Journal: ACS Chemical Biology

Article Title: Multiplex, Quantitative, High-Resolution Imaging of Protein:Protein Complexes via Hybridization Chain Reaction

doi: 10.1021/acschembio.3c00431

Figure Lengend Snippet: Imaging protein:protein complexes in human cells, mouse proT cells, and FFPE human breast tissue sections. (A,B) Imaging β-catenin:E-cadherin target complex in A-431 cells expressing β-catenin and E-cadherin (panel A) or HeLa cells expressing N-cadherin instead of E-cadherin (panel B). (C,D) Imaging RUNX1:PU.1 target complex in Scid.adh.2C2 mouse proT cells retrovirally transduced with a PU.1-expressing vector (panel C) or an empty vector (panel D). (E,F) Imaging β-catenin:E-cadherin target complex in 5 μm FFPE human breast tissue sections from the same patient: normal (panel E) or invasive lobular carcinoma (panel F). All panels: confocal image; single optical section; 0.18 × 0.18 × 0.8 μm pixels (panels A–D) or 0.57 × 0.57 × 3.3 μm pixels (panels E,F). Signal-to-backround ratio for each row (mean ± SEM for representative regions of N = 3 replicate samples). See sections S2.2–S2.4 for additional data.

Article Snippet: HCR imaging of protein:protein complexes was performed in 5 μm FFPE normal human breast tissue sections (Acepix Biosciences, HuN-06-0027) and 5 μm FFPE invasive lobular carcinoma human breast tissue sections (Acepix Biosciences, HuC-06-0101) from the same patient using the protocol detailed in section S1.11 .

Techniques: Imaging, Expressing, Transduction, Plasmid Preparation

Journal: ACS Chemical Biology

Article Title: Multiplex, Quantitative, High-Resolution Imaging of Protein:Protein Complexes via Hybridization Chain Reaction

doi: 10.1021/acschembio.3c00431

Figure Lengend Snippet: qHCR imaging: relative quantitation of protein:protein complexes with subcellular resolution in an anatomical context. (A) Two-channel redundant detection of a protein:protein complex: each target protein is detected by an unlabeled primary antibody probe and two batches of secondary antibody probes that interact with orthogonal proximity probes to colocalize full HCR initiators that trigger orthogonal spectrally distinct HCR amplifiers (Ch1, Alexa546; Ch2, Alexa647). (B) Two-channel confocal images; single optical sections. Top: β-catenin:E-cadherin complex in A-431 cells (0.18 × 0.18 × 0.8 μm pixels). Bottom: β-catenin:E-cadherin complex in a 5 μm FFPE normal human breast tissue section (0.57 × 0.57 × 3.3 μm pixels). (C) High accuracy and precision for protein:protein relative quantitation in an anatomical context. Highly correlated normalized signal (Pearson correlation coefficient, r ) for subcellular voxels in the indicated regions in panel B. Top: 2.0 × 2.0 × 0.8 μm voxels. Bottom: 2.0 × 2.0 × 3.3 μm voxels. Accuracy: linearity with zero intercept. Precision: scatter around the line. See section S2.6 for additional data.

Article Snippet: HCR imaging of protein:protein complexes was performed in 5 μm FFPE normal human breast tissue sections (Acepix Biosciences, HuN-06-0027) and 5 μm FFPE invasive lobular carcinoma human breast tissue sections (Acepix Biosciences, HuC-06-0101) from the same patient using the protocol detailed in section S1.11 .

Techniques: Imaging, Quantitation Assay

Journal: Frontiers in Neuroanatomy

Article Title: Scatterometry Measurements With Scattered Light Imaging Enable New Insights Into the Nerve Fiber Architecture of the Brain

