Journal: bioRxiv

Article Title: A myofilament lattice model of Drosophila flight muscle sarcomeres based on multiscale morphometric analysis during development

doi: 10.1101/2025.02.27.640547

Figure Lengend Snippet: (A) Schematic illustrating that indirect flight muscles (IFMs) are made up of the dorsal longitudinal muscles (DLM, shown in darker gray) and the Dorso-Ventral muscles (DVM, shown in lighter gray). The myofibrils consist of regular, uniform sarcomeres with characteristic lengths and diameters. The Z-discs (Z) at the borders of sarcomeres and the central H-zone (H) are labeled. (B) Plots displaying previously reported averages for sarcomere length and diameter, along with simulated images on the right that highlight the differences of the published measurements. (C) Panels outlining the process for measuring sarcomere length and diameter. Sarcomere length is determined from images where the Z-disc is labeled, while myofibril diameter is measured from phalloidin-labeled samples. After image acquisition, an intensity profile is recorded either along or perpendicular to the myofibril. This profile is then fitted with the appropriate Gaussian or Disc Model function to calculate sarcomere length and myofibril diameter, respectively. (D) A comparison of sarcomere length and diameter between DLM and DVM fibers reveals that while sarcomere lengths in these fibers are not significantly different (p=0.821), DVM fibers consistently exhibit larger diameters in every tested animal (p=0.031). Panels on the right show connected mean values for DLM and DVM fibers from the same animal. Statistical analysis was performed using an unpaired t-test. (E) Low- and high-resolution images illustrate commonly used methods for preparing IFM samples. F-actin is stained with phalloidin (gray or magenta), and Z-discs are labeled with α-Actinin (green). The impact of these preparation methods on sarcomere morphometrics is compared. While sarcomere lengths do not differ significantly between methods (p = 0.799), the diameter of individual myofibrils is significantly smaller in isolated myofibrils compared to sectioned or microdissected samples (p = 0.003). Statistical analysis was conducted using one-way ANOVA with Tukey’s multiple comparison test. Light gray dots represent individual measurements of sarcomere length and myofibril diameter, while the larger dots indicate the mean values from independent experiments. Error bars represent the mean and s.d. of independent experiments. “n” refers to the number of independent experiments and the number of replicates. Raw data used to generate the plots presented in this figure are available in the source data file (Fig1SourceData).

Article Snippet: The following primary antibodies were applied in blocking solution and incubated overnight at 4°C: α-Actinin (DSHB 2G3-3D7, mouse, 1:100) Cpa (gift from Florence Janody, rabbit, 1:100; ) GFP (Abcam ab13970, chicken, 1:1000) Kettin Ig16 (DSHB BB17/29.5, rat, 1:200) Myosin 3E8 (DSHB 3E8-3D3, mouse, 1:1000) Myosin MAC147 (DSHB BB7/21.14, rat, 1:1000) Obscurin Ig14-16 (gift from Belinda Bullard, rabbit, 1:400; ) Sls700 B2 (gift from Belinda Bullard, rabbit, 1:200; ) Tmod (gift from Velia Fowler, rat, 1:200) Zaps52 (DSHB 1D3-3E4, mouse, 1:400) After additional washes, secondary antibodies were applied for 2 hours at room temperature.

Techniques: Muscles, Labeling, Comparison, Staining, Isolation

Journal: bioRxiv

Article Title: A myofilament lattice model of Drosophila flight muscle sarcomeres based on multiscale morphometric analysis during development

