Amos Danielli

Label-free photoacoustic nanoscopy

Abstract. Super-resolution microscopy techniques—capable of overcoming the diffraction limit of light—have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules.

Real-time four-dimensional optical-resolution photoacoustic microscopy with Au nanoparticle-assisted subdiffraction-limit resolution

Photoacoustic microscopy (PAM) offers label-free, optical absorption contrast. A high-speed, high-resolution PAM system in an inverted microscope configuration with a laser pulse repetition rate of 100,000 Hz and a stationary ultrasonic transducer was built. Four-dimensional in vivo imaging of microcirculation in mouse skin was achieved at 18 three-dimensional volumes per second with repeated two-dimensional (2D) raster scans of 100 by 50 points. The corresponding 2D B-scan (50 A-lines) frame rate was 1800 Hz, and the one-dimensional A-scan rate was 90,000 Hz. The lateral resolution is 0.23 ± 0.03 μm for Au nanowire imaging, which is 2.0 times below the diffraction limit.

Picosecond absorption relaxation measured with nanosecond laser photoacoustics.

Picosecond absorption relaxation-central to many disciplines-is typically measured by ultrafast (femtosecond or picosecond) pump-probe techniques, which however are restricted to optically thin and weakly scattering materials or require artificial sample preparation. Here, we developed a reflection-mode relaxation photoacoustic microscope based on a nanosecond laser and measured picosecond absorption relaxation times. The relaxation times of oxygenated and deoxygenated hemoglobin molecules, both possessing extremely low fluorescence quantum yields, were measured at 576 nm. The added advantages in dispersion susceptibility, laser-wavelength availability, reflection sensing, and expense foster the study of natural-including strongly scattering and nonfluorescent-materials.

Calibration-free quantification of absolute oxygen saturation based on the dynamics of photoacoustic signals

Photoacoustic tomography (PAT) is a hybrid imaging technique that has broad preclinical and clinical applications. Based on the photoacoustic effect, PAT directly measures specific optical absorption, which is the product of the tissue-intrinsic optical absorption coefficient and the local optical fluence. Therefore, quantitative PAT, such as absolute oxygen saturation (sO₂) quantification, requires knowledge of the local optical fluence, which can only be estimated through invasive measurements or sophisticated modeling of light transportation. In this Letter, we circumvent this requirement by taking advantage of the dynamics in sO₂. The new method works when the sO₂ transition can be simultaneously monitored with multiple wavelengths. For each wavelength, the ratio of photoacoustic amplitudes measured at different sO₂ states is utilized. Using the ratio cancels the contribution from optical fluence and allows calibration-free quantification of absolute sO₂. The new method was validated through both phantom and in vivo experiments.

Optical-resolution photoacoustic endomicroscopy in vivo

Optical-resolution photoacoustic microscopy (OR-PAM) has become a major experimental tool of photoacoustic tomography, with unique imaging capabilities for various biological applications. However, conventional imaging systems are all table-top embodiments, which preclude their use in internal organs. In this study, by applying the OR-PAM concept to our recently developed endoscopic technique, called photoacoustic endoscopy (PAE), we created an optical-resolution photoacoustic endomicroscopy (OR-PAEM) system, which enables internal organ imaging with a much finer resolution than conventional acoustic-resolution PAE systems. OR-PAEM has potential preclinical and clinical applications using either endogenous or exogenous contrast agents.

Frequency stabilization of a frequency-doubled 1556-nm source to the 5S 1/2 → 5D 5/2 two-photon transitions of rubidium

Improvements in the power level of sources near 1550 nm and in the efficiency of waveguide frequency doublers enabled us to lock a frequency-doubled source directly to the 5S(1/2) ? 5D(5/2) two-photon transitions near 778 nm. We obtained a sufficiently powerful second-harmonic signal, exceeding 2 mW, by doubling an external-cavity diode laser that was amplified by an erbium-doped fiber amplifier in a periodically poled LiNbO(3) channel waveguide. Our experimental scheme can be used for realizing compact, high-performance frequency standards near 1550 nm for fiber-optic communication and sensing applications.

Single-wavelength functional photoacoustic microscopy in biological tissue

Recently, we developed a reflection-mode relaxation photoacoustic microscope, based on saturation intensity, to measure picosecond relaxation times using a nanosecond laser. Here, using the different relaxation times of oxygenated and deoxygenated hemoglobin molecules, both possessing extremely low fluorescence quantum yields, the oxygen saturation was quantified in vivo with single-wavelength photoacoustic microscopy. All previous functional photoacoustic microscopy measurements required imaging with multiple-laser-wavelength measurements to quantify oxygen saturation. Eliminating the need for multiwavelength measurements removes the influence of spectral properties on oxygenation calculations and improves the portability and cost-effectiveness of functional or molecular photoacoustic microscopy.

Functional photoacoustic microscopy of pH

pH is a tightly regulated indicator of metabolic activity. In mammalian systems, an imbalance of pH regulation may result from or result in serious illness. In this paper, we report photoacoustic microscopy (PAM) of a commercially available pH-sensitive fluorescent dye (SNARF-5F carboxylic acid) in tissue phantoms. We demonstrated that PAM is capable of pH imaging in absolute values at tissue depths of up to 2.0 mm, greater than possible with other forms of optical microscopy.

Rapid homogenous detection of the Ibaraki virus NS3 cDNA at picomolar concentrations by magnetic modulation.

Magnetic modulation biosensing (MMB) system is experimentally demonstrated for rapid and homogeneous detection of the Ibaraki virus NS3 cDNA. A novel fluorescent resonance energy transfer (FRET)-based probe discriminates the target DNA from the control. When detection is made, the FRET-based probe is cleaved using Taq-polymerase activity and fluorescent light is produced. The biotinylated probes are attached to streptavidin-coupled superparamagnetic beads and are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The beads are condensed into the detection area and their movement in and out the orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. 1.9pM of the Ibaraki virus NS3 cDNA was detected in homogeneous solution within 18min without separation or washing steps.

Detection of fluorescent-labeled probes at sub-picomolar concentrations by magnetic modulation

A sensitive and rapid method for detecting fluorescent dyes at low concentrations in homogenous solution is experimentally demonstrated. Fluorescent-labeled DNA probes are detected by attaching magnetic beads and applying alternating magnetic field gradient. This condenses the fluorescent probes into a small detection volume and eliminates the scattering noise from solution by synchronous detection. For DNA probes concentration of 1 x 10(-13) M the detection signal was 3.3 times higher than the noise, thereby implying detection sensitivity of 3 x 10(-14) M.