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Intraoperative evaluation of specimens during radical prostatectomy using frozen sections can be time and labor intensive. Nonlinear microscopy (NLM) is a fluorescence microscopy technique that can rapidly generate images that closely resemble H&E histology in freshly excised tissue, without requiring freezing or microtome sectioning. Specimens are stained with nuclear and cytoplasmic/stromal fluorophores, and NLM evaluation can begin within 3 min of grossing. Fluorescence signals can be displayed using an H&E color scale, facilitating pathologist interpretation. This study evaluates the accuracy of prostate cancer detection in a blinded reading of NLM images compared with the gold standard of formalin-fixed, paraffin-embedded H&E histology. A total of 122 freshly excised prostate specimens were obtained from 40 patients undergoing radical prostatectomy. The prostates were grossed, dissected into specimens of ~10 × 10 mm with 1-4 mm thickness, stained for 2 min for nuclear and cytoplasmic/stromal contrast, and then rinsed with saline for 30 s. NLM images were acquired and multiple images were stitched together to generate large field of view, centimeter-scale digital images suitable for reading. Specimens were then processed for standard paraffin H&E. The study protocol consisted of training, pretesting, and blinded reading phases. After a washout period, pathologists read corresponding paraffin H&E slides. Three pathologists achieved a 95% or greater sensitivity with 100% specificity for detecting cancer on NLM compared with paraffin H&E. Pooled sensitivity and specificity was 97.3% (93.7-99.1%; 95% confidence interval) and 100.0% (97.0-100.0%), respectively. Interobserver agreement for NLM reading had a Fleiss κ = 0.95. The high cancer detection accuracy and rapid specimen preparation suggest that NLM may be useful for intraoperative evaluation in radical prostatectomy.
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Microscopia de Fluorescência/métodos , Neoplasias da Próstata/diagnóstico , Humanos , Período Intraoperatório , Masculino , Prostatectomia , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Coloração e Rotulagem/métodosRESUMO
Rapid histological assessment of large areas of prostate tissue is required for many intraoperative consultation scenarios such as margin evaluation. Nonlinear microscopy (NLM) enables imaging of large (whole mount) specimens without freezing or cryotoming. This study demonstrates rapid histological imaging of unsectioned prostate cancer surgical specimens using nonlinear microscopy and compares features of prostate pathology to standard paraffin embedded H&E histology. Fresh or formalin fixed specimens were stained in 2.5 min with fluorescent nuclear and stromal dyes. Nonlinear microscopy images of unsectioned tissues were generated by nonlinear (two-photon) excitation of the fluorophores, where fluorescence is only emitted from tissue at the microscope focus, avoiding the need for physical sectioning. The images were displayed in real time using a color scale similar to H&E, then tissues were processed for standard paraffin embedded H&E histology. Seventy nonlinear microscopy and corresponding paraffin H&E images of fresh and fixed prostate specimens (15 cancer, 55 benign) from 24 patients were read by genitourinary pathologists to assess if nonlinear microscopy could achieve an equivalent evaluation to paraffin embedded H&E histology. Differences between nonlinear microscopy images and paraffin H&E slides, including cytoplasmic color and stromal density, were observed, however nonlinear microscopy images could be interpreted with minimal training. Nonlinear microscopy enabled visualization of benign, atrophic and hyperplastic glands and stroma, ejaculatory ducts, vasculature and inflammatory changes. Nonlinear microscopy enabled identification of typical and variants of adenocarcinoma, as well as Gleason patterns. Perineural invasion and extraprostatic extension could also be assessed. Nonlinear microscopy images closely resemble paraffin H&E slides and enable rapid assessment of normal prostate architecture, benign conditions, and carcinoma in freshly excised and fixed specimens. Nonlinear microscopy can image large regions of tissue, equivalent to multiple frozen section tissue blocks, within minutes because cryotoming/microtoming are not required, making it a promising technique for intraoperative consultation.
