Year: 2014
Type: conference-abstract
Abstract: Despite numerous advantages, modern renal histology techniques suffer from significant drawbacks including: low sensitivity for sparse lesions, difficult quantitation, two-dimensional perspective, slow and labor-intensive processes, and artifacts of preparation. The optical sectioning achievable with multiphoton microscopy (MPM), combined with its high contrast and resolution, make it uniquely suited to address these limitations. However, there have been few attempts to target MPM for 3D histology, largely due to its relatively shallow practical imaging depth. We have employed a new imaging approach that combines MPM with tissue clearing and staining aimed at increasing diagnostic value of renal biopsies which also avoids the most manual and technically demanding steps of tissue processing. Biopsy-sized tissue portions were extracted from formalin-fixed human autopsy specimens and kidney resections using a biopsy needle. Optical clearing was performed by replacing tissue water with benzyl alcohol benzyl benzoate (1:2 ratio) after a methanol dehydration step. Various combinations of fluorescent stains and intrinsic second harmonic generation (SHG) signals were applied to obtain diagnostically useful images. A home-built multiphoton microscope was used for image collection. An inverted logarithmic color transformation of fluorescent signals was applied for simulation of H&E and trichrome staining. Full-thickness 3D analysis of renal biopsies with high quality digital images was achievable using the clearing/MPM approach. Resolution and contrast was sufficient for detailed examination of clinically relevant glomerular features. Faithful color reproduction of traditional stains was obtained. Entire samples could be evaluated for diagnostic tubular and interstitial findings. Quantitative 3D images of collagen distribution were possible from SHG signals. The combination of MPM and clearing with fluorescent staining and SHG signal analysis improves significantly on current histology approaches for renal biopsy evaluation producing high quality images and dramatically increasing the amount of data from each sample available for examination.
Authors: Richard Torres, Eben Olson, Michael Levene
Institution: Yale University
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2890900053