The detection and quantification of circulating tumor cells (CTCs) in a patient’s bloodstream is a promising minimally invasive avenue for improving cancer diagnostics and prognostics. However, clinical utilization of CTC-based diagnostics has been limited due to the arduous nature of currently employed CTC detection strategies which largely involve tedious staining and purification procedures. We report the use of photoacoustic remote sensing microscopy to enable the label-free visualization of peripheral blood mononuclear cells for the purpose of categorizes white blood cell populations and identifying putative circulating tumor cells present in the blood stream. Image analysis of cellular characteristics is performed, and identification of cell phenotype is validated by comparison to histological staining.
We develop a dual-modality imaging system for virtual histology in breast tumor specimens, augmenting depth-resolved scattering contrast from OCT with sub-cellular resolution and label-free molecular specificity from UV photoacoustic remote sensing.
We demonstrate the use of Photoacoustic Remote Sensing (PARS) and scattering microscopy capable of acquiring virtual depth-resolved images of tissues with virtual contrast of hematoxylin using PARS & eosin using scattering microscopy.
Significance: Complementary absorption and fluorescence contrast could prove useful for a wide range of biomedical applications. However, current absorption-based photoacoustic microscopy systems require the ultrasound transducers to physically touch the samples, thereby increasing contamination and limiting strong optical focusing in reflection mode.
Aim: We sought to develop an all-optical system for imaging cells and tissues using the three combined imaging modalities: photoacoustic remote sensing (PARS), epifluorescence, and confocal laser scanning microscopy (CLSM).
Approach: A PARS subsystem with ultraviolet excitation was used to obtain label-free absorption-contrast images of nucleic acids in ex vivo tissue samples. Co-integrated epifluorescence and CLSM subsystems were used to verify the 2D and 3D nuclei distribution.
Results: Complementary absorption and fluorescence contrast were demonstrated in phantom imaging experiments and subsequent cell and tissue imaging experiments. Lateral and axial resolution of ultraviolet-PARS (UV-PARS) is shown to be 0.39 and 1.6 μm, respectively, with 266-nm light. CLSM lateral and axial resolution was measured as 0.97 and 2.0 μm, respectively. This resolution is sufficient to image individual cell layers with fine optical sectioning. UV-PARS images of cell nuclei are validated in thick tissue using CLSM.
Conclusions: Multimodal absorption and fluorescence contrast are obtained with a non-contact all-optical microscopy system for the first time and utilized to obtain images of cells and tissues with subcellular resolution.
We report the use of frequency decomposition with ultraviolet photoacoustic remote sensing (UV-PARS) microscopy that enables contact-free and label-free virtual H and E staining. Along with principal component analysis of frequency information, this image reconstruction technique allows for the incorporation of scale-specific information encoded in the frequency spectrum of generated PARS signals. The performance of this image reconstruction technique as demonstrated on histological samples achieves contrast-to-noise ratios of over 55-dB between nuclei and nearby cytoplasmic structures. This technique allows for the rapid and facile production of virtual H and E stained images without the need for additional excitation wavelengths.
Rapid, accurate intraoperative assessment of excised specimens and in vivo resection sites is a critical need in tissue-conserving cancer surgeries. We develop a multimodal imaging system integrating ultraviolet photoacoustic remote sensing (UV-PARS) microscopy with spectral-domain optical coherence tomography (SD-OCT) to provide comprehensive, co-registered information for identifying atypical tissue features. 1050-nm SD-OCT generates a widefield, depth-resolved view of subsurface tissue morphology, with 9-μm axial resolution. UV-PARS produces virtual histological C-scan images of cell nuclei, at 390-nm lateral resolution. Dual-contrast in vivo mouse imaging, and ex vivo imaging of breast and prostate specimens is demonstrated, and correlated with conventional H and E staining.