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Corantes , Amarelo de Eosina-(YS) , Hematoxilina , Microscopia de Fluorescência por Excitação Multifotônica , Próstata/patologia , Neoplasias da Próstata/patologia , Coloração e Rotulagem , Humanos , Cuidados Intraoperatórios , Masculino , Margens de Excisão , Projetos Piloto , Valor Preditivo dos Testes , Próstata/cirurgia , Prostatectomia , Neoplasias da Próstata/cirurgia , Reprodutibilidade dos Testes , Fatores de Tempo , Fluxo de TrabalhoRESUMO
BACKGROUND: This study aimed to evaluate the use of ultrahigh-speed volumetric en face and cross-sectional optical coherence tomography (OCT) with micromotor catheters for the in vivo assessment of Barrett's esophagus and dysplasia. METHODS: 74 OCT datasets with correlated biopsy/endoscopic mucosal resection histology (49 nondysplastic Barrett's esophagus [NDBE], 25 neoplasia) were obtained from 14 patients with Barrett's esophagus and a history of dysplasia and 30 with NDBE. The associations between irregular mucosal patterns on en face OCT, absence of mucosal layering, surface signalâ>âsubsurface, andâ>â5 atypical glands on cross-sectional OCT vs. histology and treatment history were assessed by three blinded readers. RESULTS: Atypical glands under irregular mucosal patterns occurred in 75â% of neoplasia (96â% of treatment-naïve neoplasia) vs. 30â% of NDBE datasets (43â% of short- and 18â% of long-segment NDBE). Mucosal layering was absent in 35â% of neoplasia and 50â% of NDBE datasets, and surface signalâ>âsubsurface occurred in 29â% of neoplasia and 30â% of NDBE datasets. CONCLUSIONS: Atypical glands under irregular mucosal patterns are strongly associated with neoplasia, suggesting potential markers for dysplasia and a role in pathogenesis.
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Esôfago de Barrett , Mucosa Esofágica , Neoplasias Esofágicas , Lesões Pré-Cancerosas , Tomografia de Coerência Óptica/métodos , Esôfago de Barrett/diagnóstico , Esôfago de Barrett/patologia , Biópsia/métodos , Mucosa Esofágica/diagnóstico por imagem , Mucosa Esofágica/patologia , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/patologia , Esofagoscopia/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/diagnóstico por imagem , Lesões Pré-Cancerosas/patologia , Reprodutibilidade dos TestesRESUMO
Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical margins by standard paraffin-embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 µm below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to contamination artifacts on the tissue surface from electrocautery, surgical ink, or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin-fixed, paraffin-embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections.
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Carcinoma de Mama in situ/patologia , Neoplasias da Mama/patologia , Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/patologia , Margens de Excisão , Laranja de Acridina/química , Mama/citologia , Mama/imunologia , Mama/cirurgia , Carcinoma de Mama in situ/diagnóstico , Carcinoma de Mama in situ/imunologia , Carcinoma de Mama in situ/cirurgia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/imunologia , Neoplasias da Mama/cirurgia , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/imunologia , Carcinoma Ductal de Mama/cirurgia , Carcinoma Lobular/diagnóstico , Carcinoma Lobular/imunologia , Carcinoma Lobular/cirurgia , Corantes/química , Feminino , Corantes Fluorescentes/química , Humanos , Imageamento Tridimensional , Período Intraoperatório , Mastectomia , Mastectomia Segmentar , Microscopia de Fluorescência , Invasividade Neoplásica , Microscopia Óptica não Linear , Tratamentos com Preservação do Órgão , Rodaminas/químicaRESUMO
Endoscopic optical coherence tomography (OCT) instruments are mostly side viewing and rely on at least one proximal scan, thus limiting accuracy of volumetric imaging and en face visualization. Previous forward-viewing OCT devices had limited axial scan speeds. We report a forward-viewing fiber scanning 3D-OCT probe with 900 µm field of view and 5 µm transverse resolution, imaging at 1 MHz axial scan rate in the human gastrointestinal tract. The probe is 3.3 mm diameter and 20 mm rigid length, thus enabling passage through the endoscopic channel. The scanner has 1.8 kHz resonant frequency, and each volumetric acquisition takes 0.17 s with 2 volumes/s display. 3D-OCT and angiography imaging of the colon was performed during surveillance colonoscopy.
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BACKGROUND AND AIMS: Angiogenesis is associated with neoplastic progression of Barrett's esophagus (BE). Volumetric optical coherence tomography angiography (OCTA) visualizes subsurface microvasculature without exogenous contrast agents. We investigated the association of OCTA microvascular features with low-grade dysplasia (LGD) and high-grade dysplasia (HGD). METHODS: Fifty-two patients undergoing BE surveillance or endoscopic eradication therapies for dysplasia were imaged using volumetric OCTA and corresponding histologic diagnoses wre obtained to yield 97 data sets (nondysplastic BE [NDBE], 74; LGD, 10; HGD, 13). After evaluating OCTA image quality, 54 datasets (NDBE, 35; LGD, 8; HGD, 11) from 32 patients were used to develop a training and reading protocol. The association of abnormal vessel branching and heterogeneous vessel size with LGD/HGD and a regular honeycomb vessel pattern with NDBE were investigated. RESULTS: Blinded OCTA reading of 41 OCTA datasets (NDBE, 27; LGD, 7; HGD, 7) was performed by readers with various levels of OCT/OCTA experience including 3 OCT trainees, 1 gastroenterologist, and 2 gastroenterology fellows. Among the 6 readers, OCTA features of abnormal vessel branching and heterogeneous vessel size had an overall 94% sensitivity (95% CI, 89-99) and 69% specificity (95% CI, 62-76) for differentiating LGD/HGD versus NDBE with a mean reading time of 45 seconds per data set and moderate (kappa = .58) interobserver agreement. CONCLUSIONS: Volumetric en face OCTA imaging enables rapid examination of depth resolved microvascular features with near-microscopic resolution. OCTA can visualize microvascular features associated with LGD/HGD with high accuracy, which motivates new technologic advances and future studies investigating the diagnostic performance of OCTA.
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Adenocarcinoma/diagnóstico por imagem , Esôfago de Barrett/diagnóstico por imagem , Neoplasias Esofágicas/diagnóstico por imagem , Microvasos/diagnóstico por imagem , Idoso , Angiografia/métodos , Progressão da Doença , Esofagoscopia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Tomografia de Coerência Óptica/métodosRESUMO
We demonstrate a computationally efficient method for correcting the nonuniform rotational distortion (NURD) in catheter-based imaging systems to improve endoscopic en face optical coherence tomography (OCT) and OCT angiography. The method performs nonrigid registration using fiducial markers on the catheter to correct rotational speed variations. Algorithm performance is investigated with an ultrahigh-speed endoscopic OCT system and micromotor catheter. Scan nonuniformity is quantitatively characterized, and artifacts from rotational speed variations are significantly reduced. Furthermore, we present endoscopic en face OCT and OCT angiography images of human gastrointestinal tract in vivo to demonstrate the image quality improvement using the correction algorithm.
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Angiografia/instrumentação , Artefatos , Catéteres , Processamento de Imagem Assistida por Computador/métodos , Rotação , Tomografia de Coerência Óptica/instrumentação , Algoritmos , Angiografia/normas , Marcadores Fiduciais , Trato Gastrointestinal/diagnóstico por imagem , Humanos , Tomografia de Coerência Óptica/normasRESUMO
Resonant scanning is critical to high speed and in vivo imaging in many applications of laser scanning microscopy. However, resonant scanning suffers from well-known image artifacts due to scanner jitter, limiting adoption of high-speed imaging technologies. Here, we introduce a real-time, inexpensive and all electrical method to suppress jitter more than an order of magnitude below the diffraction limit that can be applied to most existing microscope systems with no software changes. By phase-locking imaging to the resonant scanner period, we demonstrate an 86% reduction in pixel jitter, a 15% improvement in point spread function with resonant scanning and show that this approach enables two widely used models of resonant scanners to achieve comparable accuracy to galvanometer scanners running two orders of magnitude slower. Finally, we demonstrate the versatility of this method by retrofitting a commercial two photon microscope and show that this approach enables significant quantitative and qualitative improvements in biological imaging.
