Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media
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{"title"=>"Improving axial resolution in confocal microscopy with new high refractive index mounting media", "type"=>"journal", "authors"=>[{"first_name"=>"Coralie", "last_name"=>"Fouquet", "scopus_author_id"=>"6603407597"}, {"first_name"=>"Jean François", "last_name"=>"Gilles", "scopus_author_id"=>"57188931024"}, {"first_name"=>"Nicolas", "last_name"=>"Heck", "scopus_author_id"=>"6602148287"}, {"first_name"=>"Marc Dos", "last_name"=>"Santos", "scopus_author_id"=>"56581009300"}, {"first_name"=>"Richard", "last_name"=>"Schwartzmann", "scopus_author_id"=>"54394254300"}, {"first_name"=>"Vidjeacoumary", "last_name"=>"Cannaya", "scopus_author_id"=>"55143988900"}, {"first_name"=>"Marie Pierre", "last_name"=>"Morel", "scopus_author_id"=>"57197277641"}, {"first_name"=>"Robert Stephen", "last_name"=>"Davidson", "scopus_author_id"=>"56581126400"}, {"first_name"=>"Alain", "last_name"=>"Trembleau", "scopus_author_id"=>"7004621732"}, {"first_name"=>"Susanne", "last_name"=>"Bolte", "scopus_author_id"=>"7003433794"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84926340873", "doi"=>"10.1371/journal.pone.0121096", "pui"=>"603554707", "pmid"=>"25822785", "scopus"=>"2-s2.0-84926340873", "issn"=>"19326203"}, "id"=>"f12c5241-33e7-3ab2-8c8d-a17e739ac910", "abstract"=>"Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.", "link"=>"http://www.mendeley.com/research/improving-axial-resolution-confocal-microscopy-new-high-refractive-index-mounting-media-1", "reader_count"=>46, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>3, "Researcher"=>12, "Student > Doctoral Student"=>5, "Student > Ph. D. Student"=>12, "Student > Master"=>4, "Other"=>4, "Student > Bachelor"=>5}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>3, "Researcher"=>12, "Student > Doctoral Student"=>5, "Student > Ph. D. Student"=>12, "Student > Master"=>4, "Other"=>4, "Student > Bachelor"=>5}, "reader_count_by_subject_area"=>{"Engineering"=>5, "Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>7, "Materials Science"=>1, "Agricultural and Biological Sciences"=>19, "Neuroscience"=>5, "Physics and Astronomy"=>3, "Chemistry"=>1, "Immunology and Microbiology"=>4}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>5}, "Materials Science"=>{"Materials Science"=>1}, "Neuroscience"=>{"Neuroscience"=>5}, "Chemistry"=>{"Chemistry"=>1}, "Physics and Astronomy"=>{"Physics and Astronomy"=>3}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>4}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>19}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>7}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"United States"=>2, "France"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1996009"], "description"=>"<p>Fifty-micron thick vibratome sections of olfactory bulb from adult transgenic mice (Gg8-TTA x Tet0M72iresGFP) were processed for double immunofluorescence detection of GFP (green) and Peripherin (red) and mounted subsequently with CFM3 (A, C, E) or Vectashield (B, D, F). Confocal datasets were deconvolved. (A, B) 3D-reconstruction side view of characteristic z-stacks of brain sections mounted in CFM3 (A) or Vectashield (B) with the coverslip position on the left (arrowhead). (C, D) Orthogonal slices of z-stacks taken from sections mounted in CFM3 (C) or Vectashield (D) to demonstrate axial resolution and signal intensity throughout a single section (x, y-view, x, z-view and y, z-view) and in a maximum projection of 20 images (maximum projection). (E, F) 3D maximum projection of 8 confocal sections through olfactory bulb slices mounted in CFM3 (E) or Vectashield (F), with the insert showing a zoomed view. Note that in brain sections mounted in CFM3, 3 categories of axons can be easily distinguished: GFP+/peripherin- axons (green arrowhead), GFP-/peripherin+ axons (red arrowhead), and GFP+/peripherin+ axons (yellow arrowhead). The images obtained using Vectashield do not allow such a clear distinction between these differently labelled subpopulations of axons. Scale bars = 10 μm.