Changes in EGFP fluorescence in <i>E</i>.<i>coli</i> during dehydration and clearing.

<p>(<b>A</b>) Fluorescence of fixed, EGFP-expressing <i>E</i>. <i>coli</i> cells during dehydration and clearing. Dehydration steps: 2.5 h each (80%: 16 hours); clearing steps as indicated, all steps at RT, pH unadjusted. (<b>B</b>) Fluorescence of fixed, EGFP-expressing <i>E</i>. <i>coli</i> cells after dehydration in pH-adjusted alcohol solutions as indicated, followed by 5 d clearing at the respective pH (all steps at RT, data from different experiment). (<b>C</b>) LSFM recording (sagittal optical slice) of olfactory bulb EGFP fluorescence of a brain from a two weeks old transgenic <i>Tg</i>^{<i>CamKII-EGFP</i>} mouse. After fixation, the two hemispheres of the brain were separated. One hemisphere was dehydrated and cleared according to Dodt et al. (“E-BABB”, left) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124650#pone.0124650.ref004" target="_blank">4</a>], the other hemisphere using 1-propanol for dehydration and BABB for clearing, all steps at pH 9.5 (“1P-BABB”, right). For both samples, dehydration steps were 8h and 16h alternating; clearing step: 7 hours; all steps at RT. The brightness and contrast settings were set to two different linear ranges (indicated on the left) which were chosen to show the 1P-BABB sample intensity range (upper panels) and the E-BABB sample intensity range (lower panels). gl, glomerular layer; aob, accessory olfactory bulb. Scale bar, 1mm. (<b>D</b>) Fluorescence of fixed, EGFP-expressing <i>E</i>. <i>coli</i> cells after dehydration with 1-propanol, or <i>tert</i>-butanol, and additional five days in clearing solution, all steps at the indicated temperatures, all solutions at pH 9.5.</p

EGFP positive cell counts in the 3D skeleton dataset generated with KNOSSOS.

<p>The LSFM recorded dataset of the virus injected mouse brain shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124650#pone.0124650.g005" target="_blank">Fig 5</a> was analyzed in KNOSSOS after manual classification of the EGFP positive cells using the Allen Mouse Brain Atlas and Brain Explorer2 software. The number of EGFP positive cells and their distribution in different brain regions are given for the virus injected ipsilateral and the contralateral site of the brain. EGFP positive cells in the ipsilateral site, outside the injection region, and EGFP positive cells from the contralateral site are monosynaptically connected to RABV<i>∆G-EGFP</i> producing neurons in the ipsilateral hemisphere. Irrelevant cells were left empty.</p><p>EGFP positive cell counts in the 3D skeleton dataset generated with KNOSSOS.</p

Neuronal cells and neurites in the ipsilateral injected EC/hippocampal area.

<p>Brain sample same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124650#pone.0124650.g004" target="_blank">Fig 4</a>; original dataset (LSFM recording, horizontal): same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124650#pone.0124650.g004" target="_blank">Fig 4</a>. (<b>A</b>) Green/red overlay of LSFM 50 μm z range maximum projections from a subvolume of the EC/HC area. Green channel: RABV<i>ΔG-EGFP</i> fluorescence revealing different cell types with dendrites and axon bundles. Red channel, rAAV-labeled cells (red arrow) below the brain periphery, which shows red autofluorescence. Dotted circle encloses the red fluorescence of the injection channel, which runs almost perpendicular to the image plane. White arrows, interneurons; blue arrow, granule cell; yellow arrow, glia cell. PRh 2/3, layers 2/3 of perirhinal cortex; alv, alveus of the hippocampus; CA1, Ammons Horn field CA1; Or, oriens layer; Py, pyramidal cell layer; Rad, radiatum layer; LMol, lacunosum moleculare; DG, dentate gyrus; Mo, molecular layer, Gr, granular layer; LEnt 2/3, layers 2/3 of lateral entorhinal cortex. Colors representing fluorescence signals were partially desaturated with Adobe Photoshop. Scale bar, 200 μm. (<b>B</b>) Long-range axonal projections traced in 3D with KNOSSOS and overlaid onto a green channel maximum z projection rendered as greyscale (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124650#pone.0124650.g004" target="_blank">Fig 4C</a>, top panel). Colored axon trajectories were generated with Amira from 3D KNOSSOS datasets. Arrow points to septum. Scale bar, 1 mm.</p

Light-sheet microscope (LSFM) setup and shadow suppression.

<p>(<b>A</b>) Beam path for light sheet-generation / fluorescence excitation (blue: left panels, top views) and for fluorescence excitation (blue) and detection (green) (right panel, side view). Scanner 1 generates the light sheet, while scanner 2 controls the illumination angle rotation. During recording, both scanners are active simultaneously. (<b>B, C</b>) Suppression of shadow stripes by scanner 2. Single xy-plane Venus FP fluorescence images from a fluorescent mouse brain prepared from a <i>Tg</i>^{<i>Y5RitTA⁄Y1RVenus</i>} [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124650#pone.0124650.ref039" target="_blank">39</a>] mouse at P13, cleared with the 1P-BABB method and recorded with our LSFM (excitation. 514 nm; emission, bandpass 520–550 nm) demonstrate the effect of scanner 2 that rotates the light sheet (for technical details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124650#pone.0124650.g003" target="_blank">Fig 3A</a>). (B) scanner 1 ON, scanner 2 OFF; (C) both scanners ON. Illumination from lateral side of left hemisphere (from top of panels), imaging from dorsal (from viewer’s direction). Scale bar, 1 mm.</p

Clearing of brains using different alcohols and clearing solutions / pH adjustments.

