Photoacoustic measurement of the Grüneisen parameter of tissue.

The Grüneisen parameter, a constitutive parameter in photoacoustics, is usually measured from isobaric thermal expansion, which may not be valid for a biological medium due to its heterogeneity. Here, we directly measured the Grüneisen parameter by applying photoacoustic spectroscopy. Laser pulses at wavelengths between 460 and 1800 nm were delivered to tissue samples, and photoacoustic signals were detected by flat water-immersion ultrasonic transducers. Least-squares fitting photoacoustic spectra to molar optical absorption spectra showed that the Grüneisen parameter was 0.81±0.05 (mean±SD) for porcine subcutaneous fat tissue and 0.69±0.02 for porcine lipid at room temperature (22°C). The Grüneisen parameter of a red blood cell suspension was linearly related to hemoglobin concentration, and the parameter of bovine serum was 9% greater than that of water at room temperature.

Label-free photoacoustic microscopy of peripheral nerves.

Peripheral neuropathy is a common neurological problem that affects millions of people worldwide. Diagnosis and treatment of this condition are often hindered by the difficulties in making objective, noninvasive measurements of nerve fibers. Photoacoustic microscopy (PAM) has the ability to obtain high resolution, specific images of peripheral nerves without exogenous contrast. We demonstrated the first proof-of-concept imaging of peripheral nerves using PAM. As validated by both standard histology and photoacoustic spectroscopy, the origin of photoacoustic signals is myelin, the primary source of lipids in the nerves. An extracted sciatic nerve sandwiched between two layers of chicken tissue was imaged by PAM to mimic the in vivo case. Ordered fibrous structures inside the nerve, caused by the bundles of myelin-coated axons, could be observed clearly. With further technical improvements, PAM can potentially be applied to monitor and diagnose peripheral neuropathies.

Label-free photoacoustic microscopy of cytochromes.

Photoacoustic microscopy (PAM) has achieved submicron lateral resolution in showing subcellular structures; however, relatively few endogenous subcellular contrasts have so far been imaged. Given that the hemeprotein, mostly cytochromes in general cells, is optically absorbing around the Soret peak (~420 nm), we implemented label-free PAM of cytochromes in cytoplasm for the first time. By measuring the photoacoustic spectra of the oxidized and reduced states of fibroblast lysate and fitting the difference spectrum with three types of cytochromes, we found that the three cytochromes account for more than half the optical absorption in the cell lysate at 420 nm wavelength. Fixed fibroblasts on slides were imaged by PAM at 422 and 250 nm wavelengths to reveal cytoplasms and nuclei, respectively, as confirmed by standard staining histology. PAM was also applied to label-free histology of mouse ear sections by showing cytoplasms and nuclei of various cells. PAM of cytochromes in cytoplasm is expected to be a high-throughput, label-free technique for studying live cell functions, which cannot be accomplished by conventional histology.

Photoacoustic microscopy of bilirubin in tissue phantoms.

Determining both bilirubin's concentration and its spatial distribution are important in disease diagnosis. Here, for the first time, we applied quantitative multiwavelength photoacoustic microscopy (PAM) to detect bilirubin concentration and distribution simultaneously. By measuring tissue-mimicking phantoms with different bilirubin concentrations, we showed that the root-mean-square error of prediction has reached 0.52 and 0.83 mg/dL for pure bilirubin and for blood-mixed bilirubin detection (with 100% oxygen saturation), respectively. We further demonstrated the capability of the PAM system to image bilirubin distribution both with and without blood. Finally, by underlaying bilirubin phantoms with mouse skins, we showed that bilirubin can be imaged with consistent accuracy down to >400 µm in depth. Our results show that PAM has potential for noninvasive bilirubin monitoring in vivo, as well as for further clinical applications.

Optimal ultraviolet wavelength for in vivo photoacoustic imaging of cell nuclei.

In order to image noninvasively cell nuclei in vivo without staining, we have developed ultraviolet photoacoustic microscopy (UV-PAM), in which ultraviolet light excites nucleic acids in cell nuclei to produce photoacoustic waves. Equipped with a tunable laser system, the UV-PAM was applied to in vivo imaging of cell nuclei in small animals. We found that 250 nm was the optimal wavelength for in vivo photoacoustic imaging of cell nuclei. The optimal wavelength enables UV-PAM to image cell nuclei using as little as 2 nJ laser pulse energy. Besides the optimal wavelength, application of a wavelength between 245 and 275 nm can produce in vivo images of cell nuclei with specific, positive, and high optical contrast.

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.

The constitutive equation for membrane tether extraction.

Membrane tethers or nanotubes play a critical role in a variety of cellular and subcellular processes such as leukocyte rolling and intercellular mass transport. The current constitutive equations that describe the relationship between the pulling force and the tether velocity during tether extraction have serious limitations. In this article, we propose a new phenomenological constitutive equation that captures all known characteristics of nanotube formation, including nonlinearity, nonzero threshold force, and possible negative tether velocity. We used tether extraction from endothelial cells as a prototype to illustrate how to obtain the material constants in the constitutive equation. With the micropipette aspiration technique, we measured tether pulling forces at both positive and negative tether velocities. We also determined the threshold force of 55 pN experimentally for the first time. This new constitutive equation unites two established ones and provides us a unified platform to better understand not only the physiological role of tether extraction during leukocyte rolling and intercellular or intracellular transport, but also the physics of membrane tether growth or retraction.

