Fig 1.
Three-dimensional NLM analysis of a biopsy from a patient with advanced diabetic nephropathy.
NLM images were acquired every 4 μm in depth. Images spaced 20 μm apart in depth show advanced glomerular changes in 3 different glomeruli (labelled 1, 2, and 3). Capillary lumens and Bowman spaces are occluded with reduction of the glomerular tuft to a collapsed acellular mass of basement membranes and matrix. Exudative lesions are abundant, have a vacuolated (lipid) component (green arrow), and can be seen as both part of the glomerular tuft and Bowman’s capsule, the latter forming an encompassing ring. The hyalinized arterioles are evident in images at the 40 μm and 60 μm level (blue arrow). The depth below the biopsy surface is labelled in top right of each image. Scale bar = 100 μm.
Fig 2.
Three-dimensional NLM analysis of a biopsy from a patient diagnosed with diabetic nephropathy and IgA nephropathy.
(A) NLM images were acquired every 4 μm in depth through the biopsy and reconstructed into a 3D volume. (B) NLM images selected at different levels of a glomerulus. The depth below the tissue surface is indicated in microns (top right). (C) An NLM image and corresponding paraffin H&E slide. Hypercellularity and mesangial expansion are evident in the NLM images and paraffin H&E slide, but the Kimmelstiel-Wilson nodules are only clearly defined at the NLM levels 120 μm and 144 μm (B, green arrow). Scale bar = 100 μm.
Fig 3.
Focal and segmental glomerulosclerosis.
NLM images of a patient with focal and segmental glomerulosclerosis were acquired every 4 μm in depth. Images spaced 12 μm apart in depth show a segmental obliteration of the glomerular tuft with sclerosis (blue arrow), adhesions and obliteration of the urinary space. The relationship to the vascular pole is seen only in certain levels and an adhesion to the urinary pole is seen only in one image (at 72 μm depth). The depth below the tissue surface is indicated in microns (top right). Scale bar = 100 μm.
Fig 4.
Three-dimensional NLM analysis of FSGS with features of collapsing glomerulopathy.
NLM images spaced 4 μm in depth are shown (from left to right). Sections of glomerular collapse with associated epithelial proliferation and formation of synechia (black arrows) are seen. Fragmentation of Bowman’s capsule at the vascular pole is seen (blue arrows) associated with epithelial proliferation. Scale bar = 100 μm.
Fig 5.
Three-dimensional NLM analysis of crescentic IgA nephropathy.
NLM images spaced 4 μm in depth are shown (from left to right). In the first few images (upper left) the crescent extends into the proximal tubule. The crescent is global and associated with scattered neutrophils. Scale bar = 100 μm.
Fig 6.
Three-dimensional NLM analysis of tubular necrosis–part 1.
NLM images spaced 4 μm in depth are shown (from left to right). Proximal tubules appear intact. Distal tubules with granular debris, oxalate (star), necrosis (black arrow), and neutrophils (green arrow) are seen. Scale bar = 100 μm.
Fig 7.
Three-dimensional NLM analysis of tubular necrosis–part 2.
NLM images spaced 4 μm in depth are shown (from left to right). Proximal tubules appear intact. Deeper cortex with collecting ducts filled with granular debris, in which cellular elements can be identified. Oxalate is seen in a distal tubule (black arrow). A mitotic figure can be identified (green arrow). Scale bar = 100 μm. Inset scale bar = 25 μm.
Fig 8.
A plot of the glomerular count in the tissue analyzed using 3D NLM imaging versus the count on the original paraffin slides.
The biopsies are ordered from least to greatest glomerular count. Note that the histology and NLM counts were performed on different tissue from the same biopsy. The count on histology was performed on the original histology slides while the count on NLM was performed on the tissue from the archival block that was remaining after cutting the histology slides. The biopsy tissue thickness evaluated by NLM is also shown. Several biopsies had multiple fragments in the paraffin block and therefore multiple thicknesses are shown.
Fig 9.
Box plot of glomerular diameters and lesional volumes in biopsies of patients with diabetic nephropathy.
(A) A box plot of the diameters of all glomeruli from the biopsies shown in Figs 1 and 2. The mean glomerular diameters are 178.0 μm and 215.4 μm for the biopsy from Figs 1 and 2, respectively. (B) The volume of exudative lesions and Kimmelstiel-Wilson nodules in each glomerulus as a fraction of the volume the glomerulus is shown. The mean fraction of lesion volume:glomerular volume was 0.229 and 0.016 in the biopsy from Figs 1 and 2, respectively.
Fig 10.
Three-dimensional NLM analysis of a biopsy from a patient diagnosed with lithium nephropathy.
NLM images acquired every 4 μm in depth through the biopsy and reconstructed into a 3D volume. Individual cysts and tubules have been segmented in black and constitute 9.7% of the total biopsy volume. The 3D volume has been made partially transparent to visualize the segmented structures. A higher power view of the segmented tubules is shown in the red box. Hypertrophic proximal tubules (PT) are seen in the center and at higher power (blue box, 80 μm below biopsy surface, scale bar = 100 μm). Scale bar x, y, z = 100 μm.
Fig 11.
Histology of biopsies that were first optically cleared and evaluated with NLM.
The first column shows NLM images from 6 different biopsies that were optically cleared. After tissue optical clearing and NLM imaging, biopsies were declarified, re-embedded in paraffin, and processed for standard evaluation. H&E, PAS, Masson’s trichrome, and Jones methenamine silver staining is not affected by prior NLM analysis.