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. 2011 Jan 13;2(2):356-64.
doi: 10.1364/BOE.2.000356.

Multimodal wide-field two-photon excitation imaging: characterization of the technique for in vivo applications

Jae Youn Hwang, Sebastian Wachsmann-Hogiu, V Krishnan Ramanujan, Andreas G Nowatzyk, Yosef Koronyo, Lali K Medina-Kauwe, Zeev Gross, Harry B Gray, Daniel L Farkas

Multimodal wide-field two-photon excitation imaging: characterization of the technique for in vivo applications

Jae Youn Hwang et al. Biomed Opt Express. .

Abstract

We report fast, non-scanning, wide-field two-photon fluorescence excitation with spectral and lifetime detection for in vivo biomedical applications. We determined the optical characteristics of the technique, developed a Gaussian flat-field correction method to reduce artifacts resulting from non-uniform excitation such that contrast is enhanced, and showed that it can be used for ex vivo and in vivo cellular-level imaging. Two applications were demonstrated: (i) ex vivo measurements of beta-amyloid plaques in retinas of transgenic mice, and (ii) in vivo imaging of sulfonated gallium(III) corroles injected into tumors. We demonstrate that wide-field two photon fluorescence excitation with flat-field correction provides more penetration depth as well as better contrast and axial resolution than the corresponding one-photon wide field excitation for the same dye. Importantly, when this technique is used together with spectral and fluorescence lifetime detection modules, it offers improved discrimination between fluorescence from molecules of interest and autofluorescence, with higher sensitivity and specificity for in vivo applications.

Keywords: (110.4234) Imaging systems; (180.4315) Microscopy.

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Figures

Fig. 1
Fig. 1
Schematics of the experimental set-up for wide-field two-photon excitation: (A) multimode optical imaging with wide-field two-photon excitation (B) wide-field two-photon excitation set-up combined with scanning confocal microscopy on an Olympus Fluoview 300 platform. FR: faraday rotator, TGI: time gated intensifier, L1: a doublet lens (focal length: 200mm)
Fig. 2
Fig. 2
Optical characteristics of wide-field two-photon excited fluorescence: (A) power dependence of the fluorescence of gallium corroles (50μM) (B) fluorescence intensity versus wavelength of the mouse intestine stained with Alexa 568 phalloidin [power density: 64μW/μm2, ex: 780-910 nm (step: 10 nm), em: 620 ± 60 nm, and a 40x air objective]. (C) fluorescence intensity versus numerical aperture of the objective lens [ex: 780 nm, power density: 64μW/μm2, 10x (NA: 0.45, FOV: 256 μm x 244 μm), 40x (NA: 0.75, FOV: 64 μm x 61 μm), 40x (NA: 1.30, FOV: 64 μm x 61 μm), and 60x (NA: 1.45, FOV: 43 μm x 41 μm)]. The rectangles represent the size of FOV.
Fig. 3
Fig. 3
Comparisons of the depth dependence in scanning two-photon, wide-field two-photon, and wide-field one-photon excitation for a tissue phantom: (A) schematic of the tissue phantom (B) Intensity profiles over the selected regions (microspheres) along the axial (depth) coordinate (C) Scanning two-photon excited fluorescence image obtained with a fs pulsed laser light source at 780nm. (D) Wide-field two-photon excited fluorescence image without Gaussian correction (excitation with a fs laser at 780 nm,) (E) Wide-field one-photon excited fluorescence image (excitation at 488 ± 30 nm). The scale bar is 50μm.
Fig. 4
Fig. 4
Wide-field one photon and wide-field two-photon imaging of an eye specimen from an AD transgenic mouse model: (A) One photon excited fluorescence image (excitation at 560 nm, emission at 630 nm) (B) Wide-field two-photon excited fluorescence image without Gaussian correction (excitation at 830nm, emission at 630nm) (C) Gaussian corrected wide-field two-photon excited fluorescence image (scale bar: 25μm): The plaques can be visualized and appear as brighter white signal compared to the background; the arrows indicate plaques (D) Profile of cross-sections of the original and the corrected image along a horizontal line (a’).
Fig. 5
Fig. 5
Multimodal optical imaging with wide-field two-photon excitation of a nude mouse: (A) A nude mouse: solid circle indicates the corroles-injected tumor region; the dotted circle indicates the microsphere solution injected region. (B) One-photon and (C) wide-field two-photon excited fluorescence images (before Gaussian-correction) of microspheres at the indicated different depths (center: 10 μm, insets: 10.5 μm, 13.5 μm, and 17 μm) beneath the skin around the regions selected by the dotted circle. The insets are the depth-resolved images. The arrows indicate microspheres. (D) Spectral and (E) fluorescence lifetime images of a microsphere obtained with wide-field two-photon excitation. The spectral and fluorescence lifetime images were obtained at the same depth (10 μm) and field of view with Fig. 5(C) (center). (F) One-photon (excitation 425nm and emission 620nm, field of view: 75 μm x 75 µm) and (G) wide-field two-photon (excitation 800nm and emission 620nm) image around the tumor regions (indicated by the solid circle), indication the presence and spatial distribution of corroles. (H) Spectral classification and (I) fluorescence lifetime images of corroles.
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