<p>(<b>A</b>) Transmitted light images of whole mouse brains (age: P44) dehydrated with different alcohols as indicated (EtOH: pH unadjusted; other alcohols: pH adjusted to 9.5) and incubated in BABB clearing solution at the respective pH for 150 days (all incubations at RT). Brains were either placed on top of a transparent ruler (big panels; small ticks, 1 mm), or on the top of a 1951 USAF light-transmissive target (inserts; Scale bar, 1 mm). (<b>B</b>) Transmitted light images of forebrains and cerebella of adult mouse brains at different ages (P44, P250 and P673) dehydrated with <i>tert</i>-butanol (pH 9.5) and cleared with BABB pH 9.5 for 3 d; all incubations at RT (upper row) or 30°C (bottom row). P44 brains were recorded after additional 100 d storage at 4°C. The ages of the mice analyzed are indicated on the top of the image.</p

Long term preservation of fluorescence and sample geometry.

<p>Light sheet microscope horizontal recordings of green fluorescence (RABV<i>ΔG-EGFP</i>) from a p70 mouse brain were taken at day 32 and day 264, respectively, after the onset of clearing. Corresponding subvolumes of the entorhinal cortex / hippocampus region were selected with ImageJ and upscaled to a cubic voxelsize of 1.61 microns with AMIRA software. The day 264 dataset was 3D aligned to the day 32 dataset by affine transformation with AMIRA (Lanczos interpolation). Since the day 32 dataset images had been recorded at three times the excitation power (4.65 microjoule per mm light sheet width) compared with the day 264 dataset images (1.55 microjoule per mm light sheet width), we accounted for this difference by adjusting the image display range with ImageJ accordingly (Set Display Range (32d): 70–1269; (264d): 63–462; with the lower value of each range representing the image background outside the brain tissue signal area). All other recording and image analysis parameters were maintained. Image shows overlapping z maximum projections (50 microns z projection size) of day 32 (<b>A</b>), day 264 (<b>B</b>) and an overlay of day 32 (red) and day 264 (green) EGFP fluorescence (<b>C</b>). Bar, 100 microns.</p

Visualization of cell bodies and neurites in a tB-BABB cleared mouse brain.

<p>Brain sample same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124650#pone.0124650.g004" target="_blank">Fig 4</a>; original dataset (LSFM recording, horizontal): same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124650#pone.0124650.g004" target="_blank">Fig 4</a>, except for (A). (<b>A</b>) Confocal recording (single plane) from EC layer 3 near the injection site taken after 271 days in clearing solution. Overlay of red channel (rAAV-driven <i>mRFP1</i> fluorescence, arrows) and green channel (RABV<i>ΔG-EGFP</i> fluorescence). Double-infected cells expressing rAAV-driven <i>mRFP1</i> plus RABV<i>ΔG-EGFP</i> fluorescence are indicated by orange to green color (arrowheads). Recording direction: from brain surface (positioned perpendicular to optical axis) to interior. Scale bar, 100 μm. (<b>B</b>) LSFM 50 μm z range maximum projection from a subvolume of the PiC area (RABV<i>ΔG-EGFP</i> fluorescence). Arrowheads indicate some Layer 2 neurons, arrows some interneurons. Scale bar, 100 μm. (<b>C</b>) LSFM 50 μm z range maximum z projection from a subvolume of the auditory cortex region (RABV<i>ΔG-EGFP</i> fluorescence) showing a cortical layer 5 cell with a big apical dendrite (arrowheads), basal dendrites at the soma, and a basal axon (long arrows). Scale bar, 100 μm. (<b>D)</b> LSFM 90 μm z range maximum projection from a subvolume of the septal area (RABV<i>ΔG-EGFP</i> fluorescence) 2.3 mm below brain surface revealing neurite structures (arrowheads). Scale bar, 100 μm. (<b>E</b>) LSFM 20 μm z range maximum projection (RABV<i>ΔG-EGFP</i> fluorescence) from a subvolume of the PiC/EC/HC region, showing neurons from cortical EC and the PiC regions and glia from the HC region projecting to the injected EC neurons. Scale bar, 500 μm. (<b>F</b>) Hippocampal DG area enlarged from (E), showing glia cells in the outer granule cell layer (o-gcl) and stratum lacunosum moleculare (LMol), and neurons in the DG hilus region (arrowheads). PiC, Piriform cortex; EC, Entorhinal cortex. Scale bar, 100 μm.</p

Habitat preferences of male Corn Buntings Emberiza calandra in north-eastern Germany

Agricultural ecosystems have faced dramatic changes during past decades, resulting in a dramatic loss of farmland biodiversity. The Corn Bunting Emberiza calandra is considered a suitable indicator for the conservation value of farmland habitats, and has recently suffered strong declines throughout much of its European range. As a basis for targeted conservation measures, we investigated the habitat preferences of this species in north-eastern Germany by comparing the composition of male territories with randomly chosen control sites. A territory was defined as the area within a radius of 150 meters around the assumed centre of the territory, as the majority of nests is found within this radius. To assess food availability for nestlings, arthropod abundance within the most abundant land use types i.e. crop fields, fallows, grassland as well as within unploughed strips was investigated. In total we found 102 male Corn Bunting territories, which were mainly composed of crop fields (50%), grassland (28%), and fallows (12%). Territories compared with control sites were characterized by a lower proportion of crop fields, a higher proportion of fallows, more diverse land use types, more abundant field boundaries, unploughed strips, and tracks, and a higher availability of song posts. However, neither the number of larger (>= 1 cm), smaller ( 10%) and song posts (> 70 m 'linear song posts' or > 1 solitary post per ha) for the habitat selection of male Corn Buntings. We conclude that measures to halt population declines of Corn Buntings seem to be relatively easy to implement, provided that farmers are granted a fair compensation