Journal: Cells

Article Title: Comparison of Multiscale Imaging Methods for Brain Research

doi: 10.3390/cells9061377

Figure Lengend Snippet: Comparison of widefield microscopy methods in the imaging of tissue sections. Mouse brain sections (60 µm thick) immunofluorescently labeled with antibodies against Homer1 (Homer; red in B and C ), Shank2/ProSAP1 (Shank2; green) and MAP2 (white) imaged by 3D-Zoom microscopy ( A ), a slide scanning microscope ( B ) and a conventional widefield microscope ( C ), respectively. DNA was counter-stained with DAPI (blue). Note that fluorescence signals of Homer staining in ( A ) were not captured due to lack of an appropriate fluorescence excitation. Instead, an additional place holder fluorescence channel was captured (ND, red) to allow later comparison of acquisition speed with the other widefield methods (see ). ( A2 ) Magnified view of a cortical area boxed in ( A1 ). Individual immunofluorescence signals of this subregion are shown in monochrome in A3 to A6 . An arrow in A3 depicts an example of a cell nucleus. ( B1 ) The same sample as in A imaged in 3D using a multi-slide scanning microscope. ( B2 ) Single optical section shown as a magnified view of the subregion boxed in B1 . ( B3 to B6 ) Individual immunofluorescence channels of the image in B2 in monochrome. Arrows in B3 and B4 mark individual chromo-centers within single nuclei and MAP2-positive dendrites, respectively, and thereby highlight an improved resolution provided by the multi-slide scanning microscope. ( C1 ) The same sample imaged in 3D using a conventional widefield microscope. ( C2 ) A single optical section was selected from the dataset shown in C1 (marked with a white box) and split into individual fluorescence channels ( C3 to C6 ). Images C2d to C6d show the same subregion after deconvolution. Individual chromo-centers within single nuclei as well as MAP2-positive dendrites become clearly resolvable after deconvolution (Arrows in C3d and C4d, respectively).

Article Snippet: Images were deconvolved with the deconvolution tool of the ZEN Blue software (Zeiss).

Techniques: Comparison, Microscopy, Imaging, Labeling, Staining, Fluorescence, Immunofluorescence

Journal: Cells

Article Title: Comparison of Multiscale Imaging Methods for Brain Research

doi: 10.3390/cells9061377

Figure Lengend Snippet: Comparison of various confocal microscopy methods in tissue imaging. Comparison of 3D imaging of a 90 × 90 × 50 (x/y/z) µm 3 region of a mouse brain section immunofluorescently labeled with antibodies against Homer (red), Shank2/ProSAP1 (green) and MAP2 (white) using SIOS (ApoTome) microscopy ( A ), SPDM ( B ) and conventional point-scanning confocal microscopy with deconvolution (HyVolution) ( C ). A1 , B1 , and C1 show 3D reconstructions of deconvolved image stacks. DNA was counter-stained with DAPI (blue). ( A2 ) shows a SIOS-processed single optical section of the image stack recorded in A1 . A subregion of this optical section (white box) is shown in A3 . ( A4 ) and ( A5 ) show the same optical section and subregion, respectively, after deconvolution in ZEN software. Arrows in ( A3 ) and ( A5 ) indicate potential identification of a single synapse at a MAP2-positive dendrite. ( B1 ) The same tissue sample as in ( A1 ) imaged at an adjacent position using a spinning disc confocal microscope. ( B2 ) shows a single confocal section of this image stack. ( B3 ) shows a subregion of the same section. ( B4 ) and ( B5 ) show the same optical section after deconvolution in ZEN software. Arrows indicate identification of Homer/Shank2-positive synapses at dendrites. ( C1 ) The same specimen used in A1 and B1 was subjected to conventional point-scanning confocal microscopy but with the pinhole set to 0.5 Airy units. Furthermore the entire image stack was subjected to deconvolution by Huygens software within Leica’s HyVolution module. ( C2 ) shows a single optical section of the original image stack. ( C3 ) represents the same optical section shown in C2 after deconvolution. A subregion of the deconvolved section was selected for enlarged views ( C4 and C5 ). Arrows in C5 indicate identification of Homer/Shank2-positive synapses at dendrites.

Article Snippet: Images were deconvolved with the deconvolution tool of the ZEN Blue software (Zeiss).

