RESUMO
Current breast tumor margin detection methods are destructive, time-consuming, and result in significant reoperative rates. Dual-modality photoacoustic tomography (PAT) and ultrasound has the potential to enhance breast margin characterization by providing clinically relevant compositional information with high sensitivity and tissue penetration. However, quantitative methods that rigorously compare volumetric PAT and ultrasound images with gold-standard histology are lacking, thus limiting clinical validation and translation. Here, we present a quantitative multimodality workflow that uses inverted Selective Plane Illumination Microscopy (iSPIM) to facilitate image co-registration between volumetric PAT-ultrasound datasets with histology in human invasive ductal carcinoma breast tissue samples. Our ultrasound-PAT system consisted of a tunable Nd:YAG laser coupled with a 40 MHz central frequency ultrasound transducer. A linear stepper motor was used to acquire volumetric PAT and ultrasound breast biopsy datasets using 1100 nm light to identify hemoglobin-rich regions and 1210 nm light to identify lipid-rich regions. Our iSPIM system used 488 nm and 647 nm laser excitation combined with Eosin and DRAQ5, a cell-permeant nucleic acid binding dye, to produce high-resolution volumetric datasets comparable to histology. Image thresholding was applied to PAT and iSPIM images to extract, quantify, and topologically visualize breast biopsy lipid, stroma, hemoglobin, and nuclei distribution. Our lipid-weighted PAT and iSPIM images suggest that low lipid regions strongly correlate with malignant breast tissue. Hemoglobin-weighted PAT images, however, correlated poorly with cancerous regions determined by histology and interpreted by a board-certified pathologist. Nuclei-weighted iSPIM images revealed similar cellular content in cancerous and non-cancerous tissues, suggesting malignant cell migration from the breast ducts to the surrounding tissues. We demonstrate the utility of our nondestructive, volumetric, region-based quantitative method for comprehensive validation of 3D tomographic imaging methods suitable for bedside tumor margin detection.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Técnicas Fotoacústicas/métodos , Ultrassonografia Mamária/métodos , Feminino , Humanos , Imagens de FantasmasRESUMO
SIGNIFICANCE: Tumor heterogeneity poses a challenge for the chemotherapeutic treatment of cancer. Tissue dynamics spectroscopy captures dynamic contrast and can capture the response of living tissue to applied therapeutics, but the current analysis averages over the complicated spatial response of living biopsy samples. AIM: To develop tissue dynamics spectroscopic imaging (TDSI) to map the heterogeneous spatial response of tumor tissue to anticancer drugs. APPROACH: TDSI is applied to tumor spheroids grown from cell lines and to ex vivo living esophageal biopsy samples. Doppler fluctuation spectroscopy is performed on a voxel basis to extract spatial maps of biodynamic biomarkers. Functional images and bivariate spatial maps are produced using a bivariate color merge to represent the spatial distribution of pairs of signed drug-response biodynamic biomarkers. RESULTS: We have mapped the spatial variability of drug responses within biopsies and have tracked sample-to-sample variability. Sample heterogeneity observed in the biodynamic maps is associated with histological heterogeneity observed using inverted selective-plane illumination microscopy. CONCLUSION: We have demonstrated the utility of TDSI as a functional imaging method to measure tumor heterogeneity and its potential for use in drug-response profiling.
Assuntos
Antineoplásicos , Neoplasias , Diagnóstico por Imagem , Humanos , Neoplasias/diagnóstico por imagem , Análise EspectralRESUMO
The current gold-standard histopathology for tissue analysis is destructive, time consuming, and limited to 2D slices. Light sheet microscopy has emerged as a powerful tool for 3D imaging of tissue biospecimens with its fast speed and low photo-damage, but usually with worse axial resolution and complicated configuration for sample imaging. Here, we utilized inverted selective plane illumination microscopy for easy sample mounting and imaging, and dual-view imaging and deconvolution to overcome the anisotropic resolution. We have rendered 3D images of fresh cytology cell blocks and millimeter- to centimeter-sized fixed tissue samples with high resolution in both lateral and axial directions. More accurate cellular quantification, higher image sharpness, and more image details have been achieved with the dual-view method compared with single-view imaging.