doi: 10.1101/2025.02.27.640547

Figure Lengend Snippet: (A) This panel illustrates how simulated IFM images with predefined diameters and sarcomere lengths were generated to compare the accuracy of different measurement methods (refer to the Methods section for details). (B) Panels highlight how embedding media affect sarcomere length and myofibril diameter. While sarcomere length remains unchanged (p=0.99), myofibril diameter is highly sensitive to the medium used, showing significant differences (p<0.0001). Hardening media, such as ProlongGold, reduce myofibril diameter, whereas liquid media, like glycerol-based solutions, increase it. Micrographs (on the right) illustrate this effect: in the transmitted light image (top), a liquid media bubble is visible within the hardening medium, and in the fluorescence image (bottom), variations in myofibril diameter are apparent. F-actin is stained with phalloidin (magenta), while Z-discs are labeled with α-Actinin (green). Statistical analysis was performed using an unpaired t-test. (C) Panels display the measured differences in sarcomere length and myofibril width between sexes after eclosion. (D) Panels show the effects of formaldehyde and glutaraldehyde on sarcomere length and myofibril diameter. While fixative choice does not significantly impact sarcomere length (p=0.105), myofibrils fixed with glutaraldehyde are significantly thicker than those fixed with formaldehyde (p=0.0005). An unpaired t-test was used for analysis. (E) These panels demonstrate the reliability of morphometric measurements performed using the Individual Myofibrils Analyzer (IMA) compared to manual measurements by four individuals on simulated images with predefined sarcomere length and diameter (dark gray dots). The simulation set included 80 images with varying sarcomere lengths and widths (10 images per category), randomly rotated. (F) The micrographs display representative images of isolated myofibrils from flies with different genetic backgrounds, labeled with phalloidin (magenta) and α-Actinin (green). The accompanying plots indicate no significant differences in the IFM morphometrics of these myofibrils. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test. Light gray dots represent individual measurements of sarcomere length and myofibril diameter, while the larger dots indicate the mean values from independent experiments. Error bars represent the mean and s.d. of independent experiments. “n” refers to the number of independent experiments and the number of replicates. Raw data used to generate the plots presented in this figure are available in the source data file (SourceData).

Article Snippet: The following primary antibodies were applied in blocking solution and incubated overnight at 4°C: α-Actinin (DSHB 2G3-3D7, mouse, 1:100) Cpa (gift from Florence Janody, rabbit, 1:100; ) GFP (Abcam ab13970, chicken, 1:1000) Kettin Ig16 (DSHB BB17/29.5, rat, 1:200) Myosin 3E8 (DSHB 3E8-3D3, mouse, 1:1000) Myosin MAC147 (DSHB BB7/21.14, rat, 1:1000) Obscurin Ig14-16 (gift from Belinda Bullard, rabbit, 1:400; ) Sls700 B2 (gift from Belinda Bullard, rabbit, 1:200; ) Tmod (gift from Velia Fowler, rat, 1:200) Zaps52 (DSHB 1D3-3E4, mouse, 1:400) After additional washes, secondary antibodies were applied for 2 hours at room temperature.

Techniques: Generated, Fluorescence, Staining, Labeling, Isolation, Comparison

Journal: bioRxiv

Article Title: A myofilament lattice model of Drosophila flight muscle sarcomeres based on multiscale morphometric analysis during development

doi: 10.1101/2025.02.27.640547

Figure Lengend Snippet: (A) The plots compare previously published datasets on sarcomere length and myofibril width with the data obtained in this study. (B) Plots demonstrating that sarcomere length and myofibril diameter from young pupae (36 hours APF) are consistent whether measured in isolated individual myofibrils or within microdissected muscles. Light gray dots represent individual measurements, while larger dots show mean values from independent experiments. Error bars indicate the mean and s.d. across experiments, with “n” denoting the number of independent experiments and the number of replicates. The micrographs on the right compare the overall organization of microdissected muscles with isolated individual myofibrils from young pupae (36 hours APF). These images also display the localization of sarcomeric markers, including α-Actinin, Sls700 and Obscurin. Insets provide higher-resolution views to facilitate thorough comparisons. Raw data used to generate the plots presented in this figure are available in the source data file (SFig3SourceData).

Article Snippet: The following primary antibodies were applied in blocking solution and incubated overnight at 4°C: α-Actinin (DSHB 2G3-3D7, mouse, 1:100) Cpa (gift from Florence Janody, rabbit, 1:100; ) GFP (Abcam ab13970, chicken, 1:1000) Kettin Ig16 (DSHB BB17/29.5, rat, 1:200) Myosin 3E8 (DSHB 3E8-3D3, mouse, 1:1000) Myosin MAC147 (DSHB BB7/21.14, rat, 1:1000) Obscurin Ig14-16 (gift from Belinda Bullard, rabbit, 1:400; ) Sls700 B2 (gift from Belinda Bullard, rabbit, 1:200; ) Tmod (gift from Velia Fowler, rat, 1:200) Zaps52 (DSHB 1D3-3E4, mouse, 1:400) After additional washes, secondary antibodies were applied for 2 hours at room temperature.

