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MO‐D‐141‐09: An Investigation of the Feasibility of Volumetric Imaging of Fluorescent Bio‐Markers Using Optical‐ECT
Author(s) -
Oldham M,
Thomas A,
Dewhirst M
Publication year - 2013
Publication title -
medical physics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.473
H-Index - 180
eISSN - 2473-4209
pISSN - 0094-2405
DOI - 10.1118/1.4815256
Subject(s) - in vivo , ex vivo , green fluorescent protein , biomedical engineering , preclinical imaging , chemistry , pathology , microbiology and biotechnology , biophysics , biology , medicine , gene , biochemistry
Purpose: Toptical‐ECT is a technique with potential for high resolution 3D imaging of the distribution of fluoresent biomarkers (including reporter proteins like GFP) in un‐sectioned tissue samples. Accurate optical‐ECT data is only feasible if the biomarkers survive an optical clearing procedure. This work presents investigates this question, and the feasibility of extracting volume metrics from optical‐ECT data. Methods: 4T1 tumors were grown in window chambers on nude mice, following an approved protocol. Tumor cells constitutively expressed RFP, and endogenously expressed GFP labeling HIF‐1 transcription. Microvasculature was labeled by colloidal carbon. When the tumors were ∼5–7mm, they were imaged in‐vivo (in the chamber) using conventional epi‐fluorescent microspcopy. Tumors were then immediately removed, optically‐cleared, and imaged ex‐vivo by optical‐CT/ECT. Comparison of the in‐vivo and ex‐vivo images enabled investigation of the stability of the biomarkers through optical clearing. Volume measurements of regions expressing different markers (GFP and RFP) were generated though automatic thresholding. Results: Biomarker expressing regions (GFP and RFP) were generally consistent between comparable optical‐ECT projections and in‐vivo microscopy. In some tumors, GFP and RFP expression was observed to be partially obscured in in‐vivo images, due to absorption in overlying tissue. In optical‐ECT views, these regions became visible, due to optical clearing. In one tumor, 31% of the gross tumor was deemed viable, as determined from RFP expression. 13% of the tumor was hypoxic as inferred from HIF‐1 expression. Almost all the GFP hypoxic volume was within the viable RFP tumor volume. Conclusion: Our preliminary data supports several key concepts: fluorescent biomarkers can survive the optical clearing process representative of in‐vivo condition; the cleared tumor revealed new regions of signal that were partially obscured in in‐vivo images; and 3D quantitative metrics can be determined from optical‐CT/ECT that correspond to their 2D counterparts in standard microscopy (e.g. sub‐volume of HIF‐1 expression).