A combined method for correlative 3D imaging of biological samples from macro to nano scale.

Manuela Kellner,Marko Heidrich,Heiko Meyer,Christoph Wrede,Georgios C Antonopoulos,Roman Grothausmann,Raoul-Amadeus Lorbeer,Matthias Ochs,Mark P Kühnel,Nicole Izykowski,Tammo Ripken,Danny Jonigk,Lars Knudsen

doi:10.1038/srep35606

Abstract

Correlative analysis requires examination of a specimen from macro to nano scale as well as applicability of analytical methods ranging from morphological to molecular. Accomplishing this with one and the same sample is laborious at best, due to deformation and biodegradation during measurements or intermediary preparation steps. Furthermore, data alignment using differing imaging techniques turns out to be a complex task, which considerably complicates the interconnection of results. We present correlative imaging of the accessory rat lung lobe by combining a modified Scanning Laser Optical Tomography (SLOT) setup with a specially developed sample preparation method (CRISTAL). CRISTAL is a resin-based embedding method that optically clears the specimen while allowing sectioning and preventing degradation. We applied and correlated SLOT with Multi Photon Microscopy, histological and immunofluorescence analysis as well as Transmission Electron Microscopy, all in the same sample. Thus, combining CRISTAL with SLOT enables the correlative utilization of a vast variety of imaging techniques.

Highlights

Correlation often fails owing to various reasons: First, movement and deformation during the measurement as well as biological degradation of the sample lead to severe artifacts regarding specimen morphology and imaging data
Optical clearing is a preparation step prior to optical microscopy, which suppresses the effects of light scattering
CRISTAL is based on the infiltration of a biological sample with a liquid monomer, which is subsequently cured by UV light to produce a specimen that is optically cleared and encapsulated in a transparent polymer (Fig. 1a)

Summary

Introduction

Correlation often fails owing to various reasons: First, movement and deformation during the measurement as well as biological degradation of the sample lead to severe artifacts regarding specimen morphology and imaging data. It is nearly impossible to define distinct reference points to correlate structures from differing imaging techniques without a tool to bridge the gap between them[5,23] This is essential for the correlation itself and for the design of the experiment e.g. decisions on further analyzing steps like the direction of histological sectioning or locating regions of interest (ROI) within the sample. In addition to SLOT, we applied Multi Photon Microscopy (MPM) and Transmission Electron Microscopy (TEM) to the same sample The measurements of these highly sophisticated techniques strongly differ in terms of resolution, contrast mechanism and field of view. They were accompanied by the analysis of histological and antibody stained sections of the same sample, all correlated with each other by SLOT. We demonstrate the potential of this correlative imaging method utilizing SLOT and CRISTAL by initially presenting its application to a fibrotic lung lobe of a bleomycin-treated rat followed by a comparison of the results with correlative measurements of a second healthy control lung lobe

Methods

Results

Conclusion