Techniques: Isolation, Muscles

Journal: bioRxiv

Article Title: A myofilament lattice model of Drosophila flight muscle sarcomeres based on multiscale morphometric analysis during development

doi: 10.1101/2025.02.27.640547

Figure Lengend Snippet: (A) The top panel provides a schematic outlining of the timeline and key steps of IFM myogenesis. In the bottom left, a plot illustrates the correlation between sarcomere length and myofibril width across myofibrillogenesis. Gray dots indicate mean values for individual myofibrils, while larger blue dots represent averaged means at specific time points. The plots on the right display the average sarcomere length and myofibril width measured across 12 time points, from 36 hours APF to 96 hours AE, following the timeline depicted in the schematic. “n” indicates the number of independent experiments and the number of replicates. A yellow line highlights the theoretical resolution limit of optical microscopy, with an inset showing that conventional light microscopy (LSM) overestimates the diameter of early myofibrils. In contrast, Airyscan imaging reveals that these diameters are below the diffraction limit. (B) Images of isolated individual myofibrils showcase the growth of IFM sarcomeres during myofibrillogenesis. F-actin (magenta) is stained with phalloidin, and Z-discs (green) are labeled with α-Actinin. Scale bar: 2 µm. Raw data used to generate the plots presented in this figure are available in the source data file (Fig2SourceData).

Article Snippet: The following primary antibodies were applied in blocking solution and incubated overnight at 4°C: α-Actinin (DSHB 2G3-3D7, mouse, 1:100) Cpa (gift from Florence Janody, rabbit, 1:100; ) GFP (Abcam ab13970, chicken, 1:1000) Kettin Ig16 (DSHB BB17/29.5, rat, 1:200) Myosin 3E8 (DSHB 3E8-3D3, mouse, 1:1000) Myosin MAC147 (DSHB BB7/21.14, rat, 1:1000) Obscurin Ig14-16 (gift from Belinda Bullard, rabbit, 1:400; ) Sls700 B2 (gift from Belinda Bullard, rabbit, 1:200; ) Tmod (gift from Velia Fowler, rat, 1:200) Zaps52 (DSHB 1D3-3E4, mouse, 1:400) After additional washes, secondary antibodies were applied for 2 hours at room temperature.

Techniques: Microscopy, Light Microscopy, Imaging, Isolation, Staining, Labeling

Journal: bioRxiv

Article Title: A myofilament lattice model of Drosophila flight muscle sarcomeres based on multiscale morphometric analysis during development

doi: 10.1101/2025.02.27.640547

Figure Lengend Snippet: (A) The schematic on the left illustrates the restricted temporal activation of the duf-Gal4 and fln-Gal4 drivers during myofibrillogenesis. On the right, dSTORM reconstructions display the incorporation of GFP-labeled Act88F monomers into IFM myofibrils using these drivers. (B) Pseudo-multicolor dSTORM reconstructions reveal the localization of Tmod (in white) and Cpa (in green) in IFM myofibrils isolated from pupae (72 hours APF) and adult flies (24 hours AE). F-actin is labeled with phalloidin (magenta). (C) Pseudo-multicolor dSTORM reconstructions highlight the localization of α-Actinin (yellow) and Zasp52 (blue) within the Z-disc of IFM myofibrils from pupae (72 hours APF) and adult flies (24 hours AE). (D) The top panels show multicolor dSTORM reconstructions of myosin epitopes (MAC 147 in green and 3E8 in magenta) in IFM myofibrils from pupae (72 hours APF) and adult flies (24 hours AE). At the bottom, dSTORM images show single isolated thick filaments from adult flies (24 hours AE) labeled with the 3E8 myosin antibody. (E) A series of pseudo-multicolor dSTORM reconstructions reveal the localization of elastic filament epitopes, Kettin Ig16 (cyan) and Sls700 B2 (red), at four different time points during myofibrillogenesis. F-actin is labeled with phalloidin (gray). (F) Series of dSTORM reconstructions illustrate the localization of the central Obscurin Ig14-16 (green) epitope, across four time points of myofibrillogenesis. F-actin is labeled with phalloidin (gray).

Article Snippet: The following primary antibodies were applied in blocking solution and incubated overnight at 4°C: α-Actinin (DSHB 2G3-3D7, mouse, 1:100) Cpa (gift from Florence Janody, rabbit, 1:100; ) GFP (Abcam ab13970, chicken, 1:1000) Kettin Ig16 (DSHB BB17/29.5, rat, 1:200) Myosin 3E8 (DSHB 3E8-3D3, mouse, 1:1000) Myosin MAC147 (DSHB BB7/21.14, rat, 1:1000) Obscurin Ig14-16 (gift from Belinda Bullard, rabbit, 1:400; ) Sls700 B2 (gift from Belinda Bullard, rabbit, 1:200; ) Tmod (gift from Velia Fowler, rat, 1:200) Zaps52 (DSHB 1D3-3E4, mouse, 1:400) After additional washes, secondary antibodies were applied for 2 hours at room temperature.

