4/30/2024 0 Comments Changing scale bar PIV imagej![]() ![]() Visualization of neurons monosynaptically connected to RABV ΔG-EGFP(EnvA) infected neurons in EC. Image shows overlapping z maximum projections (50 microns z projection size) of day 32 ( A), day 264 ( B) and an overlay of day 32 (red) and day 264 (green) EGFP fluorescence ( C). All other recording and image analysis parameters were maintained. 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). The day 264 dataset was 3D aligned to the day 32 dataset by affine transformation with AMIRA (Lanczos interpolation). 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. Light sheet microscope horizontal recordings of green fluorescence (RABV ΔG-EGFP) from a p70 mouse brain were taken at day 32 and day 264, respectively, after the onset of clearing. Long term preservation of fluorescence and sample geometry. ![]() Illumination from lateral side of left hemisphere (from top of panels), imaging from dorsal (from viewer’s direction). (B) scanner 1 ON, scanner 2 OFF (C) both scanners ON. 514 nm emission, bandpass 520–550 nm) demonstrate the effect of scanner 2 that rotates the light sheet (for technical details, see Fig 3A). Single xy-plane Venus FP fluorescence images from a fluorescent mouse brain prepared from a Tg Y5RitTA⁄Y1RVenus mouse at P13, cleared with the 1P-BABB method and recorded with our LSFM (excitation. ( B, C) Suppression of shadow stripes by scanner 2. During recording, both scanners are active simultaneously. Scanner 1 generates the light sheet, while scanner 2 controls the illumination angle rotation. ( A) 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). Light-sheet microscope (LSFM) setup and shadow suppression. The ages of the mice analyzed are indicated on the top of the image.īABB clearing efficiency after dehydration with 1-propanol or tert-butanol at different temperatures. P44 brains were recorded after additional 100 d storage at 4☌. ( B) Transmitted light images of forebrains and cerebella of adult mouse brains at different ages (P44, P250 and P673) dehydrated with tert-butanol (pH 9.5) and cleared with BABB pH 9.5 for 3 d all incubations at RT (upper row) or 30☌ (bottom row). 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). ( A) 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). coli cells after dehydration with 1-propanol, or tert-butanol, and additional five days in clearing solution, all steps at the indicated temperatures, all solutions at pH 9.5.Ĭlearing of brains using different alcohols and clearing solutions / pH adjustments. ( D) Fluorescence of fixed, EGFP-expressing E. gl, glomerular layer aob, accessory olfactory bulb. 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). For both samples, dehydration steps were 8h and 16h alternating clearing step: 7 hours all steps at RT. (“E-BABB”, left), the other hemisphere using 1-propanol for dehydration and BABB for clearing, all steps at pH 9.5 (“1P-BABB”, right). One hemisphere was dehydrated and cleared according to Dodt et al. After fixation, the two hemispheres of the brain were separated. ( C) LSFM recording (sagittal optical slice) of olfactory bulb EGFP fluorescence of a brain from a two weeks old transgenic Tg CamKII-EGFP mouse. coli 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) Fluorescence of fixed, EGFP-expressing E. Dehydration steps: 2.5 h each (80%: 16 hours) clearing steps as indicated, all steps at RT, pH unadjusted. coli cells during dehydration and clearing. ( A) Fluorescence of fixed, EGFP-expressing E. ![]()
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