The selection of the best suitable tool for segmentation depends on specimen and object of interest.

<p>Five examples are given: in (A–D), the top row of each image shows a preprocessed slice and a segmentation overlay, and the bottom row shows the final 3-D visualization. Segmentation was done with MIB, and the 3-D rendering using different freeware (A–D) and commercial (E) software packages. (A) <i>Trypanosoma brucei</i> was chemically fixed and imaged with electron tomography (ET). Each Golgi cisternae (four shades of blue) was manually segmented using the brush tool, while ER and ER-derived vesicles (yellow) were segmented using a combination of the brush tool and shape interpolation. The resulting 3-D model was rendered directly in MIB. (B) A Huh-7 cell was high-pressure frozen and freeze-substituted, and a portion of the cell was subjected to ET [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002340#pbio.1002340.ref011" target="_blank">11</a>]. ER (yellow) was segmented using the brush tool with shape interpolation and microtubules (magenta) using the line tracker tool. The resulting model was exported in the IMOD-compatible format and rendered in IMOD [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002340#pbio.1002340.ref006" target="_blank">6</a>]. (C) A Huh-7 cell transiently expressing ssHRP-KDEL was cytochemically stained (dark precipitate) and imaged with a serial block-face scanning electron microscope (SB-EM) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002340#pbio.1002340.ref011" target="_blank">11</a>]. The ER network (yellow) was segmented semiautomatically using global black-and-white thresholding and further polished using quantification filtering [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002340#pbio.1002340.ref011" target="_blank">11</a>]. The model was exported in the nearly raw raster data (NRRD) format and rendered in 3D Slicer [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002340#pbio.1002340.ref008" target="_blank">8</a>]. (D) Mouse cochlea was perilymphatically fixed, and the sensory epithelium of the medial part of the cochlear duct was imaged with SB-EM [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002340#pbio.1002340.ref013" target="_blank">13</a>]. Different cell types of the organ of Corti (inner hairs in green, outer hair cells in yellow, external rod in vermilion, internal rod in sapphire blue, and phalangeal part of the Deiters’ cells in greyish blue) were segmented using local thresholding combined with shape interpolation and model rendered using 3D Slicer. (E) Stepwise segmentation workflow is needed to generate a 3-D model of a complex structure. A metaphase Huh-7 cell transiently expressing ssHRP-KDEL was cytochemically stained (dark precipitate in ER lumen) and imaged with SB-EM [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002340#pbio.1002340.ref010" target="_blank">10</a>]. The modelling workflow for ER consists of 13 steps (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002340#pbio.1002340.s002" target="_blank">S1 Table</a>). For the visualization, the model was exported in the AmiraMesh format and rendered in Amira. Scale bars: A, B 500 nm; C, D, E 5 μm.</p

Quantification and visualization of results are the final steps of the imaging workflow.

<p>Generated models may be quantified to extract different parameters of the segmented objects and visualized using a number of programs. As an example, the volumes (μm³) and numbers of segmented mitochondria were calculated (the plot in red). The quantifications results can either be plotted directly in MIB or exported to MATLAB or Microsoft Excel. The manual measurements of angles, distances, caliper, and radius complement automatic quantification. The visualization of the mitochondria model (in green) is demonstrated using six alternative programs (the lower row).</p

MIB recognizes a large array of imaging formats and offers many essential image processing tools.

<p>(A) Most common image formats can be imported to (left column) and exported from (right column) MIB. Most commonly used image processing tools are listed in the middle column (see MIB website for the full list). (B) Image segmentation workflow usually comprises a combination of several approaches. Datasets can be segmented iteratively using various manual and semiautomatic tools combined with quantification filtering of results in order to generate a model. Data in MIB are organized in four layers: Image (raw data), Selection (active layer for segmentation), Mask (an optional supporting layer for temporal storage of the segmentation results for evaluation and filtering), and Model (containing the final segmentation).</p

Semiautomatic image segmentation in MIB can dramatically decrease the time spent on modelling.

