Two-site microendoscopic imaging probe for simultaneous three-dimensional imaging at two anatomic locations in tissues

Abstract

Systems that can image in three dimensions at cellular resolution and across different locations within an organism may enable insights into complex biological processes, such as immune responses, for which a single location measurement may be insufficient. In this Letter, we describe an in vivo two-site imaging probe (TIP) that can simultaneously image two anatomic sites with a maximum separation of a few centimeters. The TIP consists of two identical bendable graded index (GRIN) lenses and is demonstrated by a two-photon two-color fluorescence imaging system. Each GRIN lens has a field of view of 162 × 162 × 170 µm³, a nominal numerical aperture of 0.5, a magnification of 0.7, and working distances of 0.2 mm in air for both ends. A blind linear unmixing algorithm is applied to suppress bleedthrough between channels. We use this system to successfully demonstrate two-site two-photon two-color imaging of two biomedically relevant samples, i.e., (1) a mixture of two autofluorescent anti-cancer drugs and (2) a live hybrid tumor consisting of two spectrally distinct fluorescent cell lines.

Fig. 1.

Illustration of the proposed TIP and the imaging system. (a) Conceptual demonstration of using a TIP for simultaneous imaging at two distinct locations, (b) schematic of the constructed two-photon two-color imaging system, and (c) an image of the demonstrated TIP made of two bendable GRIN lenses (diameter: 500 μm; length: ~110mm). TL, tube lens; DM, dichroic mirror; MMF, multimode optical fiber; PMT, photomultiplier tube.

Fig. 2.

Resolution characterization. (a) Schematic showing the lens offset, (b) typical fine-scanned image of a 1-μm-diameter bead used for resolution calculation, and (c) measured resolution versus lens offset.

Fig. 3.

Two-site two-color imaging demonstrated and verified using discrete fluorescent beads and PICASSO image unmixing. (a) Original and (b) unmixed images, and bivariate histogram for the (c) original and (d) unmixed images at site 1 and (e) original and (f) unmixed images at site 2.

Fig. 4.

Two-site imaging of anti-cancer drug mixtures. (a) Photos of the drug mixtures, (b) acquired image after unmixing, and 2D sectioning at (c) slice 1 and (d) slice 2 without (left) and with (right) unmixing.

Fig. 5.

Two-site in vivo imaging of a hybrid tumor in a living mouse. (a) Schematic and photo of the testing in the animal, (b) an acquired image (green: eYFP; red: mCherry), (c) 2D image at the middle slice in (b), and (d) image of a cryosection of the tumor, scale bar: 60 μm.