In vivo photoacoustic time-of-flight velocity measurement of single cells and nanoparticles

Optical techniques for in vivo measurement of blood flow velocity are not quite applicable for determination of velocity of individual cells or nanoparticles. Here, we describe a photoacoustic time-of-flight method to measure the velocity of individual absorbing objects by using single and multiple laser beams. Its capability was demonstrated in vitro on blood vessel phantom and in vivo on an animal (mouse) model for estimating velocity of gold nanorods, melanin nanoparticles, erythrocytes, leukocytes, and circulating tumor cells in the broad range of flow velocity from 0.1?mm/s to 14?cm/s. Object velocity can be used to identify single cells circulating at different velocities or cell aggregates and to determine a cell's location in a vessel cross-section.     

Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries

Capillaries, the smallest blood vessels, are the distal end of the vasculature where oxygen and nutrients are exchanged between blood and tissue. Hence, noninvasive imaging of capillaries and function in vivo has long been desired as a window to studying fundamental physiology, such as neurovascular coupling. Existing imaging modalities cannot provide the required sensitivity and spatial resolution. We present in vivo imaging of the microvasculature including single capillaries in mice using optical-resolution photoacoustic microscopy (OR-PAM) developed in our laboratory. OR-PAM provides a lateral resolution of 5 microm and an imaging depth >0.7 mm.     

In vivo dark-field reflection-mode photoacoustic microscopy

Reflection-mode photoacoustic microscopy with dark-field laser pulse illumination and high-numerical-aperture ultrasonic detection is designed and implemented in noninvasively imaged blood vessels in the skin in vivo. Dark-field optical illumination minimizes the interference caused by strong photoacoustic signals from superficial structures. A high-numerical-aperture acoustic lens provides high lateral resolution, 45-120 microm in this system. A broadband ultrasonic detection system provides high axial resolution, estimated to be approximately 15 microm. The optical illumination and ultrasonic detection are in a coaxial confocal configuration for optimal image quality. The system is capable of imaging optical-absorption contrast as deep as 3 mm in biological tissue.     

Solids flow measurement by correlation methods

The flow velocity of the solids content in pneumatic conveyors is determined from the transit time between two fixed points. Transducers located at these points generate random signals due to the naturally occurring random flow patterns. A special correlator is used for cross-correlating the transducer output signals and for automatically tracking the transit time. The correlating device is simple enough so that a rugged, reliable and yet precise field instrument can be built. Using optical or capacitive transducers, the average velocity of the solid content is measured without obstructing the flow profile. Under certain restrictions, the mean loading and thus the volume flow can be determined from the same sensor signals also. The effect of sensor geometry on bandwidth and the choice of optimal sensor distance is discussed. Optical and capacitive sensors are compared. Illustrative experimental results are given.     

In vivo photoacoustic flow cytometry for monitoring of circulating single cancer cells and contrast agents

A new photoacoustic flow cytometry was developed for real-time detection of circulating cells, nanoparticles, and contrast agents in vivo. Its capability, integrated with photothermal and optical clearing methods, was demonstrated using a near-infrared tunable laser to characterize the in vivo kinetics of Indocyanine Green alone and single cancer cells labeled with gold nanorods and Indocyanine Green in the vasculature of the mouse ear. In vivo applications are discussed, including selective nanophotothermolysis of metastatic squamous cells, label-free detection of melanoma cells, study of pharmokinetics, and immune response to apoptotic and necrotic cells, with potential translation to humans. The threshold sensitivity is estimated as one cancer cell in the background of 10(7) normal blood cells.     

Precise measurement of the depolarization ratio from photoacoustic Raman spectroscopy

A new method for the accurate determination of the Raman depolarization ratio is reported with an improved setup for photoacoustic Raman spectroscopy (PARS). The precise measurement is achieved by measuring the dependence of the acoustic signal intensity on the cross‐angle between the polarizations of two incident laser beams. We demonstrate this sensitive and simple method with several gaseous molecules, such as CH₄ and H₂. The measured results of depolarization ratios agree well with the theoretical values with an upper error limit of ± 0.005, which is comparable to that with polarization‐resolved CARS spectroscopy. Copyright © 2007 John Wiley & Sons, Ltd.     

Fluorescence correlation spectroscopy in vivo

This review will focus on the application and potential of FCS and FCCS in vivo. Practical issues and sources of artifacts when performing measurements in living cells are discussed. Finally, several extensions to conventional FCS, such as multiphoton excitation, scanning FCS, Fluorescence Lifetime Correlation Spectroscopy, multiplexing FCS and recent approaches to reach smaller excitation volumes are reviewed     

In vivo measurement of the local optical properties of tissue by use of differential path-length spectroscopy

We demonstrate the capability of differential path-length spectroscopy (DPS) to determine the local optical properties of tissue in vivo. DPS measurements on bronchial mucosa are analyzed and yield information on the local blood oxygenation, blood content, average microvessel diameter, and wavelength dependence of the reduced scattering coefficient. Our data collected to date show that cancerous bronchial mucosa has a lower capillary oxygenation and a larger average capillary diameter than normal bronchial mucosa.     