Techniques: Activation Assay, Labeling, Isolation

Journal: JALA: Journal of the Association for Laboratory Automation

Article Title: Transfer of Low Nanoliter Volumes between Microplates Using Focused Acoustics—Automation Considerations

doi: 10.1016/s1535-5535-03-00011-x

Figure Lengend Snippet: Figure 3. Surface tension will hold liquids and retain droplets in inverted wells. This ‘‘regime’’ map shows the propensity of water and DMSO to coalesce when impacting a dry surface or a wet well. Drops with volumes under 1 lL and velocities under 1 m/s will coalesce with a surface or liquid pool. Faster-moving drops (2 m/s) coalesce at drop volumes below 50 nL.

Article Snippet: Volumes of common assay fluids held by surface tension in a well of an inverted microplate during automated plate inversion and plate-to-plate liquid transfer operations Fluid tested for stability in inverted microplate wells 100 lL in 384-well microplate 10 lL in 1536-well microplate 100 mM PBS (potassium phosphate buffer) PS, COC PS 100 mM PBS with 0.02% CHAPS PS, COC PS 100 mM Tris buffer (Hydroxymethylaminoethane) PS, COC PS 100 mM MOPS (3-[N-Morpholino] propanesulfonic acid) PS, COC PS DMEM (cell culture media) PS, COC PS DMSO (anhydrous) PP — PP = Greiner 384 polypropylene plate, # 781 201.

Techniques:

Journal: JALA: Journal of the Association for Laboratory Automation

Article Title: Transfer of Low Nanoliter Volumes between Microplates Using Focused Acoustics—Automation Considerations

doi: 10.1016/s1535-5535-03-00011-x

Figure Lengend Snippet: Figure 2. Acoustic ejection of a 5-nL droplet (212 lm diameter) of DMSO traveling upwards at approximately 1 m/s to a glass slide with either 1, 20, 40, or 100 drops already deposited. Each droplet is captured at six points in time using a multi-flash strobe at intervals of 400 ls. The critical impact Weber numbers for the droplets are between 4 and 5 and, as expected, no evidence of splatter is found for either a dry deposition on left or for receiving into the 0.5 lL drop on right.

Article Snippet: Volumes of common assay fluids held by surface tension in a well of an inverted microplate during automated plate inversion and plate-to-plate liquid transfer operations Fluid tested for stability in inverted microplate wells 100 lL in 384-well microplate 10 lL in 1536-well microplate 100 mM PBS (potassium phosphate buffer) PS, COC PS 100 mM PBS with 0.02% CHAPS PS, COC PS 100 mM Tris buffer (Hydroxymethylaminoethane) PS, COC PS 100 mM MOPS (3-[N-Morpholino] propanesulfonic acid) PS, COC PS DMEM (cell culture media) PS, COC PS DMSO (anhydrous) PP — PP = Greiner 384 polypropylene plate, # 781 201.

Techniques:

Journal: JALA: Journal of the Association for Laboratory Automation

Article Title: Transfer of Low Nanoliter Volumes between Microplates Using Focused Acoustics—Automation Considerations

doi: 10.1016/s1535-5535-03-00011-x

Figure Lengend Snippet: Figure 5. Removable tubes with flat bottoms are suitable for acoustic ejection. Here, a 50-pL droplet of DMSO emerges from an overfilled polypropylene tube (MSP 101-1; Matrical, Spokane, WA) with a 1.5-mm inner diameter designed for a 384-tube rack system. These racks can be moved by conventional automation hardware.

Article Snippet: Volumes of common assay fluids held by surface tension in a well of an inverted microplate during automated plate inversion and plate-to-plate liquid transfer operations Fluid tested for stability in inverted microplate wells 100 lL in 384-well microplate 10 lL in 1536-well microplate 100 mM PBS (potassium phosphate buffer) PS, COC PS 100 mM PBS with 0.02% CHAPS PS, COC PS 100 mM Tris buffer (Hydroxymethylaminoethane) PS, COC PS 100 mM MOPS (3-[N-Morpholino] propanesulfonic acid) PS, COC PS DMEM (cell culture media) PS, COC PS DMSO (anhydrous) PP — PP = Greiner 384 polypropylene plate, # 781 201.

Techniques: