Correction to: A multiscale Mueller polarimetry module for a stereo zoom microscope.

[This corrects the article DOI: 10.1007/s13534-019-00116-w.].

A multiscale Mueller polarimetry module for a stereo zoom microscope.

Mueller polarimetry is a quantitative polarized light imaging modality that is capable of label-free visualization of tissue pathology, does not require extensive sample preparation, and is suitable for wide-field tissue analysis. It holds promise for selected applications in biomedicine, but polarimetry systems are often constrained by limited end-user accessibility and/or long-imaging times. In order to address these needs, we designed a multiscale-polarimetry module that easily couples to a commercially available stereo zoom microscope. This paper describes the module design and provides initial polarimetry imaging results from a murine preclinical breast cancer model and human breast cancer samples. The resultant polarimetry module has variable resolution and field of view, is low-cost, and is simple to switch in or out of a commercial microscope. The module can reduce long imaging times by adopting the main imaging approach used in pathology: scanning at low resolution to identify regions of interest, then at high resolution to inspect the regions in detail. Preliminary results show how the system can aid in region of interest identification for pathology, but also highlight that more work is needed to understand how tissue structures of pathological interest appear in Mueller polarimetry images across varying spatial zoom scales.

Monte Carlo simulation of polarization-sensitive second-harmonic generation and propagation in biological tissue.

Polarization-sensitive second harmonic generation (p-SHG) is a nonlinear optical microscopy technique that has shown great promise in biomedicine, such as in detecting changes in the collagen ultrastructure of the tumor microenvironment. However, the complex nature of light-tissue interactions and the heterogeneity of biological samples pose challenges in creating an analytical and experimental quantification platform for tissue characterization via p-SHG. We present a Monte Carlo (MC) p-SHG simulation model based on double Stokes-Mueller polarimetry for the investigation of nonlinear light-tissue interaction. The MC model predictions are compared with experimental measurements of second-order nonlinear susceptibility component ratio and degree of polarization (DOP) in rat-tail collagen. The observed trends in the behavior of these parameters as a function of tissue thickness, as well as the overall extent of agreement between MC and experimental results, are discussed. High sensitivities of the susceptibility ratio and DOP are observed for the varying tissue thickness on the incoming fundamental light propagation pathway.

Flexible polarimetric probe for 3 × 3 Mueller matrix measurements of biological tissue.

Polarimetry is a noninvasive method that uses polarised light to assess biophysical characteristics of tissues. A series of incident polarisation states illuminates a biological sample, and analysis of sample-altered polarisation states enables polarimetric tissue assessment. The resultant information can, for example, help quantitatively differentiate healthy from pathologic tissue. However, most bio-polarimetric assessments are performed using free-space optics with bulky optical components. Extension to flexible fibre-based systems is clinically desirable, but is challenging due to polarisation-altering properties of optical fibres. Here, we propose a flexible fibre-based polarimetric solution, and describe its design, fabrication, calibration, and initial feasibility demonstration in ex vivo tissue. The design is based on a flexible fibre bundle of six multimode optical fibres, each terminated with a distal polariser that ensures pre-determined output polarisation states. The resultant probe enables linear 3 × 3 Mueller matrix characterization of distal tissue. Potential in vivo Mueller matrix polarimetric tissue examinations in various directly-inaccessible body cavities are envisioned.

Polarization image segmentation of radiofrequency ablated porcine myocardial tissue.

Optical polarimetry has previously imaged the spatial extent of a typical radiofrequency ablated (RFA) lesion in myocardial tissue, exhibiting significantly lower total depolarization at the necrotic core compared to healthy tissue, and intermediate values at the RFA rim region. Here, total depolarization in ablated myocardium was used to segment the total depolarization image into three (core, rim and healthy) zones. A local fuzzy thresholding algorithm was used for this multi-region segmentation, and then compared with a ground truth segmentation obtained from manual demarcation of RFA core and rim regions on the histopathology image. Quantitative comparison of the algorithm segmentation results was performed with evaluation metrics such as dice similarity coefficient (DSC = 0.78 ± 0.02 and 0.80 ± 0.02), sensitivity (Sn = 0.83 ± 0.10 and 0.91 ± 0.08), specificity (Sp = 0.76 ± 0.17 and 0.72 ± 0.17) and accuracy (Acc = 0.81 ± 0.09 and 0.71 ± 0.10) for RFA core and rim regions, respectively. This automatic segmentation of parametric depolarization images suggests a novel application of optical polarimetry, namely its use in objective RFA image quantification.

Optimized Mass Spectrometry Analysis Workflow with Polarimetric Guidance for ex vivo and in situ Sampling of Biological Tissues.

Spatially Targeted Mass Spectrometry (MS) analysis using survey scans with an imaging modality often requires consecutive tissue slices, because of the tissue damage during survey scan or due to incompatible sample preparation requirements between the survey modality and MS. We report two spatially targeted MS analysis workflows based on polarized light imaging guidance that use the same tissue sample for survey and targeted analysis. The first workflow is applicable for thin-slice analysis, and uses transmission-polarimetry-guided Desorption ElectroSpray Ionization Mass Spectrometry (DESI-MS), and confirmatory H&E histopathology analysis on the same slice; this is validated using quantitative digital pathology methods. The second workflow explores a polarimetry-guided MS platform for thick tissue assessment by developing reflection-mode polarimetric imaging coupled with a hand-held Picosecond InfraRed Laser (PIRL) MS ablation probe that requires minimal tissue removal to produce detectable signal. Tissue differentiation within 5-10 s of sampling with the hand-held probe is shown using multivariate statistical methods of the MS profiles. Both workflows were tasked with differentiating necrotic cancer sites from viable cancers using a breast tumour model, and their performance was evaluated. The use of the same tissue surface addresses mismatches in guidance due to intrinsic changes in tissue morphology over consecutive sections.