Fast simultaneous measurement of multi-gases using quantum cascade laser photoacoustic spectroscopy

The authors developed a fast simultaneous method in detecting multi-gases using quantum cascade laser (QCL) based photoacoustic (PA) spectroscopy. We demonstrated the simultaneous measurement of CO and SO₂ concentrations using two QCLs working at 4.56 and 7.38 μm, corresponding to the absorption bands of CO and SO₂, respectively. The modulation frequencies of the two QCLs were 234 and 244 Hz. The response time was 0.6 seconds. A computer sound card was used to process the PA signals. Fast Fourier transform was an essential step to get the amplitudes of the PA signals at different frequencies. The concentration of each gas can be obtained from the PA signal amplitude at the corresponding modulation frequency.     

Open-path measurement of ozone and methane gases using quartz-enhanced photoacoustic spectroscopy

We demonstrate a standoff system based on quartz-enhanced photoacoustic spectroscopy technique for the concentration measurements of atmospheric ozone and methane. The technique is a modified version of Photoacoustic spectroscopy. Two primary features of this technique are the employment of a tunable quantum cascade laser and a resonant quartz-crystal tuning fork detector. Both of these features facilitate simultaneous sensing of multiple molecular species. External-cavity quantum cascade laser having a spectral range from 7 to 10 micron is used. Diurnal concentration variations of methane and ozone are estimated for open-path up to 25 m. The ambient methane and ozone concentration maxima were observed to have values of 3.5 parts per million by volume and 140 parts per billion by volume, respectively. Finite-element mesh-based software is used to simulate the Eigen frequency of the tuning fork sensor. High-resolution transmission molecular spectroscopic database of atmospheric gases and the real-time gas concentration data from the Delhi Pollution Control Committee have been used as references.     

In vivo photoacoustic chorioretinal vascular imaging in albino mouse

Photoacoustic ophthalmoscopy(PAOM) is a novel imaging modality, which is capable of non-invasively detecting optical absorption properties in the retina. We visualize the microvasculature in retina and choroid in albino mouse using PAOM guided by spectral-domain optical coherence tomography. Since albino mouse characterizes by lacking melanin in retinal pigment epithelium(RPE), PAOM illumination laser can penetrate through the RPE onto choroid, and consequently provides volumetric visualization of chorioretinal vasculatures as a result of strong hemoglobin optical absorption. The high-quality chorioretinal microvascular imaging acquired by PAOM implies its great potential in understanding pathological mechanisms and developing therapeutic strategies for major chorioretinal diseases that correlate with vascular disorders.     

In vivo three-dimensional photoacoustic tomography of a whole mouse head

An in vivo photoacoustic imaging system was designed and implemented to image the entire small animal head. A special scanning gantry was designed to enable in vivo imaging in coronal cross sections with high contrast and good spatial resolution for the first time to our knowledge. By use of a 2.25 MHz ultrasonic transducer with a 6 mm diameter active element, an in-plane radial resolution of approximately 312 microm was achieved. Deeply seated arterial and venous vessels in the head measuring up to 1.7 cm in diameter were simultaneously imaged in vivo with 804 nm wavelength laser excitation of photoacoustic waves.     

In vivo label-free photoacoustic microscopy of cell nuclei by excitation of DNA and RNA

Imaging of cell nuclei plays a critical role in cancer diagnosis and prognosis. To image noninvasively cell nuclei in vivo without staining, we developed UV photoacoustic microscopy (UV-PAM), in which 266 nm wavelength UV light excites unlabeled DNA and RNA in cell nuclei to produce photoacoustic waves. We applied UV-PAM to ex vivo imaging of cell nuclei in a mouse lip and a mouse small intestine and to in vivo imaging of the cell nuclei in the mouse skin. The UV-PAM images of unstained cell nuclei match the optical micrographs of the histologically stained cell nuclei. Given intrinsic optical contrast and high spatial resolution, in vivo label-free UV-PAM has potential for unique biological and clinical application.     

In vivo measurement of attenuation

We propose a new technique for the in vivo measurement of attenuation. The method is conceptually simple and can also be easily implemented on a real-time ultrasound unit. The technique is suggested by an analysis of the propagation of a Gaussian pulse in a medium which has frequency-dependent attenuation as well as dispersion (frequency-dependent velocity). If the medium has a loss factor which can be described by H(f) = exp(-eta[f]pX), where f is the frequency and O less than or equal to p less than or equal to 2 (valid for tissues and other objects of interest), then the pulse retains its Gaussian shape, shifting only its centre frequency and bandwidth. This suggests that by measuring the mean frequency of the reflected rf waveform in a window which is moved in depth we can obtain an estimate of the attenuation. Here we describe a particular hardware implementation of this technique which we have completed (based on measurement of zero-crossings) and present some preliminary in vivo measurements.     