A direct micropipette-based calibration method for atomic force microscope cantilevers.

In this report, we describe a direct method for calibrating atomic force microscope (AFM) cantilevers with the micropipette aspiration technique (MAT). A closely fitting polystyrene bead inside a micropipette is driven by precisely controlled hydrostatic pressures to apply known loads on the sharp tip of AFM cantilevers, thus providing a calibration at the most functionally relevant position. The new method is capable of calibrating cantilevers with spring constants ranging from 0.01 to hundreds of newtons per meter. Under appropriate loading conditions, this new method yields measurement accuracy and precision both within 10%, with higher performance for softer cantilevers. Furthermore, this method may greatly enhance the accuracy and precision of calibration for colloidal probes.

A Novel Technique of Quantifying Flexural Stiffness of Rod-Like Structures.

In cellular and molecular biomechanics, extensional stiffness of rod-like structures such as leukocyte microvilli can be easily measured with many techniques, but not many techniques are available for measuring their flexural stiffness. In this paper, we report a novel technique of measuring the flexural stiffness of rod-like structures. This technique is based on image deconvolution and, as an example, it was used for determining the flexural stiffness of neutrophil microvilli. The probes we used were 40-nm-diameter fluorescent beads, which were bound to the tips of neutrophil microvilli by anti-L-selectin antibody. The fluorescent images of the bead, which was positioned at the center of the cell bottom, were acquired with high magnification and long exposure time (3 s). Using a Gaussian function as the point spread function of our imaging system, we established a convolution equation based on Boltzmann's law, which yields an analytical expression that relates the bead image profile to the flexural stiffness of the microvillus. The flexural stiffness was then obtained by the least squares regression. On average, the flexural stiffness was determined to be 7 pN/mum for single neutrophil microvilli. With the resolution of our imaging system, this technique can be used for measuring any flexural stiffness smaller than 34 pN/mum and it has great potential in single molecule biomechanics.

Flexibility of single microvilli on live neutrophils and lymphocytes.

We measured the flexural stiffness of single microvilli on live human neutrophils and lymphocytes using 40-nm fluorescent beads. The beads were bound to the tips of the microvilli by anti-L-selectin antibodies. Digital bead images were acquired with an exposure time of 3 s at high magnification. Using a Gaussian point spread function, we obtained an analytical expression that relates the image profile to the flexural stiffness. We found that the flexural stiffnesses were 7 and 4 pN/microm for single microvilli on human neutrophils and lymphocytes, respectively. We also verified with live cells that 75% of neutrophil L-selectin and 72% of lymphocyte L-selectin were on the microvillus tips. Our results indicate that the leukocyte microvilli in contact with the endothelium or other surfaces will bend easily under physiological shear stresses.

[An analysis of pharyngeal respiratory pressure before and after onset of obstruction in respiratory obstructive sleep apnea hypopnea syndrome].

OBJECTIVE: To study the characteristics of pharyngeal respiratory pressure before and after onset of airflow obstruction events in obstructive sleep apnea hypopnea syndrome (OSAHS). METHODS: The respiratory pressure in nasopharynx, oropharynx, and hypopharynx in 8 patients with OSAHS and 9 normal persons were evaluated through catheter manometer. Base on mean respiratory pressure and the ratio of negative respiratory pressure persistent time, the characteristics of pharyngeal respiratory pressure before and after the event onset were analyzed. RESULTS: During sleep, the pharyngeal respiratory pressure in normal persons had similar and periodical wave shape. The ratio of negative respiratory pressure persistent time was less than 0.5. In patients with OSAHS, when the obstruction of airflow happened, the pharyngeal respiratory pressure fluctuated violently, the wave shape became irregular, and the negative expiratory pressure was evident. The mean respiratory pressure was 1 to 2 order of magnitude larger than in normal persons, even reaching -990 Pa. The ratio of persistent negative pressure time was larger than normal. During the intermittent period, the wave shape of pharyngeal respiratory pressure was regular and periodical, the pressure wave shape was different from onset of obstruction. However, in the intermittent period, the mean respiratory pressure, the ratio of negative respiratory pressure persistent time, and other characteristics were still statistically different from normal persons (P < 0.05). CONCLUSIONS: During the intermittent period, the pharyngeal respiratory pressure in patients with OSAHS during sleep is basically different from the pharyngeal respiratory pressure in normal person. The characteristics of pharyngeal respiratory pressure in intermittent period indicates that both structural and functional abnormalities in pharyngeal cavity in patients with OSAHS, which affect the respiratory airflow during sleep are inherent. The effects are more prominent during onset period, suggesting that the characteristics of the pharyngeal pressure of breathing during the onset period will be more important to the mechanism of airflow obstruction.