<p>(A) The Random Forest classifier was used to segment ER from wide-field time-lapse LM videos of Huh-7 cells. Labels were assigned (central image) to mark ER (Hsp47-GFP marker seen in green) and background (red), which were then used to train the classifier and segment ER throughout the time-lapse video (right image). (B) The semiautomatic watershed segmentation was used for segmentation of nucleus in the sieve element of <i>A</i>. <i>thaliana</i> root imaged with SB-EM [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002340#pbio.1002340.ref014" target="_blank">14</a>]. Assigning of just two labels (green for nucleus and vermilion for background) was sufficient to segment the complete nucleus in 3-D (light blue, image on the right). (C) The separation of the fused objects using the watershed segmentation. The human U251MG astrocytoma cells were loaded with oleic acid producing a large amount of lipid droplets (LDs) (left image) that tend to form clusters. LDs were segmented using marker-controlled watershed; however, because of close proximity, most of the LDs appear merged (the second image). The object separation mode of the watershed tool was used to separate individual LDs for quantitative analysis (third and fourth images). The 3-D models were rendered with 3D Slicer [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002340#pbio.1002340.ref008" target="_blank">8</a>]. Scale bars: 2 μm.</p

Mitochondrial structure and density in the flight muscles of two butterfly species with dissimilar flight behaviours.

<p>(A) Microscopic images of flight muscle of the Glanville fritillary (a, c) and the Red Admiral (b, d). Mitochondria (m and arrows), sarcoplasmic reticulum (s) and myofibrils (my) are indicated. Bars are 500 nm in a and b, 1 µm in c and d. (B) Distribution of mitochondrial profiles from thin sections of the flight muscle.</p

MIB has a user-friendly graphical user interface and is freely available from the website.

<p>(A) A screenshot of the MIB user interface. The program menu, toolbar, and panels are highlighted. A brief description of each element is provided. (B) A dedicated website includes direct links for software download and covers various topics and aspects of MIB functionality. <i>Image credit</i>: <i>Ilya Belevich</i>, <i>on behalf of MIB</i>.</p

Ultrastructural relationship of the phagophore with surrounding organelles

<div><p>Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation.</p></div

Differences in body size and ecological characteristics of the Nymphalid butterflies compared in this study.

<p><b>References.</b></p><p>¹ . <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Sutcliffe1" target="_blank">[59]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Billeter1" target="_blank">[61]</a>;</p><p>² . <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Aarnio1" target="_blank">[62]</a>;</p><p>³ . <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Hanski2" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Austin1" target="_blank">[63]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Wang1" target="_blank">[65]</a>;</p><p>⁴ . <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Hanski2" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Ovaskainen1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Hanski4" target="_blank">[23]</a>;</p><p>⁵ . <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Vandewoestijne2" target="_blank">[66]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Shreeve1" target="_blank">[67]</a>;</p><p>⁶ . <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Asher1" target="_blank">[68]</a>;</p><p>⁷ . <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Wilson1" target="_blank">[69]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Asher2" target="_blank">[70]</a>;</p><p>⁸ . <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Asher1" target="_blank">[68]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Asher2" target="_blank">[70]</a>;</p><p>⁹ . <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Stefanescu1" target="_blank">[71]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Mikkola1" target="_blank">[73]</a>;</p><p>¹⁰ . <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#pone.0078069-Brattstrom1" target="_blank">[74]</a>.</p

CytOx activity (A) and concentration (B) in flight muscles of Glanville fritillaries originating from old and new local populations.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078069#s3" target="_blank">Results</a> are shown for individuals from old (open symbols; <i>n</i> = 15 males, 13 females) and new populations (filled symbols; <i>n</i> = 19 males, 24 females), plotted against thorax wet mass. Mean CytOx activities and concentrations are shown as horizontal lines (solid lines, new populations; dashed lines, old populations). Statistics in text.</p

Reduced <i>minus</i> oxidized difference spectrum of a butterfly thorax preparation.

<p>Optical spectra of the flight muscle homogenate were collected in the ferricyanide oxidized and the dithionite reduced states. The difference spectrum has clear peaks at 550, 560 and 605<i>c</i>, <i>b</i> and <i>a</i>, respectively. This method allows highly accurate determination of tissue concentrations of the respiratory protein CytOx (also known as cytochrome <i>aa₃</i> or Complex IV).</p