Seeing Through the Surface: Non-invasive Characterization of Biomaterial-Tissue Interactions Using Photoacoustic Microscopy

At the intersection of life sciences, materials science, engineering, and medicine, regenerative medicine stands out as a rapidly progressing field that aims at retaining, restoring, or augmenting tissue/organ functions to promote the human welfare. While the field has witnessed tremendous advancements over the past few decades, it still faces many challenges. For example, it has been difficult to visualize, monitor, and assess the functions of the engineered tissue/organ constructs, particularly when three-dimensional scaffolds are involved. Conventional approaches based on histology are invasive and therefore only convey end-point assays. The development of volumetric imaging techniques such as confocal and ultrasonic imaging has enabled direct observation of intact constructs without the need of sectioning. However, the capability of these techniques is often limited in terms of penetration depth and contrast. In comparison, the recently developed photoacoustic microscopy (PAM) has allowed us to address these issues by integrating optical and ultrasonic imaging to greatly reduce the effect of tissue scattering of photons with one-way ultrasound detection while retaining the high optical absorption contrast. PAM has been successfully applied to a number of studies, such as observation of cell distribution, monitoring of vascularization, and interrogation of biomaterial degradation. In this review article, we highlight recent progress in non-invasive and volumetric characterization of biomaterial–tissue interactions using PAM. We also discuss challenges ahead and envision future directions

Melanocortin 1 receptor targeted imaging of melanoma with gold nanocages and positron emission tomography

Purpose: Melanoma is a lethal skin cancer with unmet clinical needs for targeted imaging and therapy. Nanoscale materials conjugated with targeting components have shown great potential to improve tumor delivery efficiency while minimizing undesirable side effects in vivo. Herein, we proposed to develop targeted nanoparticles for melanoma theranostics. Method: In this work, gold nanocages (AuNCs) were conjugated with α-melanocyte-stimulating hormone (α-MSH) peptide and radiolabeled with 64Cu for melanocortin 1 receptor-(MC1R) targeted positron emission tomography (PET) in a mouse B16/F10 melanoma model. Results: Their controlled synthesis and surface chemistry enabled well-defined structure and radiolabeling efficiency. In vivo pharmacokinetic evaluation demonstrated comparable organ distribution between the targeted and nontargeted AuNCs. However, micro-PET/computed tomography (CT) imaging demonstrated specific and improved tumor accumulation via MC1R-mediated delivery. By increasing the coverage density of α-MSH peptide on AuNCs, the tumor delivery efficiency was improved. Conclusion: The controlled synthesis, sensitive PET imaging, and optimal tumor targeting suggested the potential of targeted AuNCs for melanoma theranostics. </jats:sec

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

The effect of adipose-derived stem cell sheets and CTGF on early flexor tendon healing in a canine model

Oral History interview of Curt Sawyer. Interview conducted by Robin Dunn

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

Noninvasive photoacoustic sentinel lymph node mapping using Au nanocages as a lymph node tracer in a rat model

Sentinel lymph node biopsy (SLNB) has been widely performed and become the standard procedure for axillary staging in breast cancer patients. In current SLNB, identification of SLNs is prerequisite, and blue dye and/or radioactive colloids are clinically used for mapping. However, these methods are still intraoperative, and especially radioactive colloids based method is ionizing. As a result, SLNB is generally associated with ill side effects. In this study, we have proposed near-infrared Au nanocages as a new tracer for noninvasive and nonionizing photoacoustic (PA) SLN mapping in a rat model as a step toward clinical applications. Au nanocages have great features: biocompatibility, easy surface modification for biomarker, a tunable surface plasmon resonance (SPR) which allows for peak absorption to be optimized for the laser being used, and capsule-type drug delivery. Au nanocage-enhanced photoacoustic imaging has the potential to be adjunctive to current invasive SLNB for preoperative axillary staging in breast cancer patients

Quantitative Analysis of the Fate of Gold Nanocages In Vitro and In Vivo after Uptake by U87-MG Tumor Cells

Not always equal: When a mother cell that contains Au nanocages divides, the nanoparticles are unequally distributed between the two daughter cells. This unequal distribution of nanoparticles as well as their clearance from the cells (see picture) is quantitatively analyzed both in vitro and in vivo using two-photon microscopy and photoacoustic microscopy, respectively

In vivo integrated photoacoustic and confocal microscopy of hemoglobin oxygen saturation and oxygen partial pressure

We developed dual-modality microscope integrating photoacoustic microscopy (PAM) and fluorescence confocal microscopy (FCM) to noninvasively image hemoglobin oxygen saturation (sO_2) and oxygen partial pressure (pO_2) in vivo in single blood vessels with high spatial resolution. While PAM measures sO_2 by imaging hemoglobin optical absorption at two wavelengths, FCM quantifies pO_2 using phosphorescence quenching. The variations of sO_2 and pO_2 values in multiple orders of vessel branches under hyperoxic (100% oxygen) and normoxic (21% oxygen) conditions correlate well with the oxygen–hemoglobin dissociation curve. In addition, the total concentration of hemoglobin is imaged by PAM at an isosbestic wavelength

Photoacoustic Tomography of a Rat Cerebral Cortex in vivo with Au Nanocages as an Optical Contrast Agent

Poly(ethylene glycol)-coated Au nanocages have been evaluated as a potential near-infrared (NIR) contrast agent for photoacoustic tomography (PAT). Previously, Au nanoshells were found to be an effective NIR contrast agent for PAT; however, Au nanocages with their more compact sizes (100 nm for Au nanoshells) and larger optical absorption cross sections should be better suited for in vivo applications. We sequentially injected Au nanocages into the circulatory system of a rat in three administrations and in vivo PAT was conducted immediately prior to the first injection and continued until 5 h after the final injection. A gradual enhancement of the optical absorption in the cerebral cortex, by up to 81%, was observed over the course of the experiment