Practical two‐photon‐absorption cross sections and spectra of eosin and hematoxylin

Abstract

We experimentally investigate the two-photon absorption cross sections and spectra of eosin and hematoxylin for applications in nonlinear microscopy. The experiments are carried out on pure samples of the two dyes in DIwater solvent with different concentrations, in the typical range employed in standard staining procedures. Nonlinear fluorescence is excited by a line-shaped beam emitted by a Ti:Sapphire mode-locked laser in the wavelength range from 740 to 880 nm and is detected through a microscope setup. The two-photon absorption spectral response is systematically analyzed and discussed. Finally, the staining is applied on biological tissue samples, which are imaged by two-photon microscopy. Our results show that the employed dyes are fully suitable for applications in nonlinear imaging. We provide the two-photon absorption cross sections and spectra of eosin and hematoxylin for applicative purposes. The data refer to pure samples of the two dyes in water solution with different concentrations, in the typical range employed in standard staining procedures in the wavelength range from 740 to 880 nm. The obtained data are conceived as a useful tool for researchers in biophotonic, biology and all nonlinear microcopy users.

High-frequency photoacoustic microscopy of hematoxylin and eosin stained slides for veterinary oncology applications

“…Therefore, further preprocessing of initial diffuse reflectance spectra required the following steps: label-free log(1/Rd) spectra subtraction from H&E spectra; spectral data background correction to "0" at 700 nm; spectral averaging and spectral mean calculation. The resulting OD obtained from exogenous H&E contrast was described as a sum of the eosin (Eo) absorption band at 530 nm and a broadband hematoxylin absorption centered at 600 nm, which agrees to the previously published results 8,10 . The differential absorption spectra were obtained by subtracting the control (unmeasured) sample OD from OD assessed after photoacoustic scanning (or 400 MHz ultrasound impact to lipoma sample).…”

Improving the diagnosis of ductal carcinoma in situ with microinvasion without immunohistochemistry: An innovative method with H&E‐stained and multiphoton microscopy images

“…However, the low content of collagen III in the basement membrane and its weak SHG signal necessitated further investigation to visualize the myoepithelium via the TPEF mode of MPM. Eosin generates TPEF signals; however, due to the cytoplasm and stroma being stained by eosin during H&E staining, the TPEF signal from eosin does not possess the ability to independently distinguish muscle epithelium 40,41 . Furthermore, extracellular matrix elastin and intracellular NAD(P)H, lutein, retinol and tryptophan, among other tissue components, also exhibit intrinsic TPEF signals 18 .…”

Cooperative Sensitization Upconversion in Ytterbium(III)‐Based Eosin Lake Pigments

“…830 nm. [11] The cross-section value depends on the dye concentration and decreases as the concentration increases also due to aggregation.…”

“…Furthermore EOS exhibits two‐photon absorption (TPA) in the near infrared (NIR) with a band maximum at ca. 830 nm [11] . The cross‐section value depends on the dye concentration and decreases as the concentration increases also due to aggregation.…”