Multiphoton microscopy with clearing for three dimensional histology of kidney biopsies

Abstract

We present a multiphoton microscopy approach with clearing optimized for pathology evaluation producing image quality comparable to traditional histology. Use of benzyl alcohol/benzyl benzoate with 4',6-diamidino-2-phenylindole and eosin in an optimized imaging setup results in optical sections nearly indistinguishable from traditionally-cut sections. Application to human renal tissue demonstrates diagnostic-level image quality can be maintained through 1 millimeter of tissue. Three dimensional perspectives reveal changes of glomerular capsule cells not evident on single sections. Collagen-derived second harmonic generation can be visualized through entire biopsies. Multiphoton microscopy with clearing has potential for increasing the yield of histologic evaluation of biopsy specimens.

Keywords: (180.0180) Microscopy; (180.4315) Nonlinear microscopy; (180.6900) Three-dimensional microscopy.

Fig. 1

A) 3D renal biopsy reconstruction using MPM with clearing. Scale bar = 500 µm. B) Corresponding multiphoton image obtained at depth of 400 μm, pseudo-colored to mimic H&E.

Fig. 2

A) Traditionally processed kidney H&E section using 100x lens. B-D) Pseudo-colored MPM with clearing. Range of fine features recognizable include B) glomerular capillary walls, C) cytoplasmic granules, and D) nuclear membranes and chromatin patterns. Scale bar – 30 µm.

Fig. 3

Pseudo-colored MPM images on cleared renal tissue show preservation of clarity and contrast throughout entire core renal biopsies. Depths - A) 200 µm, B) 600 µm, C) 1000 µm. Scale bar – 40 µm.

Fig. 4

A). Renal glomerulus using MPM/clearing at 300 µm depth. Black inset shows capillary loop (black arrow) with luminal RBC. White inset shows visualization of mesangial cell (white arrow) facilitated by thin optical sectioning and high color contrast. B) Atubular glomerulus cross-section using MPM/clearing. Glomerular changes and surrounding connective tissue and edema are noted, but the functional status based on parietal epithelial cells and tubular connection cannot be assessed in a single section. Dimensions – 350 x 350 µm.

Fig. 5

Reconstructions in 3D of a normal (A) and atubular (B) capsule showing changes in the nuclei of the parietal epithelial cells lining the capsule that are not apparent on two dimensional histologic sections. Glomeruli have been digitally removed. Sections correspond to Fig. 4(a) and 4(b), respectively. Block dimensions are approximately 400x400x150μm. Arrows in (A) indicate proximal tubule connection.

Fig. 6

A). 3D reconstruction of collagen in human kidney biopsy using MPM/SHG in cleared tissue. B) Single MPM section showing two dimensional collagen arrangement (green) with modified background pseudo H&E coloring. (Scale bar – 500 µm) C) Higher magnification view of B showing sparse collagen SHG around glomerulus. (Scale bar – 50 µm) D) Z-projection over depth of 200 µm within the same glomerulus as in C.