Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing.

Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing. Research Abstract Details 

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Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing. Abstract Text:

H S Sakhalkar,M Dewhirst,T Oliver,Y Cao,M Oldham,

Optical emission computed tomography (optical-ECT) is a technique for imaging the three-dimensional (3D) distribution of fluorescent probes in biological tissue specimens with high contrast and spatial resolution. In optical-ECT, functional information can be imaged by (i) systemic application of functional labels (e.g. fluorophore labelled proteins) and/or (ii) endogenous expression of fluorescent reporter proteins (e.g. red fluorescent protein (RFP), green fluorescent protein (GFP)) in vivo. An essential prerequisite for optical-ECT is optical clearing, a procedure where tissue specimens are made transparent to light by sequential perfusion with fixing, dehydrating and clearing agents. In this study, we investigate clearing protocols involving a selection of common fixing (4% buffered paraformaldehyde (PFA), methanol and ethanol), dehydrating (methanol and ethanol) and clearing agents (methyl salicylate and benzyl-alcohol-benzyl-benzoate (BABB)) in order to determine a 'fluorescence friendly' clearing procedure. Cell culture experiments were employed to optimize the sequence of chemical treatments that best preserve fluorescence. Texas red (TxRed), fluorescein isothiocyanate (FITC), RFP and GFP were tested as fluorophores and fluorescent reporter proteins of interest. Fluorescent and control cells were imaged on a microscope using a DSred2 and FITC filter set. The most promising clearing protocols of cell culture experiments were applied to whole xenograft tumour specimens, to test their effectiveness in large unsectioned samples. Fluorescence of TxRed/FITC fluorophores was not found to be significantly affected by any of the test clearing protocols. RFP and GFP fluorescence, however, was found to be significantly greater when cell fixation was in ethanol. Fixation in either PFA or methanol resulted in diminished fluorescence. After ethanol fixation, the RFP and GFP fluorescence proved remarkably robust to subsequent exposure to either methyl salicylate or BABB. The optimized optical clearing procedure of ethanol fixation followed by methyl salicylate clearing preserved the fluorescence of constitutive RFP in whole xenograft tumour specimens, about 1 cc in dimension, indicating successful extension from cell plating experiments to whole tissue samples. Finally, the feasibility of imaging the 3D distribution of viable tumour cells (as indicated by the RFP emission) is demonstrated by optical-ECT imaging of cleared xenograft tumours using an in-house system.

Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing. Publishing Authors By Initials

HS Sakhalkar,M Dewhirst,T Oliver,Y Cao,M Oldham,

For similar diagnosis: diagnostic techniques and procedures: diagnostic imaging: tomography: tomography, optical research abstracts see: diagnosis: diagnostic techniques and procedures: diagnostic imaging: tomography: tomography, optical research

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Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing. Journal Published:

PUBLICATION TYPE: Research Support, N.I.H., Extr

Journal: Physics in medicine and biology

VOLUME: 52

Page Numbers: 2035-54

Journal Abbreviation:

ISSN: 0031-9155

DAY: 20

MONTH: 03

YEAR: 2007

Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing. Information

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LANGUAGE: eng

NlmUniqueID: 401220

Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing. Keywords Mesh Terms:

KEYWORDS: Tomography, Optical

MESH TERMS: methods

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Grant and Affiliation Information for Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing.

AFFILIATION: Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA.

Country: England

AGENCY: United States NCI

GRANT: R01 CA 100835

ACRONYM: CA

MEDLINETA: Phys Med Biol

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