The insensitivity of multiphoton microscopy to optical scattering enables high-resolution, high-contrast imaging deep into tissue, including in live animals. Scattering does, however, severely limit the use of spectral dispersion techniques to improve spectral resolution. In practice, this limited spectral resolution together with the need for multiple excitation wavelengths to excite different fluorophores limits multiphoton microscopy to imaging a few, spectrally distinct fluorescent labels at a time, restricting the complexity of biological processes that can be studied. Here, we demonstrate a hyperspectral multiphoton microscope that utilizes three different wavelength excitation sources together with multiplexed fluorescence emission detection using angle-tuned bandpass filters. This microscope maintains scattering insensitivity, while providing high enough spectral resolution on the emitted fluorescence and capitalizing on the wavelength-dependent nonlinear excitation of fluorescent dyes to enable clean separation of multiple, spectrally overlapping labels, in vivo. We demonstrated the utility of this instrument for spectral separation of closely overlapped fluorophores in samples containing 10 different colors of fluorescent beads, live cells expressing up to seven different fluorescent protein fusion constructs, and in multiple in vivo preparations in mouse cortex and inflamed skin, with up to eight different cell types or tissue structures distinguished.

BackgroundTo determine the effect of hydration as well as prone versus supine positioning on the pelvic veins during cardiovascular magnetic resonance (CMR) venography.MethodsUnder institutional review board approval, 8 healthy subjects were imaged with balanced steady state free precession, non-contrast CMR venography to measure common and external iliac vein volumes and common femoral vein cross-sectional area in the supine, prone and decubitus positions after dehydration and again following re-hydration. CMR venography from 23 patients imaged both supine and prone were retrospectively reviewed and measurements of common femoral and iliac veins areas were compared using Wilcoxon test.ResultsCommon femoral vein area on CMR venography increased with prone positioning (83 ± 35 mm2) compared to supine positioning (59 ± 21 mm2) (p = 0.02) and further increased with hydration to 123 ± 44 mm2 (p < 0.01). With right and left side down decubitus positioning, the common femoral vein area on dehydration increased from 29 ± 17 mm2 in the ante-dependent position to 134 ± 36 mm2 in the dependent position (p < 0. 001). Similarly, common and external iliac veins increased in volume with prone, 5.4 ± 1.9 cm3 and 5.8 ± 1.9 cm3 compared to supine positioning 4.6 ± 1.8 cm3 and 4.5 ± 1.9 cm3 (p = 0.01) and further increase with hydration to 6.7 ± 2.1 cm3 and 6.3 ± 1.9 cm3 (p = 0.01). CMR venography on patients also demonstrated an increase in mean common femoral vein luminal area from 103 ± 44 mm2 in supine position to 151 ± 52 mm2 with prone positioning (p < 0.001) as well as increases in common and external iliac vein volumes from 6.5 ± 2.6 cm3 and 8.0 ± 3.4 cm3 in the supine position to 7.5 ± 2.5 cm3 and 9.3 ± 3.6 cm3 with prone positioning (p < 0.01).ConclusionsCommon femoral and common/external iliac vein size on CMR venography may be affected by position and hydration status. Routine clinical CMR venography of the pelvis could include prone positioning and avoiding dehydration to maximize pelvic vein distension.Electronic supplementary materialThe online version of this article (10.1186/s12968-018-0503-6) contains supplementary material, which is available to authorized users.