</p>", "links"=>[], "tags"=>["signal intensity", "New High Refractive Index Mounting Media Resolution", "confocal microscopy", "Axial Resolution", "snr", "Spherical aberration results", "index mismatches", "peak signal intensity", "psf", "180 nm laterally", "index media", "uk", "subcellular levels", "500 nm", "confocal microscopes", "medium CFM 3", "imaging depth", "noise ratio", "mouse brain tissue"], "article_id"=>1361708, "categories"=>["Biological Sciences"], "users"=>["Coralie Fouquet", "Jean-François Gilles", "Nicolas Heck", "Marc Dos Santos", "Richard Schwartzmann", "Vidjeacoumary Cannaya", "Marie-Pierre Morel", "Robert Stephen Davidson", "Alain Trembleau", "Susanne Bolte"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0121096.g004", "stats"=>{"downloads"=>9, "page_views"=>26, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Improvement_of_axial_resolution_in_fixed_mouse_olfactory_bulbs_/1361708", "title"=>"Improvement of axial resolution in fixed mouse olfactory bulbs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-03-30 04:37:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1996002"], "description"=>"<p>170 nm green beads have been mounted in CFM1, CFM3, 2’2-Thiodiethanol (TDE), Vectashield (VS), Fluoromount (FM), Mowiol (MW) or Prolong Gold (PG) mounting media as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121096#pone.0121096.g001\" target=\"_blank\">Fig. 1A</a>, and measured directly at the coverslips (A, A’, B, B’) or in depth (B, B’, B”). Imaging was carried out with the 488 nm laser line collecting green emission. (A, A’) X, Z-projections of fluorescent beads are shown in A. Similar beads have been used to measure the lateral (dots) and axial (rhombus) resolution of the confocal microscope. Beads located at the coverslip show no significant difference in lateral (A’, mean value = 255 nm) and axial (A’, mean value = 523 nm) resolution through the different mounting media. Scale bar = 1 μm. (B, B’, B”) X, Z-projection of 170 nm beads mounted in CFM3 (B) or in Vectashield (B’) at the level of the coverslip, or at 58 μm or 116 μm depth. Axial resolution in high refractive index medium CFM3 (B”, triangles) remains constant with focussing depth, whereas axial resolution of beads increased with increasing focusing depth in Vectashield (B”, squares). Note that the lateral resolution remains unaffected in the two mounting media (B”, squares = CFM3 and circles = VS).</p>", "links"=>[], "tags"=>["signal intensity", "New High Refractive Index Mounting Media Resolution", "confocal microscopy", "Axial Resolution", "snr", "Spherical aberration results", "index mismatches", "peak signal intensity", "psf", "180 nm laterally", "index media", "uk", "subcellular levels", "500 nm", "confocal microscopes", "medium CFM 3", "imaging depth", "noise ratio", "mouse brain tissue"], "article_id"=>1361702, "categories"=>["Biological Sciences"], "users"=>["Coralie Fouquet", "Jean-François Gilles", "Nicolas Heck", "Marc Dos Santos", "Richard Schwartzmann", "Vidjeacoumary Cannaya", "Marie-Pierre Morel", "Robert Stephen Davidson", "Alain Trembleau", "Susanne Bolte"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0121096.g002", "stats"=>{"downloads"=>2, "page_views"=>32, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Lateral_and_axial_resolution_as_a_function_of_the_mounting_medium_and_focusing_depth_/1361702", "title"=>"Lateral and axial resolution as a function of the mounting medium and focusing depth.