Resonance photoacoustic spectroscopy and gas analysis of gaseous flow at reduced pressure

The results of theoretical and experimental studies of sensitivity of a resonant photoacoustic Helmholtz resonator detector for gas flowing through a photoacoustic cell under reduced pressure are presented. The measurements of the sensitivity and ultimate sensitivity of the differential photoacoustic cell were performed with a near-IR room-temperature diode laser using the well-known H₂O absorption line (12496.1056 cm^-1) as a reference. The measured value of the sensitivity (6–17 Pa W m^-1) is in satisfactory agreement with the calculated one, which equals 6–35 Pa W m^-1. The obtained value of the ultimate sensitivity ((3–5)×10^-7 W m^-1 Hz^-1/2) provides measurements of the concentration of molecules at the ppb–ppm level. Received: 19 April 2001 / Revised version: 18 September 2001 / Published online: 7 November 2001     

Theory of X-ray photoacoustic spectroscopy

PhotoAcoustic Spectroscopy (PAS) in the X-ray region is becoming a new field in PAS research and poses some new problems, such as heat production mechanisms and non-exponential heat distribution due to multi-excitation processes, additional signals caused by escaped fluorescence and electrons, special backing and fronting setup for comparison with absorption experiment, etc. In this paper we treat these problems and include them in an extended theoretical model which can be reduced to the RG model in a special case. The results indicate that the non-exponential heat distribution has no effect on the PA phase. The contribution by gas heating of the escaped electrons can be neglected. The thermally thin gas layers between the sample and the Be windows are sensitive factors to both amplitude and phase. The PA signal generated in the backing gas layer by the backing surface of the sample should be considered to understand the frequency dependence of the PA signal. The expansion of the backing gas layer is the reason for the phase change in PA-EXAFS.     

A quartz-enhanced photoacoustic spectroscopy sensor for measurement of water vapor concentration in the air

A compact and highly linear quartz-enhanced photoacoustic spectroscopy(QEPAS)sensor for the measurement of water vapor concentration in the air is demonstrated.A cost-effective quartz tuning fork(QTF)is used as the sharp transducer to convert light energy into an electrical signal based on the piezoelectric effect,thereby removing the need for a photodetector.The short optical path featured by the proposed sensing system leads to a decreased size.Furthermore,a pair of microresonators is applied in the absorbance detection module(ADM)for QTF signal enhancement.Compared with the system without microresonators,the detected QTF signal is increased to approximately 7-fold.Using this optimized QEPAS sensor with the proper modulation frequency and depth,we measure the water vapor concentration in the air at atmospheric pressure and room temperature.The experimental result shows that the sensor has a high sensitivity of 1.058parts-per-million.     

In vivo measurement of basilar membrane stiffness

Basilar membrane stiffness measurements were made in the base of the gerbil cochlea. Basilar membrane stiffness was determined by contacting the basilar membrane with a stainless steel needle (tip diameter 25 microns) attached to a force transducer, putting the needle/transducer structure through a low-frequency sinusoidal excursion with amplitude 5 or 25 nm, and measuring the restoring force exerted on the needle by the basilar membrane at the applied frequency. Stiffness was calculated as the amplitude of the restoring force divided by the amplitude of the excursion. Stiffness was measured over a 24-microns range of static displacements of the basilar membrane and is presented as stiffness versus static displacement. In cochleas that were not damaged during surgery the stiffness versus displacement characteristic usually had the following features: (1) an initial stiffness plateau with average stiffness 0.6 N/m; (2) a second plateau or level off with average stiffness 9.1 N/m; and (3) an increase in stiffness beyond the second plateau that was consistent with the theoretical stiffness-vs-displacement function of a beam. These features were present both pre- and post-mortem.     

Analysis of liquid surface films by pulsed laser photoacoustic spectroscopy

The photoacoustic generation of plane acoustic waves in strongly absorbing or opaque liquids by pulsed laser radiation is discussed both experimentally and theoretically. The regimes of a confined and a free surface of the liquid are considered. The model which takes the temporal shape of the laser pulses applied in the experiments into account, implies that spectroscopic studies are feasible with direct photoacoustic generation and detection also for opaque liquids. The experiments are performed with a tunable hybrid CO₂ laser and piezoelectric detection. For the first time liquid/liquid interfaces are studied by this technique. We demonstrate that the presence of an absorbing liquid film with a thickness of >1 m on the surface of another liquid amplifies the acoustic signal which is detected in the bottom liquid. The enhancement depends on the thickness and the optical and thermal properties of the film medium. The surface layer can be analyzed on the basis of the photoacoustic spectrum. It is also shown that this non-contact method is surface-film selective and should thus prove useful for pollution analysis of liquid surfaces.