Hematoxylin and Eosin (H and E) staining is the gold standard for the majority of histopathological diagnostics but requires lengthy processing times not suitable for point-of-care diagnosis. Here we demonstrate a 266-nm excitation Ultraviolet Photoacoustic Remote Sensing (UV-PARS) and Scattering Microscopy system capable of virtual H and E 3D imaging of tissues in conjunction with with confocal fluorescence microscopy (CFM) for validation in thick tissues. We demonstrate the capabilities of this dual-contrast system for en-face planar and volumetric imaging of human tissue samples exhibiting high concordance with the gold standard of H and E staining procedures as well as confocal fluorescence microscopy. To our knowledge, this is the first near real-time microscopy approach capable of volumetric imaging unstained thick tissues with virtual H and E contrast.
Photoacoustic remote sensing (PARS) microscopy with multi-wavelength excitation and spectral unmixing was recently reported for non-contact imaging of hemoglobin oxygen saturation. In this work, we aim to extend this technique to provide additional functional information on oxygen metabolic activity. Stimulated Raman scattering provides multi-wavelength PARS excitation for SO2 estimation, and is integrated with Doppler OCT for quantitative blood flow rate determination. Structural imaging is demonstrated in phantoms and in vivo angiography. To evaluate system performance, tube phantoms containing blood of varied oxygenation and flow rates were imaged. This represents progress towards deploying the technique for in vivo oxygen consumption monitoring.
KEYWORDS: Microscopy, In vivo imaging, Signal to noise ratio, Photoacoustic spectroscopy, Absorption, Ultraviolet radiation, Tissue optics, Image resolution, Remote sensing
Photoacoustic remote sensing (PARS) is a non-contact imaging modality that is based on the optical absorption contrast of endogenous molecules. PARS has shown promise in vascular imaging, blood oxygenation estimation, and virtual biopsy without the need for exogenous labels. Here we demonstrate simultaneous imaging of cell nuclei and blood using UV and visible excitation wavelengths. This is important for decoupling blood signals from cell nuclei signals in removed tissue and resection beds. A 532nm fiber laser is split with one light path frequency doubled using a CLBO crystal to 266nm. These two wavelength lasers are co-aligned and co-focused with a 1310nm interrogation beam and using a reflective objective to image microvasculature and cell nuclei with intrinsic optical absorptions at 532nm and 266nm, respectively. These images are taken serially and co-registered with lateral resolutions of 1.2μm and 0.44μm respectively. Co-alignment using multiple wavelengths is demonstrated using carbon fiber phantoms. We imaged both paraffin embedded tissue and in vivo mouse ear. Cell nuclei in sectioned tissues were clearly visualized with a SNR of 42dB while hemoglobin demonstrated an SNR of 39dB. In vivo cell nuclei and vasculature images produced an SNR up to 40dB and 35dB, respectively.
Despite advancements in imaging and surgical methodology surgeons continue to face challenges in differentiating suitable margins in tumor resection sites and assessing the extent of cancer proliferation. Presently, histology is the gold standard used for determining margins post-operatively. However, currently no intraoperative tools can analyze entire resection sites. This results in unnecessary repeated surgeries and is especially critical to patient outcome for certain cancers. To address this critical need, we have developed a next-generation microscopy system intended to allow accurate intraoperative virtual histopathology for margin assessment. The modality, named ultraviolet photoacoustic remote sensing microscopy (UV-PARS), takes advantage of the intrinsic optical absorption contrast of DNA at 266 nm and a non-contact PARS technique. This approach measures time-dependent refractive index modulations at their subsurface origin resulting from thermo-elastic excitation from a pulsed excitation source. This enables the possibility of real time non-contact label-free visualization of cell nuclei in vivo. Preliminary results are presented including studies with live cell cultures, excised tissue samples, phantoms, and characterization of system parameters. Lateral-resolution was found to be 0.7 µm with a signal-to-noise ratio of 50 dB achieved in phantoms and 30 dB for HeLa cells. Simultaneously collected confocal reflectance microscopy using 1310 nm light provided cell body morphology. HeLa and PC3 cell cultures were imaged with good agreement to conventional H&E stained images of the same samples.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.