In vivo and simultaneous multimodal imaging: Integrated multiplex coherent anti-Stokes Raman scattering and two-photon microscopy
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Schematic of the multimodal NLO microscopy. BS: beam splitter; M: mirror; BP: band pass filter; LP: long pass filter; DM1: dichroic mirror (900 DCXRU); DM2: dichroic mirror (600 DCXRU); SP: short pass filter; F: single fiber; FB: fiber bundle, M-PMT: multichannel PMT. Inset: CARS images of diameter polystyrene beads measured in horizontal plane (upper panel) and vertical plane (lower panel) for the measurements of lateral and axial resolutions. The intensity profiles along the line indicated in the figure show the lateral and axial resolution are about 0.4 and , respectively.
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Representative multimodal NLO images of living C.elegans. (a) Pure nonresonant spectrum measured from a glass coverslip. N: nonresonance vibrational band; L: aliphatic C–H vibrational band; W: water O–H vibrational band; (b) pure nonresonant image measured from C.elegans; (c) aliphatic C–H bond CARS image before NRB subtraction; (d) after NRB subtraction; (e) intensity profile of C–H bond CARS signals; (f) water O–H bond CARS image before NRB subtraction; (g) after NRB subtraction; (h) intensity profile of O–H bond CARS signals; (i) subtracted features in gray scale and residual lipid droplets colored in red; (j) SHG image; (k) TPEF image; (l) merged image; (m) TPEF spectrum of lipofuscin granules; (n) the CARS spectra of lipid droplets and water content after NRB subtraction.
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