A hierarchical model of the organization of fibrillar collagen is developed and its implications on polarization-resolved
second harmonic generation (SHG) microscopy are investigated. A “ground-up” approach is employed to develop the
theory for understanding of the origin of SHG from fibrillar collagen. The effects of fibril ultrastructure and fibril
macroscopic organization on the second-order polarization properties of fibrillar collagen are presented in conjunction
with recent ab initio results performed on a collagen triple-helix model (-GLY-PRO-HYP-)n. Various tissues containing
fibrillar collagen are quantified using a polarization-resolved SHG technique, termed polarization-in, polarization-out
(PIPO) and interpreted in light of the aforementioned theory. The method involves varying the incident laser
polarization, while monitoring the SHG intensity through an analyzer. From the SHG polarization data the orientation of
the fibers, in biological tissue, can be deduced. Unique PIPO signatures are observed for different rat tissues and
interpreted in terms of the collagen composition, fibril ultrastructure, and macroscopic organization. Similarities and
discrepancies in the second-order polarization properties of different collagen types and ultrastructures will be presented.
PIPO SHG microscopy shows promise in its ability to quantify the organization of collagen in various tissues. The
ability to characterize the structure of collagen in various tissue microenvironments will aid in the study of numerous
collagen related biological process, including tissue diseases, wound repair, and tumor development and progression.
Imaging hematoxylin-and-eosin-stained cancerous histological sections with multicontrast nonlinear excitation fluorescence, second- and third-harmonic generation (THG) microscopy reveals cellular structures with extremely high image contrast. Absorption and fluorescence spectroscopy together with second hyperpolarizability measurements of the dyes shows that strong THG appears due to neutral hemalum aggregation and is subsequently enhanced by interaction with eosin. Additionally, fluorescence lifetime imaging microscopy reveals eosin fluorescence quenching by hemalums, showing better suitability of only eosin staining for fluorescence microscopy. Multicontrast nonlinear microscopy has the potential to differentiate between cancerous and healthy tissue at a single cell level.
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.
We report on the development and successful application of a femtosecond Yb:KGd(WO4)2 laser for multimodal imaging of various biological samples. Its operation at longer wavelengths, 1029 nm, provided efficient excitation and greatly reduced sample photobleaching. The laser produced ~300 fs pulses with up to 100 nJ of energy at 14.3 MHz repetition rate. This laser system enabled continuous imaging of various live samples for prolonged periods of time. The details of laser development and fluorescence imaging of isolated chloroplasts are reported.
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