focus light in a highly scattering medium with a round-trip optical penetration thickness (extinction coefficient multiplied by round-trip depth) as large as 160.
In turbid media such as biological tissues, light undergoes multiple scattering. Consequently, it is not
possible to focus light at depths beyond one transport mean free path in such media. To break through this
limit, we proposed and experimentally demonstrated a novel technique, based on ultrasonic encoding of
diffused laser light and optical time reversal, which effectively focuses light into a turbid medium. In the
experimental implementation of the Time-Reversed Ultrasonically Encoded (TRUE) optical focusing, a
turbid medium was illuminated by a laser beam with a long coherence length. The incident light was
multiply scattered inside the medium and ultrasonically encoded within the ultrasonic focal zone. The
wavefront of the ultrasonically encoded light was then time reversed by a Phase Conjugate Mirror (PCM)
outside the medium. The time-reversed (or phase conjugated) optical wavefront traced back the trajectories
of the ultrasonically encoded diffused light, and converged to the ultrasonic focal zone. With a
commercially available photorefractive crystal as the PCM, the main approaches for increasing focusing
depth are to improve the efficiencies of ultrasonic encoding and time reversal. Our recent experiments
showed that light can be focused into a 5-mm thick tissue-mimicking phantom (optical thickness = 50, i.e.,
geometric thickness = 50 mean free paths) with a dynamically adjustable focus. The TRUE optical focusing
opens a door to focusing light into turbid media or manipulating light-matter interactions.
Ultrasound-modulated optical tomography is a hybrid imaging technique based on detection of the diffused light
modulated by a focused ultrasonic wave inside a scattering medium. With the combined advantages of ultrasonic
resolution and optical contrast, UOT is ideal for deep tissue imaging. Its growth in popularity and application, however,
is hindered by the low efficiency in detecting the modulated diffused photons. Research activities on UOT have
therefore been centered on improving its signal detection efficiency by exploring various technical solutions. A prime
example is the use of spectral-hole burning (SHB) in UOT. By applying SHB crystal as a spectral filter, one modulation
sideband of the diffused light can be efficiently selected while all the other frequency components are strongly
suppressed. Immune to both the spatial and temporal incoherence of the signal with a high enough on/off ratio, SHB can
boost the UOT imaging ability dramatically and push it towards practical applications. We compare SHB with the other
technologies that have been applied to UOT, and identify the unique features that make SHB a preferable tool for UOT.
We also discuss the desired improvements from the SHB side, which will help UOT pave the way from research to
everyday life.
We apply spectral hole burning (SHB)-aided detection in ultrasound-modulated optical tomography (UOT) to image optical heterogeneities in thick tissue-mimicking phantom samples and chicken breast tissue. The efficiency of SHB is improved by using a Tm3+:YAG crystal of higher doping concentration (2.0-atomic%) and a double-pass pumping configuration, in which the pump beam is transmitted through the crystal twice to burn a deeper spectral hole with the available optical intensity. With the improved SHB-UOT system, we image absorbing, scattering, and phase objects that are embedded in the middle plane of a 30-mm-thick phantom sample. The imaging resolution was 0.5 mm in the lateral direction, as defined by the focal width of the ultrasonic transducer, and 1.5 mm in the axial direction, as determined by the ultrasonic burst length. We also image two absorbing objects embedded in a 32-mm-thick chicken breast sample. The results suggest that the improved SHB-UOT system is one step closer to the practical optical imaging application in biological and clinical studies.
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