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Optical Clearing and Imaging of Immunolabeled Kidney Tissue
Published on: July 22, 2019
Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during
H S Sakhalkar1, M Dewhirst, T Oliver
1Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA.
Physics in Medicine and Biology
|April 4, 2007
Summary
This study optimized optical clearing protocols for optical emission computed tomography (optical-ECT) to preserve fluorescence. Ethanol fixation followed by methyl salicylate clearing best maintained fluorescence in reporter proteins like red fluorescent protein (RFP) for 3D imaging.
Area of Science:
- Biomedical Imaging
- Optical Engineering
- Cell Biology
Background:
- Optical emission computed tomography (optical-ECT) enables 3D imaging of fluorescent probes in biological tissues.
- Optical clearing is crucial for optical-ECT, rendering tissues transparent to light.
- Preserving fluorescence during clearing is essential for accurate functional imaging.
Purpose of the Study:
- To determine an optimal 'fluorescence friendly' optical clearing protocol for optical-ECT.
- To evaluate the impact of various fixing, dehydrating, and clearing agents on common fluorophores and fluorescent proteins.
- To validate the chosen protocol on whole xenograft tumor specimens.
Main Methods:
- Investigated various combinations of fixing (paraformaldehyde, methanol, ethanol), dehydrating (methanol, ethanol), and clearing agents (methyl salicylate, BABB).
- Optimized chemical treatment sequences using cell culture experiments with Texas red, FITC, red fluorescent protein (RFP), and green fluorescent protein (GFP).
- Applied promising protocols to whole xenograft tumor specimens and performed optical-ECT imaging.
Main Results:
- Texas red and FITC fluorescence were unaffected by tested clearing protocols.
- Red fluorescent protein (RFP) and green fluorescent protein (GFP) fluorescence were significantly higher with ethanol fixation compared to PFA or methanol.
- Ethanol fixation followed by methyl salicylate or BABB clearing robustly preserved RFP and GFP fluorescence.
- The optimized protocol successfully preserved constitutive RFP fluorescence in whole xenograft tumors for optical-ECT.
Conclusions:
- Ethanol fixation followed by methyl salicylate clearing is an effective protocol for preserving fluorescence in optical-ECT.
- This optimized clearing method enables 3D imaging of fluorescent reporter proteins in whole tissue samples.
- The study demonstrates the feasibility of imaging viable tumor cells using optical-ECT after optimized clearing.