doi: 10.3389/fnana.2021.767223

Figure Lengend Snippet: Optimized smoothing of SLI profiles. (A) Averaged scattered light intensity of a coronal vervet monkey brain section (section 493, top) and three crossing sections of human optic tracts (sections 32/33, bottom). The white rectangles mark the evaluated regions containing mostly (I) in-plane, (II) out-of-plane, (III) two times crossing, (IV) three times crossing nerve fibers. Relevant anatomical structures are labeled: corpus callosum (cc), cingulum (cg), corona radiata (cr), fornix (f). (B) Examples of original (blue) and smoothed (orange) SLI profiles (normalized by their maximum value) with Δϕ = 1°-steps, obtained from scattering patterns measured at locations indicated by the yellow asterisks in A. The SLI scatterometry measurements were performed with one illuminated LED and 10 s illumination. For the vervet brain section, the measurement was performed 16 months after tissue embedding with 64 × 64 kernels and a gain factor of 27. For the three sections of optic tracts, the measurement was performed 20 months after tissue embedding with 50 × 50 kernels and a gain factor of 10. (C) Detection rate (average over the four selected tissue types) for different parameters of the Fourier low pass filter (different cutoff frequencies and windows widths) applied to the SLI profiles generated with Δϕ = 1°-steps (top) and 5°-steps (bottom). The magenta asterisks mark the set of parameters (shown in magenta numbers) for which the maximum detection rate is reached: 83.4% for 1°-steps and 80.8% for 5°-steps. (D) Detection rates were evaluated separately for the different regions in (A) . The black numbers show the detection rates for SLI profiles with 15°-steps (without smoothing) as well as for SLI profiles with 1°- and 5°-steps when using the optimum smoothing parameters (magenta numbers in C ). The gray numbers show the detection rates before applying the smoothing.

Article Snippet: This asymmetric illumination at the image borders leads to asymmetries in the resulting SLI profiles so that peaks might not be detected and wrong/perpendicular fiber orientations are computed. shows angular SLI measurements of a coronal vervet monkey brain section (section 512, as shown in Menzel et al., , Figure 8C) for different fields of view.

Techniques: Labeling, Generated

Journal: Frontiers in Neuroanatomy

Article Title: Scatterometry Measurements With Scattered Light Imaging Enable New Insights Into the Nerve Fiber Architecture of the Brain

doi: 10.3389/fnana.2021.767223

Figure Lengend Snippet: Angular SLI measurements of a coronal vervet monkey brain section (section 512) for different fields of view (left/right hemisphere). The measurements were performed 1 day after tissue embedding with Δϕ = 15°-steps, 0.5 s illumination, and px = 13.7 μm. (A) Maximum scattered light intensity. (B) In-plane fiber orientations are displayed for each image pixel in different colors. (C) Fiber orientation distribution maps of the regions highlighted in (B) : fiber orientations are displayed on top of each other as colored lines for every 30 × 30 image pixels. The arrows mark artifacts caused by asymmetric illumination of the respective regions.

Article Snippet: This asymmetric illumination at the image borders leads to asymmetries in the resulting SLI profiles so that peaks might not be detected and wrong/perpendicular fiber orientations are computed. shows angular SLI measurements of a coronal vervet monkey brain section (section 512, as shown in Menzel et al., , Figure 8C) for different fields of view.

Techniques:

Journal: Frontiers in Neuroanatomy

Article Title: Scatterometry Measurements With Scattered Light Imaging Enable New Insights Into the Nerve Fiber Architecture of the Brain

doi: 10.3389/fnana.2021.767223

Figure Lengend Snippet: SLI scatterometry measurements of a coronal vervet monkey brain section (section 493). (A) Averaged scattered light intensity with labeled anatomical structures: corpus callosum (cc), cingulum (cg), corona radiata (cr), fornix (f). (B) Scattering patterns for two crossing fiber bundles (left) and an out-of-plane fiber bundle (right). The top images show the simulated scattering patterns obtained from finite-difference time-domain simulations of two 90°-crossing, interwoven fiber bundles and a 50°-inclined fiber bundle (adapted from Menzel et al., , Figure 7). The bottom images show the measured scattering patterns for an image pixel in the corona radiata (1) and in the fornix (2), indicated by the red asterisks in (A) . The SLI scatterometry measurement was performed 10 months after tissue embedding with 4 × 4 illuminated LEDs, 40 × 40 kernels, gain factor 10, and illumination 10 s. (C) Scattering patterns of the rectangular region in (A) , shown for every 150 th image pixel (px = 3 μm). The SLI scatterometry measurement was performed 15 months after tissue embedding with one illuminated LED, 50 × 50 kernels, gain factor 27, and illumination 10 s. (D) Fiber orientation distribution map of the same region: the fiber orientations were computed with SLIX from every 15 th scattering pattern and displayed on top of each other as colored lines for every 10 × 10 scattering patterns.

Article Snippet: This asymmetric illumination at the image borders leads to asymmetries in the resulting SLI profiles so that peaks might not be detected and wrong/perpendicular fiber orientations are computed. shows angular SLI measurements of a coronal vervet monkey brain section (section 512, as shown in Menzel et al., , Figure 8C) for different fields of view.

Techniques: Labeling