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Processamento de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Microscopia Confocal/instrumentação , Artefatos , Desenho de Equipamento , Animais , Algoritmos , Imagens de FantasmasRESUMO
Advanced fluorescence imaging modalities such as confocal microscopy and two photon fluorescence microscopy can provide rapid, real-time histology images, but the mounting of fresh tissue specimens in standard orientations required for diagnosis without embedding and sectioning remains an unsolved problem. Here, we introduce a piston-based specimen holder designed for consistent, even pressure distribution. We improve upon previous designs by incorporating an air piston system with a flexible membrane and wick that extracts fluid during compression. We combine this with support fixtures to aid in the distribution of pressure, enabling imaging of specimens with small surface areas relative to their thickness, such as bisected shave skin biopsies in standard orientation without embedding or sectioning. We image both fresh biopsy specimens and diagnostic Mohs first stage specimens during clinical procedures, demonstrating improved visualization of the tissue surface in real time. Finally, we show that conventional cryosectioning can exaggerate the extent of margin positivity, which can be avoided using the piston-based holder.
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Tissue clearing methods render biological tissues transparent while maintaining tissue structure, enabling visualization of entire tissues. Recent developments in tissue clearing have predominantly emphasized preserving intrinsic fluorescent proteins or aqueous-based tissue clearing and so typically involve complex procedures and long processing times. The utilization of tissue clearing protocols in standard of care histology settings has been less well explored, and protocols for rapid clearing of human tissue specimens are limited. This study presents a novel rapid clearing protocol and demonstrates a low-cost tissue processor for high volume rapid tissue clearing that can be intergraded into standard histology workflow. We demonstrate rapid clearing in dermatological specimens, including both nonmelanoma and melanoma excisions.
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We have implemented multispectral multiple scattering low coherence interferometry (ms2/LCI) with Fourier domain data collection. The ms2/LCI system is designed to localize features with spectroscopic contrast with millimeter resolution up to 1 cm deep in scattering samples by using photons that have undergone multiple low-angle (forward) scattering events. Fourier domain detection both increases the data acquisition speed of the system and gives access to rich spectroscopic information, compared to the previous single channel, time-domain implementation. Separate delivery and detection angular apertures reduce collection of the diffuse background signal in order to isolate localized spectral features from deeper in scattering samples than would be possible with traditional spectroscopic optical coherence tomography. Light from a supercontinuum source is used to acquire absorption spectra of chromophores in the visible range within a tissue-like scattering phantom. An intensity modulation and digital lock-in detection scheme is implemented to mitigate relative intensity and spectral noise inherent in supercontinuum sources. The technical parameters of the system and comparative analysis are presented.
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Interferometria/instrumentação , Nefelometria e Turbidimetria/instrumentação , Refratometria/instrumentação , Análise Espectral/instrumentação , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Fourier , Luz , Espalhamento de RadiaçãoRESUMO
High-speed laser scanning microscopy frequently relies on resonant scanners due to their order of magnitude increase in imaging rate compared to conventional galvanometer scanners. However, the use of a nonlinear scan trajectory introduces distortion that must be corrected. This manuscript derives a new algorithm based on filtered Hermite polynomial interpolation that provides the optimal shot-noise-limited SNR for a fixed number of photons and provides higher spatial accuracy than previous methods. An open-source library is presented using the Intel advanced vector instruction set (AVX) to process up to 32 samples in parallel. Using this approach, I simultaneously demonstrate lower shot noise variance, moderately higher spatial accuracy and greater than 1 gigapixel per second interpolation rate on a desktop CPU.
