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Proceedings Paper

Enhancement of third harmonic contrast with harmonophores in multimodal non-linear microscopy of histological sections
Author(s): Adam Tuer; Ludmila Bakueva; Richard Cisek; Jennifer Alami; Daniel J. Dumont; John Rowlands; Virginijus Barzda
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Paper Abstract

Histological investigations of biological tissue benefited tremendously from staining different cellular structures with various organic dyes. With the introduction of new imaging modalities such as second harmonic generation (SHG) and third harmonic generation (THG) microscopy, the demand for novel dyes that enhance the harmonic signals has arisen. The new labels with high molecular hyperpolarizability have recently been termed harmonophores. In this study, we demonstrate that hematoxylin, the standard histological stain used in H&E (hematoxylin and eosin) staining, enhances the microscopic THG signal. Hematoxylin has an affinity for basophilic structures such as the cell nucleus, ribosomes and mitochondria, while eosin stains structures such as the cytoplasm, collagen and red blood cells. The histological sections of H&E stained cancerous prostate tissue found in transgenic adenocarcinoma of the mouse prostate (TRAMP) have been investigated with the multimodal SHG, THG and multiphoton excitation fluorescence (MPF) microscope. Strong THG signal revealed intracellular structures originating where the hematoxylin stain resides, while SHG imaging of the tissue showed the presence of collagen fibrils in the extracellular matrix. The MPF was mostly present in the extracellular matrix. The spectrally and temporally resolved MPF revealed that most of the fluorescence originates from the eosin. The THG image did not correlate with MPF confirming that the harmonic signal originates from hematoxylin. Multimodal nonlinear microscopy adds invaluable information about cellular structures to the widely used bright field investigations of H&E stained histological sections, and can be efficiently used for morphological studies as well as cancer diagnostics.

Paper Details

Date Published: 12 March 2008
PDF: 6 pages
Proc. SPIE 6860, Multiphoton Microscopy in the Biomedical Sciences VIII, 686005 (12 March 2008); doi: 10.1117/12.763509
Show Author Affiliations
Adam Tuer, Univ. of Toronto (Canada)
Ludmila Bakueva, Sunnybrook Health Sciences Ctr. (Canada)
Univ. of Toronto (Canada)
Richard Cisek, Univ. of Toronto (Canada)
Jennifer Alami, Sunnybrook Health Sciences Ctr. (Canada)
Univ. of Toronto (Canada)
Daniel J. Dumont, Sunnybrook Health Sciences Ctr. (Canada)
Univ. of Toronto (Canada)
John Rowlands, Sunnybrook Health Sciences Ctr. (Canada)
Univ. of Toronto (Canada)
Virginijus Barzda, Univ. of Toronto (Canada)


Published in SPIE Proceedings Vol. 6860:
Multiphoton Microscopy in the Biomedical Sciences VIII
Ammasi Periasamy; Peter T. C. So, Editor(s)

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