matlab image-processing pipelines  (MathWorks Inc)

Journal: The Analyst

Article Title: Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies

doi: 10.1039/d0an00030b

Figure Lengend Snippet: Overview of workflow for the quad microraft arrays with biopsy of colony fragments. (A) Single cells (red and green objects) were seeded onto a quad microraft array. Shown is a side view through the array. Each quad site contains four magnetic microrafts (only two are shown in brown on this side view). The light tan marks the PDMS substrate. (B) The cells were cultured into clonal micro-colonies (2 red colonies and 1 green colony are depicted). (C) A PDMS-on-glass microwell collection plate (light gray) was mated directly above the quad array, and colonies were biopsied by microneedle-based release of a single microraft within a quad site. The microrafts with their attached biopsied cells or colony fragment were magnetically collected into the overlying collection plate and cultured for 2 days. (D) After colony biopsy expansion, the collection plate is detached from the quad array. Immunofluorescence staining of select intracellular proteins (marked by yellow sunburst) is performed on cells in the microwell collection plate after fixation. (E) The mother colonies which when biopsied demonstrated the selected trait are released from the array. (F) The collected mother colonies are assayed or expanded for later use.

Article Snippet: Quad Microraft Image Analysis Customized MATLAB image-processing pipelines ( Fig. S1 ) were used to detect cellular material on quad microraft arrays from the bright field microscopy images.

Techniques: Cell Culture, Immunofluorescence, Staining

Journal: The Analyst

Article Title: Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies

doi: 10.1039/d0an00030b

Figure Lengend Snippet: Colony growth across quad microraft arrays. (A) Image of a quad microraft array mounted in a culture cassette (left-side) and a close-up view of a quad microraft array (right-side), where the scale bars are 20 mm and 400 μm, respectively. (B) Example of clonal colony expansion across a quad site where a single cell resides on the site immediately after cell seeding. Scale bar is 100 μm. (C) Semi-logarithmic plot showing the number of quad colony sites located on an array (306 colonies located over 10,000 total quad sites) plotted against the number of microrafts with cells in each quad colony site. The threshold area for a microraft to be considered as occupied by cells was ≥25% surface area.

Article Snippet: Quad Microraft Image Analysis Customized MATLAB image-processing pipelines ( Fig. S1 ) were used to detect cellular material on quad microraft arrays from the bright field microscopy images.

Techniques:

Journal: The Analyst

Article Title: Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies

doi: 10.1039/d0an00030b

Figure Lengend Snippet: Platform for retrieval and assay of biopsied colony fragments. (A) Microwell collection plate components: i) base plate, ii) microwell collection array, iii) snap-on fastener, and iv) assembled microwell collection plate. Scale bar is 3 cm. (B) Exploded view schematic of the entire assembly used during colony biopsy and collection (left side). From top to bottom: disk magnet, EL light sheet, inverted collection plate housing the microwell collection array (blue), and quad microraft array (orange). Top right panels: ESEM micrographs of microwell collection array and a close-up of a sectioned microwell. Lower right panels: ESEM micrographs of quad microraft array and a close-up of sectioned quad sites. Scale bars are 200 μm. (C) Steps involved in assembly of the mated device: i) quad microraft array with cultured microcolonies, ii) microwell collection plate inverted and situated on top of the quad microraft array, iii) the magnet and EL light sheet mounted in their holder is secured on top of the collection plate and quad microraft array, and iv) microwell collection array detached from the mated components (panel iii.) and transferred into another chamber for assay. Scale bar is 3 cm.

Article Snippet: Quad Microraft Image Analysis Customized MATLAB image-processing pipelines ( Fig. S1 ) were used to detect cellular material on quad microraft arrays from the bright field microscopy images.

Techniques: Cell Culture

Journal: The Analyst

Article Title: Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies

doi: 10.1039/d0an00030b

Figure Lengend Snippet: Automated micro-colony biopsy and cell collection into a microwell collection plate for immunofluorescence assays. (A) Microraft X-Y travel trajectory from biopsy location on quad microraft array to collected location in the microwell collection plate. Total length of arrow from tip-to-tail is representative of the total distance traveled by one microraft. Scale bar is 1 cm. (B) Microraft X-Y travel trajectory from initial collected position in the microwell collection plate to the post assay location in the microwell array top-piece. Scale bar is 1 cm. (C) Representative quad colony site pre-biopsy and the vacant microraft location post-biopsy on the quad microraft array (left two panels). Image of the biopsied microraft immediately after collection into and after the assay into the microwell array (right two panels). In the post-biopsy and post-collection images, EL light sheet illumination (due to light obstruction by the magnet) was needed for visualization, under which the cell and microwell walls were not visible. Scale bar is 400 μm.

