Fig 1.
Experimental setup for evaluating axial resolution at different focusing depth with sub-resolution beads.
(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.
Fig 2.
Lateral and axial resolution as a function of the mounting medium and focusing depth.
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 Fig. 1A, 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).
Fig 3.
Axial resolution as a function of the mounting medium and focusing depth under brain sections.
(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 Fig. 1B. 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 Fig. 1B. 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.
Fig 4.
Improvement of axial resolution in fixed mouse olfactory bulbs.
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.
Fig 5.
Depth penetration and resolution improvement in VGLUT1-Venus mouse brain slices.
100 μm vibratome sections of brain of adult transgenic mice (VGLUT1Venus) 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 VGLUT1Venus-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.
Table 1.
Comparison of mounting media.