Imaging deformation of adherent cells due to shear stress using quantitative phase imaging.

We present a platform for detecting cellular deformations from mechanical stimuli, such as fluid shear stress, using rapid quantitative phase imaging. Rapid quantitative phase imaging was used to analyze changes in the optical path length of adherent skin cancer cells during mechanical displacement. Both the whole-cell phase displacement and the resultant shift of the cellular center of mass were calculated over the duration of the stimulus. Whole-cell phase displacement images were found to match expectation. Furthermore, center-of-mass shifts of adherent cells were found to resemble that of a one-dimensional Kelvin-Voigt (KV) viscoelastic solid. Cellular steady-state displacements from step fluid shear stimuli were found to be linearly related to the shear stress. Shear stiffness constants for cells exposed to a cytoskeletal disrupting toxin were found to be significantly lower than unexposed cells. This novel technique allows for elastographic analysis of whole-cell effective shear stiffness without the use of an exogenous force applicator, a specialized culture substrate, or tracking net perimeter movement of the cell.

Quantitative phase imaging with molecular sensitivity using photoacoustic microscopy with a miniature ring transducer.

We present a dual-modality system for both structural and molecular cell imaging based on coregistered quantitative phase imaging (QPI) and photoacoustic microscopy (PAM). The QPI system was based on off-axis holography, whereas the PAM system comprised a sinusoidally modulated optical source for excitation and a narrow-band low profile and low-cost ring ultrasonic transducer for detection. This approach facilitated a simple confocal alignment of the excitation beams of both modalities and the ultrasonic detector. This system was demonstrated by imaging endogenous molecules in red blood cells (RBCs) as well as by imaging exogenous molecular labels on cancer cells using gold nanoparticles (GNPs) functionalized to target epidermal growth factor receptor. QPI provided high resolution imaging of the cellular structures while PAM provided molecular contrast. This dual-modality microscopy method can potentially be implemented as a compact and low cost cellular diagnostic assay.

Fast wide-field photothermal and quantitative phase cell imaging with optical lock-in detection.

We present a fast, wide-field holography system for detecting photothermally excited gold nanospheres with combined quantitative phase imaging. An interferometric photothermal optical lock-in approach (POLI) is shown to improve SNR for detecting nanoparticles (NPs) on multiple substrates, including a monolayer of NPs on a silanized coverslip, and NPs bound to live cells. Furthermore, the set up allowed for co-registered quantitative phase imaging (QPI) to be acquired in an off-axis holographic set-up. An SNR of 103 was obtained for NP-tagging of epidermal growth factor receptor (EGFR) in live cells with a 3 second acquisition, while an SNR of 47 was seen for 20 ms acquisition. An analysis of improvements in SNR due to averaging multiple frames is presented, which suggest that residual photothermal signal can be a limiting factor. The combination of techniques allows for high resolution imaging of cell structure via QPI with the ability to identify receptor expression via POLI.

Photoacoustic thermal diffusion flowmetry.

Thermal Diffusion Flowmetry (TDF) (also called Heat Clearance Method or Thermal Clearance Method) is a longstanding technique for measuring blood flow or blood perfusion in living tissues. Typically, temperature transients and/or gradients are induced in a volume of interest and the temporal and/or spatial temperature variations which follow are measured and used for calculation of the flow. In this work a new method for implementing TDF is studied theoretically and experimentally. The heat deposition which is required for TDF is implemented photothermally (PT) and the measurement of the induced temperature variations is done by photoacoustic (PA) thermometry. Both excitation light beams (the PT and the PA) are produced by directly modulated 830 nm laser diodes and are conveniently delivered to the volume under test by the same optical fiber. The method was tested experimentally using a blood-filled phantom vessel and the results were compared with a theoretical prediction based on the heat and the photoacoustic equations. The fitting of a simplified lumped thermal model to the experimental data yielded estimated values of the blood velocity at different flow rates. By combining additional optical sources at different wavelengths it will be possible to utilize the method for non-invasive simultaneous measurement of blood flow and oxygen saturation using a single fiber probe.

Photoacoustic Doppler measurement of flow using tone burst excitation.

In this paper a novel technique for flow measurement which is based on the photoacoustic (PA) Doppler effect is described. A significant feature of the proposed approach is that it can be implemented using tone burst optical excitation thus enabling simultaneous measurement of both velocity and position. The technique, which is based on external modulation and heterodyne detection, was experimentally demonstrated by measurement of the flow of a suspension of carbon particles in a silicon tube and successfully determined the particles mean velocity up to values of 130 mm/sec, which is about 10 times higher than previously reported PA Doppler set-ups. In the theoretical part a rigorous derivation of the PA response of a flowing medium is described and some important simplifying approximations are highlighted.

Simultaneous spatial and spectral mapping of flow using photoacoustic Doppler measurement.

We demonstrate the use of tone-burst excitation and time-gated spectral analysis for photoacoustic Doppler mapping of flow in an unperturbed vessel phantom and in a vessel with a spatially varying lumen. The method, which mimics pulsed Doppler ultrasound, enables simultaneous measurement of axial position and flow as well as complete characterization of the Doppler spectrum over a wide range of mean velocities (3.5 to 200 mm∕s). To generate the required optical excitation, a continuous cw laser source followed by an external electro-optic modulator is used. Stenoses at various levels are emulated in a C-flex tube with a flowing suspension of micrometer-scale carbon particles. Two-dimensional maps of spectral content versus axial position at different points along the vessel and for various levels of perturbations demonstrate the potential use of the method for characterization of flow irregularities.

The use of pulse synthesis for optimization of photoacoustic measurements.

In this paper the use of pulse shaping in photoacoustic (PA) measurements is presented. The benefits of this approach are demonstrated by utilizing it for optimization of either the responsivity or the sensitivity of PA measurements. The optimization is based on the observation that the temporal properties of the PA effect can be represented as a linear system which can be fully characterized by its impulse response. Accordingly, the response of the PA system to an input optical pulse, whose instantaneous power is arbitrarily shaped, can be analytically predicted via a convolution between the pulse envelope and the PA impulse response. Additionally, the same formalism can be used to show that the response of the PA system to a pulse whose instantaneous power is a reversed version of the impulse response, i.e. a matched pulse, would exhibit optimal peak amplitude when compared with all other pulses with the same energy. Pulses can also be designed to optimize the sensitivity of the measurement to a variation in a specific system parameter. The use of the matched pulses can improve SNR and enable a reduction in the total optical energy required for obtaining a detectable signal. This may be important for applications where the optical power is restricted or for dynamical measurements where long integration times are prohibited. To implement this new approach, a novel PA optical setup which enabled synthesis of excitation waveforms with arbitrary temporal envelopes was constructed. The setup was based on a tunable laser source, operating in the near-IR range, and an external electro-optic modulator. Using this setup, our approach for system characterization and response prediction was tested and the superiority of the matched pulses over other common types of pulses of equal energy was demonstrated.