Super-resolution microscopy techniques—capable of overcoming the diffraction limit of light—have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules.
The scanning mechanism is a major technical focus in optical-resolution photoacoustic microscopy. Flexible scanning access with fast scanning speed is desired to monitor biological and physiological dynamics with high temporal resolution. We developed random-access optical-resolution photoacoustic microscopy (RA-OR-PAM) using a digital micromirror device (DMD). Each micromirror on the DMD can be independently controlled, allowing imaging of regions of interest with arbitrary user-selected shapes without extraneous information. A global structural image is first acquired, and the regions of interest are selected. The laser beam then scans these regions exclusively, resulting in a faster frame rate than in a conventional raster scan. This system can rapidly scan arbitrarily shaped regions of interest with a lateral resolution of 3.6 μm within a 40×40 μm2 imaging area, a size comparable to the focal spot size of a 50 MHz ultrasound transducer. We demonstrated the random-access ability of RA-OR-PAM by imaging a monolayer of red blood cells. This system was then used to monitor blood flow in vivo within user-selected capillaries in a mouse ear. By imaging only the capillary of interest, the frame rate was increased by up to 13.3 times.
The fundamental limitations of photoacoustic microscopy for detecting optically absorbing molecules are investigated both theoretically and experimentally. We experimentally demonstrate noise-equivalent detection sensitivities of 160,000 methylene blue molecules (270 zeptomol or 2.7×10 −19 mol ) and 86,000 oxygenated hemoglobin molecules (140 zeptomol) using narrowband continuous-wave photoacoustics. The ultimate sensitivity of photoacoustics is fundamentally limited by thermal noise, which can present in the acoustic detection system as well as in the medium itself. Under the optimized conditions described herein and using commercially available detectors, photoacoustic microscopy can detect as few as 100s of oxygenated hemoglobin molecules. Realizable improvements to the detector may enable single molecule detection of select molecules.
Recently, a number of optical imaging modalities have achieved single molecule sensitivity, including photothermal
imaging, stimulated emission microscopy, ground state depletion microscopy, and transmission microscopy. These
optical techniques are based on optical absorption contrast, extending single-molecule detection to non-fluorescent
chromophores. Photoacoustics is a hybrid technique that utilizes optical excitation and ultrasonic detection, allowing it to
scale both the optical and acoustic regimes with 100% sensitivity to optical absorption. However, the sensitivity of
photoacoustics is limited by thermal noise, inherent in the medium itself in the form of acoustic black body radiation. In
this paper, we investigate the molecular sensitivity of photoacoustics in the context of the thermal noise limit. We show
that single molecule sensitivity is achievable theoretically at room temperature for molecules with sufficiently fast
relaxation times. Hurdles to achieve single molecule sensitivity in practice include development of detection schemes
that work at short working distance, <100 microns, high frequency, <100 MHz, and low loss, <10 dB.
Optical coherence tomography (OCT) can provide new insight into disease progression and therapy by enabling nondestructive, serial imaging of in vivo cancer models. In previous studies, we have shown the utility of endoscopic OCT for identifying adenomas in the azoxymethane-treated mouse model of colorectal cancer and tracking disease progression over time. Because of improved imaging speed made possible through Fourier domain imaging, three-dimensional imaging of the entire mouse colon is possible. Increased amounts of data can facilitate more accurate classification of tissue but require more time on the part of the researcher to sift through and identify relevant data. We present quantitative software for automatically identifying potentially diseased areas that can be used to create a two-dimensional "disease map" from a three-dimensional Fourier domain OCT data set. In addition to sensing inherent changes in tissue that occur during disease development, the algorithm is sensitive to exogeneous highly scattering gold nanoshells that can be targeted to disease biomarkers. The results of the algorithm were compared to histological diagnosis. The algorithm was then used to assess the ability of gold nanoshells targeted to epidermal growth factor receptor in vivo to enable functional OCT imaging.
Polarimetric glucose sensing is a promising method for noninvasive estimation of blood glucose concentration.
Published methods of polarimetric glucose sensing generally rely on measuring the rotation of light as it traverses
the aqueous humor of the eye. In this article, an interferometer is described that can detect polarization changes
due to glucose without the use of polarization control or polarization analyzing elements. Without polarizers,
this system is sensitive to optical activity, inherent to glucose, but minimally sensitive to linear retardance,
inherent to the cornea. The underlying principle of the system was experimentally verified using spectral domain
optical coherence tomography. A detection scheme involving amplitude modulation was simulated, demonstrating
sensitivity to clinically relevant glucose concentrations and an acceptable error due to time varying linear
birefringence of the cornea using Clarke Error Grid Analysis.