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A design for a low-cost, heterodyne, frequency domain-diffuse optical spectroscopy system is presented and validated. The system uses a single wavelength of 785â nm and a single detector to illustrate the capability, but is built in a modular fashion to make it easily expandable to additional wavelengths and detectors. The design incorporates methods to allow software-based control over the system operating frequency, laser diode output amplitude, and detector gain. Validation methods include characterization of electrical designs as well as determination of the system stability and accuracy using tissue-mimicking optical phantoms. The system requires only basic equipment for its construction and can be built for under $600.
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SIGNIFICANCE: Two-photon and confocal microscopy can obtain high frame rates; however, mosaic imaging of large tissue specimens remains time-consuming and inefficient, with higher imaging rates leading to a larger fraction of time wasted translating between imaging locations. Strip scanning obtains faster mosaic imaging rates by translating a specimen at constant velocity through a line scanner at the expense of more complex stitching and geometric distortion due to the difficulty of translating at completely constant velocity. AIM: We aim to develop an approach to mosaic imaging that can obtain higher accuracy and faster imaging rates while reducing computational complexity. APPROACH: We introduce an approach based on scanner-synchronous position sampling that enables subwavelength accurate imaging of specimens moving at a nonuniform velocity, eliminating distortion. RESULTS: We demonstrate that this approach increases mosaic imaging rates while reducing computational complexity, retaining high SNR, and retaining geometric accuracy. CONCLUSIONS: Scanner synchronous strip scanning enables accurate, high-speed mosaic imaging of large specimens by reducing acquisition and processing time.
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Microscopia Confocal , CintilografiaRESUMO
Importance: Nonmelanoma skin cancers (NMSCs) are primarily diagnosed through paraffin section histologic analysis of skin biopsy specimens that requires days to weeks before a formal diagnosis is reported. Two-photon fluorescence microscopy (TPFM) has the potential for point-of-care diagnosis of NMSC and other dermatologic conditions, which could enable same-visit diagnosis and treatment. Objective: To demonstrate that TPFM imaging of NMSC can occur within minutes of obtaining biopsies and provide similar histological features to those of conventional histology and evaluate TPFM diagnostic performance with respect to conventional histology. Design, Setting, and Participants: This comparative effectiveness pilot study examined 29 freshly excised biopsies from confirmed NMSC lesions in patients presenting for treatment. Biopsies underwent imaging immediately with TPFM on site at Rochester Dermatologic Surgery (Victor, New York) between October 2019 and August 2021. The imaged biopsies were subsequently submitted for paraffin histology to produce coregistered images. Twelve of these coregistered image pairs (41.4%) were used as a training set. Fifteen (51.7%) were used in a masked evaluation by a board-certified dermatopathologist. Two (6.9%) were excluded from the study before evaluation because they could not be coregistered. Main Outcomes and Measures: Sensitivity, specificity, and accuracy of TPFM for NMSC biopsies were evaluated compared with conventional histology. Results: Fourteen of the 15 biopsy specimens (93.3%) in the evaluation set were identically diagnosed with TPFM and paraffin histology. The TPFM had 100% sensitivity (95% CI, 48%-100%), 100% specificity (95% CI, 69%-100%), and 100% accuracy (95% CI, 78%-100%) for basal cell carcinoma diagnosis. For squamous cell carcinoma diagnosis, TPFM had 89% sensitivity (95% CI, 52%-100), 100% specificity (95% CI, 54%-100%), and 93% accuracy (95% CI, 68%-100%). For overall NMSC diagnosis, TPFM had a 93% sensitivity (95% CI, 66%-100%), 100% specificity (95% CI, 3%-100%), and 93% accuracy (95% CI, 68%-100%). Examination of the 1 discordant pair revealed mismatched imaging planes as the source of error. Conclusions and Relevance: The results of this comparative effectiveness pilot study suggest that TPFM captures histological characteristics of NMSC that are present in conventional histology, which reveals its potential as a rapid, point-of-care diagnostic alternative that does not need extensive sample preparation or retraining for image evaluation. Further validation of TPFM imaging performed for a larger cohort is needed to fully evaluate its diagnostic accuracy and potential effect within the field.