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-03-30 04:37:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1996007"], "description"=>"<p>(A-I) 100 μm vibratome brain sections were mounted in different mounting media and bright field images were taken in relief-contrast mode at equal light levels with a 1.0 objective (Zeiss Axiozoomer). Overview image, taken with black and white pattern as background (A-C) and zoomed images (A’-C’, D-I) were carried out to visualise contrast changes in the striatum (S), corpus callosum (CC) and cortex (C). Sections were mounted in CFM3 (A, A’, H), ri = 1.518), Vectashield (B, B’, ri = 1.458), Mowiol (C, C’), glycerol (D, ri = 1.390), and CFM3–variants with tuned refractive indices, such as CFM3-1 (E, ri = 1.390), CFM3-2 (F, ri = 1.399), CFM3-3 (G, ri = 1.515) and CFM3-4 (I, ri = 1.522). Note that with fine-tuning of the refractive index to a value of 1.518, contrast of the brain section is very low. Scale bars = 500 μm. (J, J’) 170 nm red fluorescent beads were mounted in CFM3 (ri = 1.518) and Vectashield (VS), as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121096#pone.0121096.g001\" target=\"_blank\">Fig. 1B</a>. Imaging was carried out with the 561 nm laser line collecting red emission. X, Z-projections (J) and axial resolution (J’) were assessed directly at the coverslip level (left), or at 60 μm depth, adjacent to brain sections (middle) and under the cortical region of the brain section (right). For beads mounted in CFM3 little variations in axial resolution were observed (J, upper panel, J’ circles, ri = 1.518). However, for beads mounted in Vectashield (J, lower panel, J’ triangles, ri = 1.454) a two-fold loss of resolution at 60 μm was observed. Scale bar = 1 μm. (K, K’) Red fluorescent beads were mounted in Vectashield (VS), as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121096#pone.0121096.g001\" target=\"_blank\">Fig. 1B</a>. X, Z-projections (K) and axial resolution (K’) was measured directly at the coverslip level (left) and below the cortical region of brain sections, at 20 μm, 30 μm and 60 μm depth. Note that a considerable loss of axial resolution is already observed at 20 μm depth. Scale bar = 1 μm.</p>", "links"=>[], "tags"=>["signal intensity", "New High Refractive Index Mounting Media Resolution", "confocal microscopy", "Axial Resolution", "snr", "Spherical aberration results", "index mismatches", "peak signal intensity", "psf", "180 nm laterally", "index media", "uk", "subcellular levels", "500 nm", "confocal microscopes", "medium CFM 3", "imaging depth", "noise ratio", "mouse brain tissue"], "article_id"=>1361706, "categories"=>["Biological Sciences"], "users"=>["Coralie Fouquet", "Jean-François Gilles", "Nicolas Heck", "Marc Dos Santos", "Richard Schwartzmann", "Vidjeacoumary Cannaya", "Marie-Pierre Morel", "Robert Stephen Davidson", "Alain Trembleau", "Susanne Bolte"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0121096.g003", "stats"=>{"downloads"=>4, "page_views"=>39, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Axial_resolution_as_a_function_of_the_mounting_medium_and_focusing_depth_under_brain_sections_/1361706", "title"=>"Axial resolution as a function of the mounting medium and focusing depth under brain sections.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-03-30 04:37:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1996016"], "description"=>"<p>*fluorochrome compatibility was experimentally evaluated only for CFM3, data for other mounting media were taken out of the manufacturers datasheets.</p><p><sup>#</sup> GFP shows enhanced photobleaching in CFM3</p><p>Comparison of mounting media.</p>", "links"=>[], "tags"=>["signal intensity", "New High Refractive Index Mounting Media Resolution", "confocal microscopy", "Axial Resolution", "snr", "Spherical aberration results", "index mismatches", "peak signal intensity", "psf", "180 nm laterally", "index media", "uk", "subcellular levels", "500 nm", "confocal microscopes", "medium CFM 3", "imaging depth", "noise ratio", "mouse brain tissue"], "article_id"=>1361715, "categories"=>["Biological Sciences"], "users"=>["Coralie Fouquet", "Jean-François Gilles", "Nicolas Heck", "Marc Dos Santos", "Richard Schwartzmann", "Vidjeacoumary Cannaya", "Marie-Pierre Morel", "Robert Stephen Davidson", "Alain Trembleau", "Susanne Bolte"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0121096.t001", "stats"=>{"downloads"=>9, "page_views"=>35, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparison_of_mounting_media_/1361715", "title"=>"Comparison of mounting media.