Continuously tracing brain-wide long-distance axonal projections in mice at a one-micron voxel resolution

Self Cite

Abstract:To resolve fine structures of biological systems like neurons, it is required to realize microscopic imaging with sufficient spatial resolution in three dimensional systems. With regular optical imaging systems, high lateral resolution is accessible while high axial resolution is hard to achieve in a large volume. We introduce an imaging system for high 3D resolution fluorescence imaging of large volume tissues. Selective plane illumination was adopted to provide high axial resolution. A scientific CMOS working in sub-array mode kept the imaging area in the sample surface, which restrained the adverse effect of aberrations caused by inclined illumination. Plastic embedding and precise mechanical sectioning extended the axial range and eliminated distortion during the whole imaging process. The combination of these techniques enabled 3D high resolution imaging of large tissues. Fluorescent bead imaging showed resolutions of 0.59 μm, 0.47μm, and 0.59 μm in the x, y, and z directions, respectively. Data acquired from the volume sample of brain tissue demonstrated the applicability of this imaging system. Imaging of different depths showed uniform performance where details could be recognized in either the near-soma area or terminal area, and fine structures of neurons could be seen in both the xy and xz sections.

Section: Imaging Speedmentioning

confidence: 99%

“…In brain science research, combining mechanical sectioning with regular imaging methods, researchers could obtain high resolution images in large volume [1][2][3][4][5]. However, limited by optics principle, the axial resolution of these methods, which mostly above 1 micron, would not be equally high as lateral resolution.…”

Section: Introductionmentioning

confidence: 99%

High axial resolution imaging system for large volume tissues using combination of inclined selective plane illumination and mechanical sectioning

Zhang

Yang

et al. 2017

Self Cite

“…Micro-optical sectioning tomography (MOST) technology automatically and simultaneously performs precise serial slicing and high resolution optical imaging [15]. Using MOST technique, we have successfully obtained 3D high-resolution imaging data sets of Golgi stained or the green fluorescent protein (GFP)-labeled whole brain [16][17][18].…”

Section: Introductionmentioning

confidence: 99%

“…However, procedures and resins for immunostaining and embedding the thin sample are unsuitable for large-volume sample. For large sample embedding, preservation of the fluorescent signal brightness and fine structures is a major challenge [17].…”

Section: Introductionmentioning

confidence: 99%

Plastic embedding immunolabeled large-volume samples for three-dimensional high-resolution imaging

Gang¹,

Liu²,

Wang³

et al. 2017

Self Cite

High-resolution three-dimensional biomolecule distribution information of large samples is essential to understanding their biological structure and function. Here, we proposed a method combining large sample resin embedding with iDISCO immunofluorescence staining to acquire the profile of biomolecules with high spatial resolution. We evaluated the compatibility of plastic embedding with an iDISCO staining technique and found that the fluorophores and the neuronal fine structures could be well preserved in the Lowicryl HM20 resin, and that numerous antibodies and fluorescent tracers worked well upon Lowicryl HM20 resin embedding. Further, using fluorescence MicroOptical sectioning tomography (fMOST) technology combined with ultra-thin slicing and imaging, we were able to image the immunolabeled large-volume tissues with high resolution. Miyawaki, "Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain," Nat. Neurosci. 14(11), 1481-1488 (2011). 11. J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sørensen, R. Baldock, and D. Davidson, "Optical projection tomography as a tool for 3D microscopy and gene expression studies," Science 296 (

“…In strip imaging, the entire sample is divided into several strips and samples are imaged one strip at a time rather than the conventional mosaic imaging method. Recently, stage-scanning confocal microscopy combined with strip imaging was applied for rapid imaging of large specimens [12][13][14][15]. In these systems, line illumination is generated by scanning a focused beam on the sample.…”

Section: Introductionmentioning

confidence: 99%

Rapid imaging of large tissues using high-resolution stage-scanning microscopy

Yang

Zheng

Shang

et al. 2015

Self Cite

Rapid and high-resolution imaging of large tissues is essential in biological research, like brain neuron connectivity research and cancer margins imaging. Here a novel stage-scanning confocal microscopy was developed for rapid imaging of large tissues. Line scanning methods and strip imaging strategy were used to increase the imaging speed. The scientific CMOS was used as line detector in sub-array mode and the optical sectioning ability can be easily adjusted by changing the number of line detectors according to different samples. Fluorescent beads imaging showed resolutions of 0.47 μm, 0.56 μm, and 1.56 μm in the X, Y, and Z directions, respectively, with a 40 × objective lens. A 10 × 10 mm 2 coronal plane with enough signal intensity could be imaged in about 88 sec at a sampling resolution of 0.16 μm/pixel. Rapid imaging of mouse brain slices demonstrated the applicability of this system in visualizing neuronal details at high frame rate.