Coherence-gated photoacoustic remote sensing microscopy.

Photoacoustic remote sensing microscopy (PARS) represents a new paradigm within the optical imaging community by providing high sensitivity (>50 dB in vivo) non-contact optical absorption contrast in scattering media with a reflection-mode configuration. Unlike contact-based photoacoustic modalities which can acquire complete A-scans with a single excitation pulse due to slow acoustic propagation facilitating the use of time-gated collection of returning acoustic signals, PARS provides depth resolution only through optical sectioning. Here we introduce a new approach for providing coherence-gated depth-resolved PARS imaging using a difference between pulsed-interrogation optical coherence tomography scan-lines with and without excitation pulses. Proposed methods are validated using simulations which account for pulsed-laser induced initial-pressures and accompanying refractive index changes. The changes in refractive index are shown to be proportional to optical absorption. It is demonstrated that to achieve optimal image quality, several key parameters must be selected including interrogation pulse duration and delay. The proposed approach offers the promise of non-contact depth-resolved optical absorption contrast at optical-resolution scales and may complement the scattering contrast offered by optical coherence tomography.

Scattering cross-sectional modulation in photoacoustic remote sensing microscopy.

Modeling and observations of large scattering cross-sectional modulations in absorbing optical scatterers due to a pulsed laser excitation are reported. Rapid laser-induced thermo-elastic expansion produces nontrivial perturbations to the local refractive indices. This mechanism forms the basis of a recent non-contact photoacoustic technique known as photoacoustic remote sensing microscopy. A time-evolution model is constructed and discussed, comparing it with existing planar models, time-independent models, and experiments. Fractional scattering cross-sectional modulations greater than 20 times that of the unperturbed particles are predicted and observed for the first time, to the best of our knowledge. A nonlinear acoustic enlargement effect is likewise predicted and observed. Implications of system and material properties are explored.

Temporal evolution of low-coherence reflectrometry signals in photoacoustic remote sensing microscopy.

Recently, a new noncontact reflection-mode imaging modality called photoacoustic remote sensing (PARS) microscopy was introduced providing optical absorption contrast. Unlike previous modalities, which rely on interferometric detection of a probe beam to measure surface oscillations, the PARS technique detects photoacoustic initial pressures induced by a pulsed laser at their origin by monitoring intensity modulations of a reflected probe beam. In this paper, a model describing the temporal evolution from a finite excitation pulse is developed with consideration given to the coherence length of the interrogation beam. Analytical models are compared with approximations, finite-difference time-domain (FDTD) simulations, and experiments with good agreement.

Non-interferometric photoacoustic remote sensing microscopy.

Elasto-optical refractive index modulation due to photoacoustic initial pressure transients produced significant reflection of a probe beam when the absorbing interface had an appreciable refractive index difference. This effect was harnessed in a new form of non-contact optical resolution photoacoustic microscopy called photoacoustic remote sensing microscopy. A non-interferometric system architecture with a low-coherence probe beam precludes detection of surface oscillations and other phase-modulation phenomenon. The probe beam was confocal with a scanned excitation beam to ensure detection of initial pressure-induced intensity reflections at the subsurface origin where pressures are largest. Phantom studies confirmed signal dependence on optical absorption, index contrast and excitation fluence. In vivo imaging of superficial microvasculature and melanoma tumors was demonstrated with ~2.7±0.5 µm lateral resolution.

In vivo optical resolution photoacoustic microscopy using glancing angle-deposited nanostructured Fabry-Perot etalons.

In this Letter, reflection-mode optical resolution photoacoustic microscopy (OR-PAM) using glancing angle-deposited (GLAD) nanostructured Fabry-Perot interferometers (FPI) for in vivo applications is reported. GLAD is a single-step physical vapor deposition (PVD) technique used to fabricate porous nanostructured thin films. Using titanium dioxide, a transparent semiconductor with a high refractive index (n=2.4), the GLAD technique can be employed to fabricate samples with tailored nano-porosity, refractive index periodicities, and high Q-factor reflectance spectra. The OR-PAM in vivo images of chorioallantoic membrane (CAM) of 5-day chicken embryo model are demonstrated. The phantom study shows lateral resolution and signal-to-noise ratio better than 7 µm and 35 dB, respectively. The sensitive GLAD FPI allows photoacoustic imaging down to a few-nJ pulse energy. To the best of our knowledge, this is the first time that a FPI-based reflection mode optical resolution photoacoustic imaging technique is demonstrated for in vivo applications.

In-Vivo functional optical-resolution photoacoustic microscopy with stimulated Raman scattering fiber-laser source.

In this paper a multi-wavelength optical-resolution photoacoustic microscopy (OR-PAM) system using stimulated Raman scattering is demonstrated for both phantom and in vivo imaging. A 1-ns pulse width ytterbium-doped fiber laser is coupled into a single-mode polarization maintaining fiber. Discrete Raman-shifted wavelength peaks extending to nearly 800 nm are generated with pulse energies sufficient for OR-PAM imaging. Bandpass filters are used to select imaging wavelengths. A dual-mirror galvanometer system was used to scan the focused outputs across samples of carbon fiber networks, 200µm dye-filled tubes, and Swiss Webster mouse ears. Photoacoustic signals were collected in transmission mode and used to create maximum amplitude projection C-scan images. Double dye experiments and in vivo oxygen saturation estimation confirmed functional imaging potential.

Multifocus optical-resolution photoacoustic microscopy using stimulated Raman scattering and chromatic aberration.