Article Snippet: Quad Microraft Image Analysis Customized MATLAB image-processing pipelines ( Fig. S1 ) were used to detect cellular material on quad microraft arrays from the bright field microscopy images.

Techniques: Immunofluorescence

Journal: The Analyst

Article Title: Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies

doi: 10.1039/d0an00030b

Figure Lengend Snippet: Immunofluorescence assay of biopsied clones and identification of clones to target mother colony resampling. (A) Image locations across the microwell collection array, where the orange circles depict the microraft and colony fragment locations identified at 4× magnification, the open black squares marked the FOVs used to image the microwell array at 10× magnification, and the blue line is the order of 10× FOV image collection identified by the nearest-neighbor imaging algorithm (scale bar is 1 cm). The right-side panels show a 10× FOV of microwells as marked on the microwell collection array, where the top FOV has 2 microrafts each in a different microwell and the bottom FOV shows a single microraft in a microwell (scale bar is 400 μm). (B) The total average immunofluorescence of pSTAT3, STAT3, and pSTAT3/STAT3 for the 25 clonal fragments collected. Data points for fragments identified as having the 3 lowest (L1, L2, L3) and 3 highest (H1, H2, H3) pSTAT3/STAT3 are plotted as solid blue squares and pink triangles respectively. The median value among the measurements is plotted as a horizontal line. (C) Representative fluorescence images of immunostained fragments in the microwell collection array for Hoechst 33342 (DNA), pSTAT3, STAT3, and a composite image of pSTAT3 and STAT3. Of the 6 target fragments identified, representative images shown are for clonal fragment L3. Scale bar is 100 μm. (D) Representative bright field images of the resampled mother colony for clone L3 immediately after collection (day 0) and the expanded colony (day 5) before and after rinsing the well with media which removes the microraft. Scale bar is 300 μm.

Article Snippet: Quad Microraft Image Analysis Customized MATLAB image-processing pipelines ( Fig. S1 ) were used to detect cellular material on quad microraft arrays from the bright field microscopy images.

Techniques: Immunofluorescence, Clone Assay, Imaging, Fluorescence

Journal: The Analyst

Article Title: Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies

doi: 10.1039/d0an00030b

Figure Lengend Snippet: Nuclear pSTAT3/STAT3 and β-hex release among RBL-2H3 clones. (A) pSTAT3/STAT3 measured per cell for the initial fragments’ biopsied after 7 days in culture (5 days on quad microraft array and 2 days’ post-biopsy in microwell collection array): i.) for the 6 individual target clones where each data point represents a single cell’s pSTAT3/STAT3 value and the mean value is plotted as a horizontal line (7–53 cells per fragment). ii.) the total distribution of pSTAT3/STAT3 per cell in the colony fragments pooled as low (L1, L2, and L3) and high (H1, H2, and H3). (B) Distribution of pSTAT3/STAT3 measured per cell for the resampled and expanded mother colonies after 18 days in culture (18 days’ post-cell seeding on microraft arrays): i.) for the 6 individual resampled clones (2,369 – 7,072 cells per clone) and ii.) for the resampled clones pooled based on their initial sort criteria of low (L1, L2, and L3) and high (H1, H2, and H3) pSTAT3/STAT3. (C) Percent β-hex release measured after activation via IgE cross-linking of FcεRI: i) in unsorted bulk RBL-2H3 cells without treatment and with activation of FcεRI cross-linking (N=12 wells per condition, 100,000 cells per well), and ii) in the expanded mother clones with activation of FcεRI cross-linking after 24–28 days in culture (N=12 wells per colony type, 100,000 cells per well). For each violin plot, the solid horizontal line represents the median value, while dotted horizontal lines below and above the medium value represent the 25th and 75th percentile values, respectively. p values are represented as p<0.05 = *, p<0.01 = **, p<0.001 = ***.

Article Snippet: Quad Microraft Image Analysis Customized MATLAB image-processing pipelines ( Fig. S1 ) were used to detect cellular material on quad microraft arrays from the bright field microscopy images.

Techniques: Clone Assay, Activation Assay