Until recently, researchers
have used confocal microscopy to estimate cell populations in Glomeruli. This
study demonstrates a novel technique for Confocal microscopy with cellular
resolution on tissue cleared with Benzyl alcohol, benzyl benzoate (BABB), utilising
the Leica BABB-resistant dipping objective mounted on a fixed-stage Upright SP8
confocal microscope.
Previously kidney tissue samples were
dehydrated and mounted for microtomy, producing thin sections for
immuno-histochemical staining. Sections were imaged on a large-area tiling
fluorescent microscope to generate a map of glomeruli, these individual
glomeruli were located using a confocal microscope and imaged in three
dimensions for cell counting. The microtomy, large-area tiling and continual repositioning
of the sample during confocal imaging was both time-consuming and laborious.
By using optical clearing, with the reagent BABB,
we have removed the need for thin sections and instead can image large (~2 to
4mm) tissue samples. Samples are fixed, incubated for extended periods to
promote antibody penetration, dehydrated and then treated with BABB. Imaging is
carried out with a long working distance, wide field of view objective
(x20/0.95 NA, Leica), and an xy scanning stage for region tiling. Deep imaging
into kidney sections (up to 1mm deep) with a wide collection area allowed for
the collection of multiple, whole glomeruli per single xy stage position.
Multiple xy stage positions were set using Mark and Find software,
substantially increasing the number of glomeruli imaged in a single session.
This
method substantially increased the number of glomeruli sampled using a
relatively simple automation, thus reducing operator workload, and increasing
throughput more than twenty-five fold.