Techniques: Comparison, Confocal Microscopy, Imaging, Labeling, Microscopy, Staining, Software

Journal: Cells

Article Title: Comparison of Multiscale Imaging Methods for Brain Research

doi: 10.3390/cells9061377

Figure Lengend Snippet: Comparison of optical sectioning and confocal/deconvolution microscopy in synapse imaging of mouse brain tissue. Image stacks of mouse brain labeled to detect Homer (red), Shank2/ProSAP1 (green) and MAP2 (white) were acquired with SIOS (ApoTome) microscopy ( A ) or SPDM/deconvolution ( B ). DNA was counterstained with DAPI (blue). Images show orthogonal views of each image stack. Single accumulations of colocalized Homer and Shank2 fluorescence, likely representing PSDs, were selected (white box in B ) and shown as enlarged x/z views on the left side of B .

Article Snippet: Images were deconvolved with the deconvolution tool of the ZEN Blue software (Zeiss).

Techniques: Comparison, Microscopy, Imaging, Labeling, Fluorescence

Journal: Cells

Article Title: Comparison of Multiscale Imaging Methods for Brain Research

doi: 10.3390/cells9061377

Figure Lengend Snippet: Comparison of various super-resolution microscopy methods in tissue imaging. A 90 × 90 × 50 ( x / y / z ) µm 3 3D region of mouse brain immunofluorescently labeled with antibodies against Homer1 (Homer; red), Shank2/ProSAP1 (Shank2; green) and MAP2 (white) was imaged using an array detector (Airyscan) microscope ( A1 ), a Lattice-SIM super-resolution microscope ( B1 ) and a STED super-resolution microscope ( C1 ), respectively. DNA was counter-stained with DAPI (blue). Please note that A1 , B1 , and C1 show 3D reconstructions of deconvolved image stacks. ( A2 ) shows a single optical section of the A1 image stack after processing and deconvolution. A subregion of this image slice (white box in A2 ) is shown enlarged as a merged view as well as in monochrome individual channels ( A3 – A6 ). ( B1 ) The same tissue sample used in A1 was imaged at an adjacent position employing Lattice-SIM super-resolution microscopy. ( B2 ) shows a single optical section of the image stack shown in B1. A subregion of this section was selected (white box) for display as enlarged views of the merged as well as individual channels ( B3 – B6 ). ( C1 ) The same tissue sample used in A1 and B1 was imaged at an adjacent position employing STED super-resolution microscopy. ( C2 ) shows a single optical section of the image stack shown in C1 after deconvolution. A subregion of this section was selected (white box) for display as enlarged views of the merged as well as individual channels ( C3 – C6 ). Note the higher resolution of the STED microscopy (bar in C6 , 200 nm instead of 500 nm, as in A6 and B6 ).

Article Snippet: Images were deconvolved with the deconvolution tool of the ZEN Blue software (Zeiss).

Techniques: Comparison, Super-Resolution Microscopy, Imaging, Labeling, Microscopy, Staining

Journal: Cells

Article Title: Comparison of Multiscale Imaging Methods for Brain Research

doi: 10.3390/cells9061377

Figure Lengend Snippet: Methods to improve deep tissue imaging and image processing. ( A – C ) Influence of an objective correction ring on image quality. Mouse brain tissue immunofluorescently labeled for Homer (red) and Shank2 (green). DNA was counterstained with DAPI (white). Imaging was performed in an area 40 µm deep within this tissue section employing point-scanning confocal microscopy ( A ). The same optical section was imaged sequentially with increased settings of a motorized correction ring of a 93× glycerol objective. The signal intensity at a selected synapse of Homer fluorescence (boxed in A ) dependent on correction ring setting is shown in B . ( C ) Quantitation of signal intensity vs. motorized correction ring setting (motCORR) as shown in B ( n = 5 per data point). ( D ) Effect of AO: Representative z-STED microscopy images of Homer (magenta, gp-anti-Homer-STAR 635P immunolabeled) in the mouse brain tissue without (left, AO off) and with (right, AO on) AO correction on the bottle-shaped STED laser (added wavefront distortion in the upper right insets, arbitrary color scale from 0 (blue) to maximum (yellow)) with intensity profiles along the lines in-between the arrows. Scale bar, 1 µm. ( E ) Acceleration of deconvolution by CUDA graphics card. Image stacks of various thickness of the mouse brain tissue as shown in A were acquired on a confocal microscope. Deconvolution of the stacks was performed with or without employment of a CUDA graphics card. The time required for deconvolution was measured ( n = 3) and plotted versus the file size of the image stack.

Article Snippet: Images were deconvolved with the deconvolution tool of the ZEN Blue software (Zeiss).

Techniques: Imaging, Labeling, Confocal Microscopy, Fluorescence, Quantitation Assay, Microscopy, Immunolabeling