In this Letter, multifocus optical-resolution photoacoustic microscopy is demonstrated using wavelength tuning and chromatic aberration for depth scanning. Discrete focal zones at several depth locations were created by refocusing light from a polarization-maintaining single-mode fiber pumped by a nanosecond fiber laser. The fiber and laser parameters were chosen to take advantage of stimulated Raman scattering (SRS) in the fiber to create a multiwavelength output that could then be bandpass filtered. The collimator lens and objective lens are chosen to take advantage of chromatic aberration in which each generated SRS wavelength peak focuses at a slightly different depth. The maximum amplitude of photoacoustic signals is mapped to form C-scan images. Additionally, all wavelength peaks fired simultaneously offers improved depth-of-field structural imaging at the cost of slight degradation of mainlobe-to-sidelobe ratios. Wavelength-tuned depth scanning over more than 440 µm is demonstrated, significantly greater than the ~100 µm depth of field predicted from our focused Gaussian beams. The improved depth of focus could be valuable for structural imaging of microvascular morphology without the need for mechanical scanning in the depth direction.

Glancing angle deposited nanostructured film Fabry-Perot etalons for optical detection of ultrasound.

In this paper a new class of optical Fabry-Perot-based ultrasound detectors using low acoustic impedance glancing angle deposited (GLAD) films is demonstrated. GLAD is a single-step physical vapor-deposition (PVD) technique used to fabricate porous nanostructured thin films. Using titanium dioxide (TiO(2)), a transparent semiconductor with a high refractive index (n = 2.4), the GLAD technique can be employed to fabricate samples with tailored nano-porosity, refractive index periodicities, and high Q-factor reflectance spectra. The average acoustic impedance of the porous films is lower than bulk materials which will improve acoustic coupling, especially for high acoustic frequencies. For this work, two filters with high reflection in the C-band range and high transparency in the visible range (~80%) using GLAD films were fabricated. A 23 µm Parylene C layer was sandwiched between these two GLAD films in order to form a GLAD Fabry Perot Interferometer (GLAD-FPI). A high speed tunable continuous wavelength C-band laser was focused at the FPI and the reflection was measured using a high speed photodiode. The ultrasound pressure modulated the optical thickness of the FPI and hence its reflectivity. The fabricated sensor was tested using a 10 MHz unfocused transducer. The ultrasound transducer was calibrated using a hydrophone. The minimum detectable acoustic pressure was measured as 80 ± 20 Pa and the -3dB bandwidth was measured to be 18 MHz. This ultra-sensitive sensor can be an alternative to piezoelectric ultrasound transducers for any techniques in which ultrasound waves need to be detected including ultrasonic and photoacoustic imaging modalities. We demonstrate our GLAD-FPI for photoacoustic signal detection in optical-resolution photoacoustic microscopy (OR-PAM). To the best of our knowledge, this is the first time that a FPI fabricated using the GLAD method has been used for ultra-sensitive ultrasound detection.

In vivo dynamic process imaging using real-time optical-resolution photoacoustic microscopy.

The authors demonstrate in vivo dynamic process imaging using a label-free real-time optical-resolution photoacoustic microscope (OR-PAM). This reflection-mode system takes advantage of a 532-nm fiber laser source with a high pulse repetition rate of up to 600 kHz combined with a fast-scanning mirror system. Microvasculature in SCID mouse ears is imaged at near real-time (0.5 fps) for a 1×1 mm2 field of view (FOV) with micron-scale lateral resolution. We also demonstrate imaging of cardiac-induced microhemodynamics in murine microvasculature at real-time frame-rates (30 fps) over a 250×250 µm2 FOV using real-time C-scan OR-PAM with ability to provide sustained imaging with near real-time feedback for focusing and positioning.

Integrated micro-endoscopy system for simultaneous fluorescence and optical-resolution photoacoustic imaging.

We present a new integrated micro-endoscopy system combining label-free, fiber-based, real-time C-scan optical-resolution photoacoustic microscopy (F-OR-PAM) and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the F-OR-PAM sub-system is able to reach a resolution of ∼7 µm. The fluorescence subsystem, which does not require any mechanical scanning, consists of a 447.5-nm-centered diode laser as the light source, an objective lens, and a CCD camera. Proflavine is used as the fluorescent contrast agent by topical application. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single-mode fibers. The absorption of proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural information given by F-OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping visualize angiogenesis and the effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

Real-time handheld optical-resolution photoacoustic microscopy.

In this paper a new generation of optical-resolution photoacoustic microscopy (OR-PAM) with a wide range of potential clinical applications is demonstrated. Using fast scanning mirrors, an image guide with 30,000 fiber pixels, a refocusing lens and a unique probe we managed to reduce the footprint of an OR-PAM system from a stationary table-top system to a portable, 4 cm by 6 cm, probe weighing ~500 g tethered to a scanning unit. The phantom studies show that the handheld optical-resolution photoacoustic microscope is able to image with ~7 µm resolution. For in vivo studies images of the microvasculature in a Swiss Webster mouse ear are shown. The compact, flexible nature of the proposed design and the small footprint of the apparatus increase the usability of OR-PAM for potential clinical applications such as in dermatology.

In vivo near-realtime volumetric optical-resolution photoacoustic microscopy using a high-repetition-rate nanosecond fiber-laser.

Optical-resolution photoacoustic microscopy (OR-PAM) is capable of achieving optical-absorption-contrast images with micron-scale spatial resolution. Previous OR-PAM systems have been frame-rate limited by mechanical scanning speeds and laser pulse repetition rate (PRR). We demonstrate OR-PAM imaging using a diode-pumped nanosecond-pulsed Ytterbium-doped 532-nm fiber laser with PRR up to 600 kHz. Combined with fast-scanning mirrors, our proposed system provides C-scan and 3D images with acquisition frame rate of 4 frames per second (fps) or higher, two orders of magnitude faster than previously published systems. High-contrast images of capillary-scale microvasculature in a live Swiss Webster mouse ear with ~6-µm optical lateral spatial resolution are demonstrated.