Da-Kang Yao

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.

Label-free photoacoustic microscopy of cytochromes

Abstract. 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 measurement of the Grüneisen parameter of tissue

Abstract. 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

Abstract. 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.

Photoacoustic microscopy of bilirubin in tissue phantoms

Abstract. 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.

Measurement of Grüneisen parameter of tissue by photoacoustic spectrometry

The Grüneisen parameter of tissue is a constitutive parameter in photoacoustic tomography. Here, we applied photoacoustic spectrometry (PAS) to directly measure the Grüneisen parameter. In our PAS system, laser pulses at wavelengths between 460 and 1600 nm were delivered to tissue samples, and photoacoustic signals were detected by a 20 MHz flat water-immersion ultrasonic transducer. By fitting photoacoustic spectra to light absorption spectra, we found that the Grüneisen parameter was 0.73 for porcine subcutaneous fat tissue, and 0.15 for oxygenated bovine red blood cells at room temperature (24°C).

Label-free photoacoustic microscopy of cytochrome c in cells

Cytochrome c is a heme protein normally bound to mitochondria and is important for mitochondrial electron transport and apoptosis initiation. Since cytochrome c is nonfluorescent, it is always labeled with fluorescent molecules for imaging, which, however, may affect normal cellular functions. Here, label-free photoacoustic microscopy (PAM) of mitochondrial cytochrome c was realized for the first time by utilizing the optical absorption around the Soret peak. PAM was demonstrated to be sensitive enough to image mitochondrial cytochrome c at 422 nm wavelength. Mitochondrial cytochrome c in the cytoplasm of fixed fibroblasts was clearly imaged by PAM as confirmed by fluorescent labeling. By showing mitochondrial cytochrome c in various cells, we demonstrated the feasibility of PAM for label-free histology of mouse ear sections. Therefore, PAM can sensitively image cytochrome c in unstained cells at 422 nm wavelength and has great potential for functional imaging of cytochrome c in live cells or in vivo.

In vivo imaging of cell nuclei by photoacoustic microscopy without staining

Ultraviolet photoacoustic microscopy (UVPAM) can image cell nuclei in vivo with high contrast and resolution noninvasively without staining. Here, we used UV light at wavelengths of 210-310 nm for excitation of DNA and RNA to produce photoacoustic waves. We applied the UVPAM to in vivo imaging of cell nuclei in mouse skin, and obtained UVPAM images of the unstained cell nuclei at wavelengths of 245-282 nm as ultrasound gel was used for acoustic coupling. The largest ratio of contrast to noise was found for the images of cell nuclei at a 250 nm wavelength.

Quantitative imaging of bilirubin by photoacoustic microscopy

Noninvasive detection of both bilirubin concentration and its distribution is important for disease diagnosis. Here we implemented photoacoustic microscopy (PAM) to detect bilirubin distribution. We first demonstrate that our PAM system can measure the absorption spectra of bilirubin and blood. We also image bilirubin distributions in tissuemimicking samples, both without and with blood mixed. Our results show that PAM has the potential to quantitatively image bilirubin in vivo for clinical applications.

Photoacoustic microscopy of myocardial sheet architecture in unfixed and unstained mammalian hearts

The laminar myocardial sheet architecture and its dynamic change play a key role in myocardial wall thickening. Histology, confocal optical microscopy (COM), and diffusion tensor MRI (DTI) have been used to unveil the structures and functions of the myocardial sheets. However, histology and COM require fixation, sectioning, and staining processes, which dehydrate and deform the sheet architecture. Although DTI can delineate sheet architecture nondestructively in viable hearts, it cannot provide cellular-level resolution. Here we show that photoacoustic microscopy (PAM), with high resolution (~1 μm) and label-free detection, is appropriate for imaging 3D myocardial architecture. Perfused half-split mouse hearts were also imaged by PAM in vitro without fixation, dehydration, nor staining. The laminar myocardial sheet architecture was clearly visualized within a 0.15 mm depth range. Two populations of oppositely signed sheet angles were observed. Therefore, PAM promises to access dynamic changes of myocardial architectures in ex vivo perfused-viable hearts.