Rapid Detection of Necrosis in Breast Cancer with Desorption Electrospray Ionization Mass Spectrometry.

Identification of necrosis in tumors is of prognostic value in treatment planning, as necrosis is associated with aggressive forms of cancer and unfavourable outcomes. To facilitate rapid detection of necrosis with Mass Spectrometry (MS), we report the lipid MS profile of necrotic breast cancer with Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) imaging validated with statistical analysis and correlating pathology. This MS profile is characterized by (1) the presence of the ion of m/z 572.48 [Cer(d34:1) + Cl]- which is a ceramide absent from the viable cancer subregions; (2) the absence of the ion of m/z 391.25 which is present in small abundance only in viable cancer subregions; and (3) a slight increase in the relative intensity of known breast cancer biomarker ions of m/z 281.25 [FA(18:1)-H]- and 303.23 [FA(20:4)-H]-. Necrosis is accompanied by alterations in the tissue optical depolarization rate, allowing tissue polarimetry to guide DESI-MS analysis for rapid MS profiling or targeted MS imaging. This workflow, in combination with the MS profile of necrosis, may permit rapid characterization of necrotic tumors from tissue slices. Further, necrosis-specific biomarker ions are detected in seconds with single MS scans of necrotic tumor tissue smears, which further accelerates the identification workflow by avoiding tissue sectioning and slide preparation.

Rapid wide-field Mueller matrix polarimetry imaging based on four photoelastic modulators with no moving parts.

A new polarimetry method is demonstrated to image the entire Mueller matrix of a turbid sample using four photoelastic modulators (PEMs) and a charge coupled device (CCD) camera, with no moving parts. Accurate wide-field imaging is enabled with a field-programmable gate array (FPGA) optical gating technique and an evolutionary algorithm (EA) that optimizes imaging times. This technique accurately and rapidly measured the Mueller matrices of air, polarization elements, and turbid phantoms. The system should prove advantageous for Mueller matrix analysis of turbid samples (e.g., biological tissues) over large fields of view, in less than a second.

Polarimetric assessment of healthy and radiofrequency ablated porcine myocardial tissue.

Radiofrequency (RF) ablation offers a potential treatment for cardiac arrhythmia, where properly titrated energy delivered at critical sites can destroy arrhythmogenic foci. The resulting ablation lesion typically consists of a core (coagulative necrosis) surrounded by a rim of mixed viable and non-viable cells. The extent of the RF lesion is difficult to delineate with current imaging techniques. Here, we explore polarization signatures of ten ex-vivo samples from untreated (n = 5) and RF ablated porcine hearts (n = 5), in backscattered geometry through Mueller matrix polarimetry. Significant differences (p < 0.01) in depolarization, ΔT , were observed between the healthy, RF ablated and rim regions. Linear retardance, δ, was significantly lower in the core and rim regions compared to healthy regions (p < 0.05). The results demonstrate a novel application of polarimetry, namely the characterization of RF ablation extent in myocardium, including the visualization of the important lesion rim region. White light photo (top) of porcine myocardium tissue with radiofrequency ablation lesion and corresponding depolarization map (bottom). Depolarization is useful for visualizing the lesion core and rim.

Wide-field tissue polarimetry allows efficient localized mass spectrometry imaging of biological tissues.

While mass spectrometers can detect chemical signatures within milliseconds of data acquisition time, the non-targeted nature of mass spectrometry imaging (MSI) necessitates probing the entire surface of the sample to reveal molecular composition even if the information is only sought from a sample subsection. This leads to long analysis times. Here, we used polarimetry to identify, within a biological tissue, areas of polarimetric heterogeneity indicative of cancer. We were then able to target our MS analysis using polarimetry results to either the cancer region itself or to the cancer margin. A tandem of polarimetry and Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI) enables fast (10 fold compared to non-targeted imaging), and accurate pathology assessment (cancer typification in less than 2 minutes compared to 30 minutes for histopathology) of ex vivo tissue slices, without additional sample preparation. This workflow reduces the overall analysis time of MSI as a research tool.

Assessment of local structural disorders of the bladder wall in partial bladder outlet obstruction using polarized light imaging.

Partial bladder outlet obstruction causes prominent morphological changes in the bladder wall, which leads to bladder dysfunction. In this paper, we demonstrate that polarized light imaging can be used to identify the location of obstruction induced structural changes that other imaging modalities fail to detect. We induced 2-week and 6-week partial outlet obstruction in rats, harvested obstructed bladders, then measured their retardances while distended to high pressures and compared them to controls. Our results show that the retardance of the central part of the ventral side (above the ureters) closer to the urethra can be used as a potential metric of the distending bladder obstruction.

Experimental validation of optimum input polarization states for Mueller matrix determination with a dual photoelastic modulator polarimeter.

Dual photoelastic modulator polarimeters can measure light polarization, which is often described as a Stokes vector. By evaluating changes in polarization when light interacts with a sample, the sample Mueller matrix also can be derived, completely describing its interaction with polarized light. The choice of which and how many input Stokes vectors to use for sample investigation is under the experimenter's control. Previous work has predicted that sets of input Stokes vectors forming the vertices of platonic solids on the Poincaré sphere allow for the most robust Mueller matrix determination. Further, when errors specific to the dual photoelastic modulator polarimeter are considered, simulations revealed that one specific shape and orientation of Stokes vectors (cube on the Poincaré sphere with vertices away from principal sphere axes) allows for the most robust Mueller matrix determination. Here we experimentally validate the optimum input Stokes vectors for dual photoelastic modulator Mueller polarimetry, toward developing a robust polarimetric platform of increasing relevance to biophotonics.