Introduction: The development of new and improved cancer diagnostic and therapeutic methods relies on a deep understanding of the protein expression profile in the tumor microenvironment. Conventional immunohistochemistry (IHC) methods only provide insights into a limited number of markers of interest, prompting the need for the development of multiplexed methods to assess and characterize multiple cell phenotypes and their spatial context in a single sample. We have developed an approach to enable high-multiplexing detection of protein markers in FFPE tumor samples using an integrated workflow with the Leica Bond RX autostainer and Leica Versa whole slide scanner. Methods: A cocktail of primary antibodies of interest, modified with unique, addressable DNA barcodes, is applied to tissue samples in a single staining step. The barcodes are then amplified simultaneously to improve sensitivity. Finally, a cocktail of fluorescently labeled oligonucleotides, complementary to the barcodes on the antibodies, is used to tag the different targets of interest for multiplexed imaging. Formalin-fixed, paraffin-embedded samples from both human tonsil and primary tumor biopsies were stained manually and automatically on the BondRX, using a panel of antibodies specific to cancer immuno-profiling markers. Images were acquired using the Leica Versa whole slide scanner, and analyzed with Leica Aperio Image Analysis and Indica Labs Halo software. Results: Compared to typical multiplex IHC methods, the InSituPlex single staining step offers multiple advantages. First, the workflow preserves the integrity and antigenicity of the samples by avoiding any antibody stripping steps and streamlining the assay development process by eliminating the need to optimize staining order. In addition, the total assay time stays constant regardless of the multiplexing level. Adding targets to the panel does not increase staining time, facilitating the optimization of higher multiplex assays, as well as the development of streamlined automated assays. A fully automated staining protocol was developed on the BondRX by modifying assay parameters such as reagent concentrations and volumes, optimizing the timing of each step, and evaluating the impact on the resulting images in terms of specificity, sensitivity and overall signal to background ratio. After optimization, the automated staining protocol delivered similar or better results compared to the manual assay protocol, while decreasing the total assay time by approximately 40%. The optimized protocol was subsequently used to carry out a multiplexed analysis of immune cell phenotypes in tumor samples. Conclusions: the InSituPlex technology enables multiplexed immune-profiling in tumor FFPE sections, and provides a streamlined, high-throughput workflow with existing automated staining and imaging instrumentation. Citation Format: Mael Manesse, Amanda Bares, Heike Boisvert, Stephanie Walter. From staining to analysis: End-to-end application of InSituPlexTM technology for multiplexed immuno-profiling in FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5658.

Background: Immuno-oncology research and predictive biomarkers discovery requires the ability to rapidly identify, quantify, and spatially map many cell sub-types of the tumor environment in FFPE tissue sections. Multiplexed immunofluorescence (IF) enables identification of multiple targets within separate spectral, or color, channels, which in turn enables more in-depth analysis of rare tissue material relative to traditional methods. Although many methods for multiplexed IF exist, they are often costly and require time-consuming assay development and long imaging times. The Ultivue® InSituPlex® technology enables rapid, optimized staining of multiple targets utilizing widely-used immunohistochemistry (IHC) staining and fluorescence imaging instrumentation. Here, we demonstrate the application of InSituPlex technology to image 6 targets and a nuclear counterstain in a 7-color single whole-slide scan, without linear unmixing-based image reconstruction. This technique expands the subtyping depth that can be achieved in a single imaging round and on a single section, and also increases sample-to-answer throughput with a streamlined, single-day workflow. Methods: InSituPlex technology was used to perform 6-plex immune profiling of different FFPE tumor sections. Slides were stained with a cocktail of primary antibodies using a Leica® Bond® RX autostainer and imaged on commercially available fluorescent slide scanners. Subsequent image analysis of the whole slide scans was carried out using Indica Labs HALO® software. Results: In this poster, we present an assay for pathology research that enables fast immuno-profiling in tumor tissues, using a streamlined staining workflow and 7-color imaging. Newly selected fluorophores were used with corresponding optical filter pairs, which increased the number of spectral channels available on commercially available slide scanners while limiting channel cross-talk. Analysis led to the identification of key immune cell phenotypes through marker colocalization without significant ambiguity between targets tagged with spectrally adjacent fluorescent labels. Conclusion: The newly-expanded fluorophore configuration of the InSituPlex technology enables high throughput identification of 6 targets and a nuclear counterstain on whole tumor sections, empowering translational and immuno-oncology research. Citation Format: Amanda J. Bares, Eloise M. Wheeler, Maël Manesse. Toward high-throughput, high-multiplex FFPE tumor tissue assays for translational research: 7-color, whole slide imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1187.

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