Ovarian cancer is the fourth leading cause of cancer-related death among women. If diagnosed at early stages, 5-year survival rate is 94%, but drops to 68% for regional disease and 29% for distant metastasis; only 19% of cases are diagnosed at early, localized stages. Optical coherence tomography is a recently emerging non-destructive imaging technology, achieving high axial resolutions (10-20 µm) at imaging depths up to 2 mm. Previously, we studied OCT in normal and diseased human ovary ex vivo. Changes in collagen were suggested with several images that correlated with changes in collagen seen in malignancy. Areas of necrosis and blood vessels were also visualized using OCT, indicative of an underlying tissue abnormality. We recently developed a custom side-firing laparoscopic OCT (LOCT) probe fabricated for in vivo imaging. The LOCT probe, consisting of a 38 mm diameter handpiece terminated in a 280 mm long, 4.6 mm diameter tip for insertion into the laparoscopic trocar, is capable of obtaining up to 9.5 mm image lengths at 10 µm axial resolution. In this pilot study, we utilize the LOCT probe to image one or both ovaries of 17 patients undergoing laparotomy or transabdominal endoscopy and oophorectomy to determine if OCT is capable of differentiating normal and neoplastic ovary. We have laparoscopically imaged the ovaries of seventeen patients with no known complications. Initial data evaluation reveals qualitative distinguishability between the features of undiseased post-menopausal ovary and the cystic, non-homogenous appearance of neoplastic ovary such as serous cystadenoma and endometroid adenocarcinoma.
Strong vascular endothelial growth factor (VEGF) receptor expression has been found at the sites of angiogenesis,
particularly in tumor growth areas. An increase in VEGF receptor-2 is associated with colon cancer progression. The in
vivo detection of VEGF receptor is of interest for the purposes of studying carcinogenesis, the efficacy of
chemopreventive and therapeutic agents, clinical diagnosis, and therapeutic monitoring. In this study, a novel single
chain (sc) VEGF-based molecular probe is utilized in the AOM-treated mouse model of colorectal cancer to study
delivery route and specificity for disease. The probe was constructed by site-specific conjugation of a near-infrared dye,
Cy5.5, to scVEGF and detected in vivo with a dual-modality optical coherence tomography / laser-induced fluorescence
(OCT/LIF) endoscopic system. The LIF excitation source was a 633 nm He:Ne laser and red/near-infrared fluorescence
was detected with a spectrometer. OCT was used to obtain two-dimensional longitudinal tomograms at eight rotations in
the distal colon. Fluorescence emission levels were correlated with OCT-detected disease in vivo and H&E stained
histology slides ex vivo. Specificity for disease was found to be highly dependent on the delivery route. Intravenous
injection resulted in poor specificity due to many extra-colon confounders, while colon lavage eliminated most of these.
High fluorescence emission intensity was correlated with tumor presence as detected using OCT. Results suggest
potential for clinical use to facilitate earlier diagnosis of cancer.
In previous work we have demonstrated the utility of laser-induced fluorescence (LIF) and optical coherence
tomography (OCT) to identify adenoma in mouse models of colorectal cancer with high sensitivity and specificity.
However, improved sensitivity to early disease, as well as the ability to distinguish confounders (e.g. fecal
contamination, natural variations in mucosal thickness), is desired. In this study, we investigated the signal enhancement
of fluorescent and scattering contrast agents in the colons of AOM-treated mice. The fluorescent tracer scVEGF/Cy,
targeted to receptors for vascular endothelial growth factor, was visualized on a dual modality OCT/LIF endoscopic
system with 1300-nm center wavelength OCT source and 635-nm LIF excitation. Scattering agents were tested with an
890-nm center wavelength endoscopic OCT system. Agents included nanoshells, 120-nm in diameter, and nanorods, 20-nm in diameter by 80-nm in length. Following imaging, colons were excised. Tissue treated with fluorophore was
imaged on an epifluorescence microscope. Histological sections were obtained and stained with H&E and silver
enhancer to verify disease and identify regions of gold uptake, respectively. Non-specific signal enhancement was
observed with the scattering contrast agents. Specificity for adenoma was seen with the scVEGF/Cy dye.
Depth dependent broadening of the axial point spread function due to dispersion in the imaged media, and algorithms for postprocess correction have been previously described for both time domain and frequency domain optical coherence tomography. Homogeneous media dispersion artifacts disappear when frequency domain samples are uniformly spaced in circular wavenumber, as opposed to uniform sampling in optical frequency. In this paper, we explicate the source of this point spread broadening and simulate its magnitude in aqueous media. We conclude with a suggestion for interferometric k-triggering which accounts for dispersion in the media.