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Carcinoma Basocelular , Dermatologia , Neoplasias Cutâneas , Humanos , Projetos Piloto , Parafina , Carcinoma Basocelular/diagnóstico , Carcinoma Basocelular/patologia , Neoplasias Cutâneas/patologia , Biópsia , Microscopia de FluorescênciaRESUMO
We present an imaging system based on low coherence interferometric detection of multiply scattered light for extended depth imaging into highly scattering media. By incorporating angle-resolved detection, coherence imaging with multiply scattered photons is shown to be both feasible and potentially superior to existing techniques for performing time-resolved measurements of scattered light. Imaging is demonstrated through nearly 100 mean free paths of scattering phantom in a single-ended geometry. The resolution and imaging contrast are compared to those obtained with conventional OCT systems which chiefly detect singly scattered light.
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Aumento da Imagem/instrumentação , Interferometria/instrumentação , Nefelometria e Turbidimetria/instrumentação , Tomografia de Coerência Óptica/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Luz , Espalhamento de RadiaçãoRESUMO
Microscopy with ultraviolet surface excitation (MUSE) typically has an optical sectioning thickness significantly larger than standard physical sectioning thickness, resulting in increased background fluorescence and higher feature density compared to formalin-fixed, paraffin-embedded physical sections. We demonstrate that high-index immersion with angled illumination significantly reduces optical sectioning thickness through increased angle of refraction of excitation light at the tissue interface. We present a novel objective dipping cap and waveguide-based MUSE illuminator design with high-index immersion and quantify the improvement in optical sectioning thickness, demonstrating an e-1 section thickness reduction to 6.67 µm in tissue. Simultaneously, the waveguide illuminator can be combined with high or low magnification objectives, and we demonstrate a 6 mm2 field of view, wider than a conventional 10x pathology objective. Finally, we show that resolution and contrast can be further improved using deconvolution and focal stacking, enabling imaging that is robust to irregular surface profiles on surgical specimens.
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Conventional two-photon microscopes use photomultiplier tubes, which enable high sensitivity but can detect relatively few photons per second, forcing longer pixel integration times and limiting maximum imaging rates. We introduce novel detection electronics using silicon photomultipliers that greatly extend dynamic range, enabling more than an order of magnitude increased photon detection rate as compared to state-of-the-art photomultiplier tubes. We demonstrate that this capability can dramatically improve both imaging rates and signal-to-noise ratio (SNR) in two-photon microscopy using human surgical specimens. Finally, to enable wider use of more advanced detection technology, we have formed the OpenSiPM project, which aims to provide open source detector designs for high-speed two-photon and confocal microscopy.
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Eletrônica/métodos , Microscopia Confocal/métodos , Fótons , Cintilografia/métodos , Eletrônica/instrumentação , Humanos , Microscopia Confocal/instrumentação , Cintilografia/instrumentação , Razão Sinal-Ruído , Silício/químicaAssuntos
Angiografia/métodos , Esôfago de Barrett/diagnóstico , Endoscopia Gastrointestinal/métodos , Imageamento Tridimensional/métodos , Microcirculação , Tomografia de Coerência Óptica/métodos , Angiografia/instrumentação , Endoscopia Gastrointestinal/instrumentação , Humanos , Imageamento Tridimensional/instrumentação , Tomografia de Coerência Óptica/instrumentaçãoRESUMO
We present a fiber-optic interferometric system for measuring depth-resolved scattering in two angular dimensions using Fourier-domain low-coherence interferometry. The system is a unique hybrid of the Michelson and Sagnac interferometer topologies. The collection arm of the interferometer is scanned in two dimensions to detect angular scattering from the sample, which can then be analyzed to determine the structure of the scatterers. A key feature of the system is the full control of polarization of both the illumination and the collection fields, allowing for polarization-sensitive detection, which is essential for two-dimensional angular measurements. System performance is demonstrated using a double-layer microsphere phantom. Experimental data from samples with different sizes and acquired with different polarizations show excellent agreement with Mie theory, producing structural measurements with subwavelength accuracy.