Label-free highly multimodal nonlinear endoscope

Abstract

We demonstrate a 2 mm diameter highly multimodal nonlinear micro-endoscope allowing label-free imaging of biological tissues. The endoscope performs multiphoton fluorescence (3-photon, 2-photon), harmonic generation (second-SHG and third-THG) and coherent anti-Stokes Raman scattering (CARS) imaging over a field of view of 200 µm. The micro-endoscope is based on a double-clad antiresonant hollow core fiber featuring a high transmission window (850 nm to 1800 nm) that is functionalized with a short piece of graded-index (GRIN) fiber. When combined with a GRIN micro-objective, the micro-endoscope achieves a 1.1 µm point spread function (PSF). We demonstrate 3-photon, 2-photon, THG, SHG, and CARS high resolution images of unlabelled biological tissues.

Fig. 1.

(a) Scanning electron micrographs of the double clad antiresonant hollow core fiber (DC-HCF). Bottom: close-up view on the core region. (b) Measured attenuation spectrum (blue line), GVD (red dots) and simulated GVD (red solid line) over the first transmission band, for a 2 m long DC-HCF.

Fig. 2.

(a) Measured mode size at the output of the hollow core fiber alone (blue dots) and with the GRIN fiber spliced (red dots) at $\lambda$ = 900 nm. Inset: image of the mode. (b) Measured (blue dots) and simulated (gray line) FWHM spot size as a function of the spliced GRIN fiber length, and simulated distance between the focal spot and the GRIN fiber endface. The vertical black dashed line corresponds to half the self-imaging period. The vertical red dashed line corresponds to the actual length of the GRIN fiber used to suppress parasitic THG. (c) Parasitic THG spectrum at the output of the fiber functionalized with a silica bead (blue) and with a 125 $\mu$ m GRIN fiber of optimized length (red) (see corresponding insights), for a pump wavelength of $\lambda$ = 1300 nm, with 60 fs and 1.2 $\mu$ J pulses.

Fig. 3.

(a) Endoscopy setup scheme, with an inside view and (b) photo of the endoscopic head. (c) Measured points and Gaussian fit (gray line) of the PSF at the output of the full endoscope (hollow-core fiber, spliced GRIN fiber and GRIN micro-objective) for a wavelength $\lambda$ = 920 nm. (d) Measured pulse duration before (blue) and after (red) propagation in a 2 m long fiber with a spliced GRIN lens, and at the output of the endoscope (hollow-core fiber, GRIN fiber and GRIN objective, green).

Fig. 4.

2PEF ( ${\lambda }_{\text{exc}}$ = 920 nm, represented in green on all sub-figures) and 3PEF ( ${\lambda }_{\text{exc}}$ = 1300 nm, represented in red on all sub-figures) comparison: (a) Dependence of fluorescence signal of 2 $\mu$ m diameter beads on the excitation power. (b) Axial resolution: measured fluorescence signal when moving the endoscope closer to a 2 $\mu$ m fluorescent bead and Gaussian fit (gray line). Comparison images of 2 $\mu$ m fluorescent beads ((c), (d), scale bar is 10 $\mu$ m, mean power on the sample is 10 mW for 2PEF and 12 mW for 3PEF) with an intensity plot along the white line (e), oak leaves ((f), (g), scale bar is 50 $\mu$ m, mean power on the sample is 16 mW for 2PEF and 20 mW for 3PEF) and optical cleaning paper ((h), (i), scale bar is 50 $\mu$ m, mean power on the sample is 15 mW for 2PEF and 14 mW for 3PEF). All images have been acquired with a 2 fps acquisition rate and averaged over 5 images.

Fig. 5.

3PEF images of (a) GFP labeled mouse brain ( ${\lambda }_{\text{exc}}$ = 1300 nm), scale bar is 50 $\mu$ m and mean power on the sample is 25 mW, (b) unstained mouse retina ( ${\lambda }_{\text{exc}}$ = 1300 nm) radial section, scale bar is 80 $\mu$ m and mean power on the sample is 29 mW. (c) and (d) are transverse sections in the ganglion cell layer (GCL) and inner plexiform layer (IPL) respectively, scale bar is 50 $\mu$ m. (e) unstained human colon ( ${\lambda }_{\text{exc}}$ = 1700 nm), scale bar is 50 $\mu$ m and mean power on the sample is 13 mW. All images have been acquired with a 2 fps acquisition rate and averaged over 10 images.

Fig. 6.

(a) Composite 3PEF (red), THG (blue) and SHG (green) image of unstained mouse skin ( ${\lambda }_{\text{exc}}$ = 1300 nm and mean power on the sample is 17 mW for 3PEF and THG, and ${\lambda }_{\text{exc}}$ = 920 nm and mean power on the sample is 11 mW for SHG), (b) THG ( ${\lambda }_{\text{exc}}$ = 1300 nm and mean power on the sample is 32 mW), and (c) CARS ( ${\lambda }_{\text{pump}}$ = 874 nm, and ${\lambda }_{\text{Stokes}}$ = 1164 nm, mean power on the sample is 12 mW) images of fresh unstained ox liver at a depth of 3mm (endoscope inserted into the tissue). All images have been acquired with a 2 fps acquisition rate and averaged over 10 images, scale bar is 50 $\mu$ m.