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2015-03-30 04:37:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1996000"], "description"=>"<p>(A, B) 170 nm green or red fluorescent beads have been immobilised on the coverslip and slide surface prior to mounting. One or two spacers of scotch tape (58 μm thick) have been introduced to obtain different depths. Coverslips have been assembled with mounting medium (A) or with brain slices incubated in mounting medium (B) and imaged with a 63x, 1.4 NA objective at 488 nm or 561 nm respectively.</p>", "links"=>[], "tags"=>["signal intensity", "New High Refractive Index Mounting Media Resolution", "confocal microscopy", "Axial Resolution", "snr", "Spherical aberration results", "index mismatches", "peak signal intensity", "psf", "180 nm laterally", "index media", "uk", "subcellular levels", "500 nm", "confocal microscopes", "medium CFM 3", "imaging depth", "noise ratio", "mouse brain tissue"], "article_id"=>1361700, "categories"=>["Biological Sciences"], "users"=>["Coralie Fouquet", "Jean-François Gilles", "Nicolas Heck", "Marc Dos Santos", "Richard Schwartzmann", "Vidjeacoumary Cannaya", "Marie-Pierre Morel", "Robert Stephen Davidson", "Alain Trembleau", "Susanne Bolte"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0121096.g001", "stats"=>{"downloads"=>2, "page_views"=>25, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Experimental_setup_for_evaluating_axial_resolution_at_different_focusing_depth_with_sub_resolution_beads_/1361700", "title"=>"Experimental setup for evaluating axial resolution at different focusing depth with sub-resolution beads.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-03-30 04:37:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/1996015"], "description"=>"<p>100 μm vibratome sections of brain of adult transgenic mice (VGLUT1<sup>Venus</sup>) were mounted in CFM3, Vectashield or Mowiol and imaged using the 514 nm laser line. Imaging was carried out using an optimal z-section. 3D-reconstructions of the entire z-stack (A) with the coverslip position at the top (arrowhead) and the first 20 μm (A’) of VGLUT1<sup>Venus</sup>-labelled synaptic boutons were carried out to demonstrate depth penetration in the different media. (A”) is a magnified view of the insert in (B) showing synaptic boutons in a depth of 5–15 μm. Note that the boutons mounted in Vectashield appear elongated in comparison to CFM3 mounted sections. Scalebars are 10 and 3 μm.</p>", "links"=>[], "tags"=>["signal intensity", "New High Refractive Index Mounting Media Resolution", "confocal microscopy", "Axial Resolution", "snr", "Spherical aberration results", "index mismatches", "peak signal intensity", "psf", "180 nm laterally", "index media", "uk", "subcellular levels", "500 nm", "confocal microscopes", "medium CFM 3", "imaging depth", "noise ratio", "mouse brain tissue"], "article_id"=>1361714, "categories"=>["Biological Sciences"], "users"=>["Coralie Fouquet", "Jean-François Gilles", "Nicolas Heck", "Marc Dos Santos", "Richard Schwartzmann", "Vidjeacoumary Cannaya", "Marie-Pierre Morel", "Robert Stephen Davidson", "Alain Trembleau", "Susanne Bolte"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0121096.g005", "stats"=>{"downloads"=>4, "page_views"=>51, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Depth_penetration_and_resolution_improvement_in_VGLUT1_Venus_mouse_brain_slices_/1361714", "title"=>"Depth penetration and resolution improvement in VGLUT1-Venus mouse brain slices.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-03-30 04:37:03"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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