RESUMO
Inverted selective plane illumination microscopy (iSPIM) enables fast, large field-of-view, long term imaging with compatibility with conventional sample mounting. However, the imaging quality can be deteriorated in thick tissues due to sample scattering. Three strategies have been adopted in this paper to optimize the imaging performance of iSPIM on thick tissue imaging: electronic confocal slit detection (eCSD), structured illumination (SI) and the two combined. We compared the image contrast when using SPIM, confocal SPIM (using eCSD alone), SI SPIM (using SI alone) or confocal-SI SPIM (combining both methods) on images of gelatin phantom and highly-scattering fluorescently-stained human tissue. We demonstrate that all the three methods showed remarkable contrast enhancement on both samples compared to iSPIM alone, and SI SPIM and the combined confocal-SI mode outperformed confocal SPIM in contrast enhancement. Moreover, the use of SI at high pattern frequencies outperformed confocal SPIM in terms of optical sectioning capability. However, image signal-to-noise ratio (SNR) was decreased at high pattern frequencies when imaging scattering samples with SI SPIM. By combining eCSD with SI to reduce background signal and noise, the superior optical sectioning performance of SI could be achieved while also maintaining high image SNR.
RESUMO
The precise annotation and accurate identification of neural structures are prerequisites for studying mammalian brain function. The orientation of neurons and neural circuits is usually determined by mapping brain images to coarse axial-sampling planar reference atlases. However, individual differences at the cellular level likely lead to position errors and an inability to orient neural projections at single-cell resolution. Here, we present a high-throughput precision imaging method that can acquire a co-localized brain-wide data set of both fluorescent-labelled neurons and counterstained cell bodies at a voxel size of 0.32 × 0.32 × 2.0 µm in 3 days for a single mouse brain. We acquire mouse whole-brain imaging data sets of multiple types of neurons and projections with anatomical annotation at single-neuron resolution. The results show that the simultaneous acquisition of labelled neural structures and cytoarchitecture reference in the same brain greatly facilitates precise tracing of long-range projections and accurate locating of nuclei.
Assuntos
Encéfalo/diagnóstico por imagem , Conectoma/métodos , Imageamento Tridimensional/métodos , Vias Neurais/diagnóstico por imagem , Neurônios/ultraestrutura , Animais , Encéfalo/citologia , Cor , Estudos de Viabilidade , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia/métodos , Modelos Animais , Análise de Célula Única/métodos , Tomografia/métodosRESUMO
High-throughput optical imaging is critical to obtain large-scale neural connectivity information of brain in neuroscience. Using a digital mirror device and a scientific complementary metal-oxide semiconductor camera, we report a significant speed improvement of structured illumination microscopy (SIM), which produces a maximum SIM net frame rate of 133 Hz. We perform three-dimensional (3-D) imaging of mouse brain slices at diffraction-limited resolution and demonstrate the fast 3-D imaging capability to a large sample with an imaging rate of 6.9×10(7) pixel/s of our system, an order of magnitude faster than previously reported.
Assuntos
Iluminação/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Vídeo/instrumentação , Animais , Encéfalo/patologia , Diagnóstico por Imagem , Desenho de Equipamento , Processamento de Imagem Assistida por Computador , Luz , Iluminação/métodos , Metais/química , Camundongos , Microscopia de Fluorescência/métodos , Microscopia de Vídeo/métodos , Microesferas , Neurônios/metabolismo , Óptica e Fotônica , Óxidos/química , SemicondutoresRESUMO
Fluorescent proteins serve as important biomarkers for visualizing both subcellular organelles in living cells and structural and functional details in large-volume tissues or organs. However, current techniques for plastic embedding are limited in their ability to preserve fluorescence while remaining suitable for micro-optical sectioning tomography of large-volume samples. In this study, we quantitatively evaluated the fluorescence preservation and penetration time of several commonly used resins in a Thy1-eYFP-H transgenic whole mouse brain, including glycol methacrylate (GMA), LR White, hydroxypropyl methacrylate (HPMA) and Unicryl. We found that HMPA embedding doubled the eYFP fluorescence intensity but required long durations of incubation for whole brain penetration. GMA, Unicryl and LR White each penetrated the brain rapidly but also led to variable quenching of eYFP fluorescence. Among the fast-penetrating resins, GMA preserved fluorescence better than LR White and Unicryl. We found that we could optimize the GMA formulation by reducing the polymerization temperature, removing 4-methoxyphenol and adjusting the pH of the resin solution to be alkaline. By optimizing the GMA formulation, we increased percentage of eYFP fluorescence preservation in GMA-embedded brains nearly two-fold. These results suggest that modified GMA is suitable for embedding large-volume tissues such as whole mouse brain and provide a novel approach for visualizing brain-wide networks.