High resolution Fluorescence lifetime IMaging Micro-Endoscopy (FLIMME) using a single multimode fiber

TL;DR

A fluorescence lifetime imaging microscopy (FLIM) modality to the MMF endomicroscope is introduced and the capability of the ultrathin endomicroscope to perform label-free imaging in thick ex vivo murine submandibular gland tissue is experimentally demonstrated.

Abstract

Endoscopic optical imaging using a single multimode fiber (MMF) has emerged as a promising approach for highly compact, minimally invasive, and high-resolution imaging. Unlike conventional fiber bundles, MMF-based endomicroscopes exploit the controlled excitation of multiple spatially overlapping modes in a single MMF. of core diameters of tens of micrometers. to deliver and collect light to form images with sub-micrometer resolution. Here, we introduce a fluorescence lifetime imaging microscopy (FLIM) modality to the MMF endomicroscope. We use amplitude modulation of a 405 nm single-mode light source at radio frequency (RF) and lock-in detection of autofluorescence to obtain intensity and lifetime images at sub-micrometer resolution. We experimentally demonstrate the capability of the ultrathin endomicroscope to perform label-free imaging in thick ex vivo murine submandibular gland tissue. With a temporal resolution of 0.03 ns, the FLIM images show distinguished structures of lifetime differences down to 0.5 ns. The combination of sub-micrometer fluorescence intensity and lifetime images in a minimally invasive endomicroscope opens new avenues for label-free cancer detection.

Figures (5)

• Figure 1: High-resolution FLIM endoscopy using a single MMF: The wavefront of an amplitude-modulated near-UV laser at high frequency is shaped to obtain a tight, diffraction-limited focal spot at the output of a MMF. Endogenous fluorescence is emitted from the focus spot with a small delay as a function of its lifetime compared with the modulated excitation light. The photomultiplier detects the fluorescence signal returning from the MMF, whose phase shift from the modulation reference, and therefore the fluorescence lifetime, is extracted. MMF: multimode fiber. DMD: Digital Micromirror Device. DM: dichroic mirror. PMT: photomultiplier.
• Figure 2: Minimally invasive endomicroscope: (a) Schematic of the optical setup. A 405 nm laser is externally modulated at 80 MHz using an electro-optic modulator and delivered to the system via a single-mode fiber. After wavefront shaping with a digital micromirror device, the light is coupled into the proximal side of the multimode fiber (MMF) and then focused at its distal side. Fluorescence collected through the same fiber is spectrally filtered to match the spectral bands of NADH and FAD and detected by a photomultiplier tube (PMT). (b) Tabletop MMF FLIMME system
• Figure 3: Focusing performance of the endomicroscope: (a) Histogram of focal spots enhancement in a $1225~\mu\mathrm{m}^2$ area using a $30~\mathrm{mm}$ long MMF. (b) 2D maps of focal-spot intensities (0–255; 8-bit) showing less-intense spots in the center (lower enhancement). (c,d) Example of two focal spots illustrating a round and slightly elliptical shape. Scale bar: $1~\mu\mathrm{m}$.
• Figure 4: FLIM images after calibration: (a) Coumarin C314 in solution. The average lifetime is measured at 2.89 ns, close to the value of 2.92 ns obtained with a commercial FLIM microscope on the same sample (b) Coumarin C102 dye in agarose gel. The average lifetime is 3.20 ns, close to the 3.22 ns value measured with the commercial FLIM microscope. Out of focus YG beads can be seen in the FLIM image but not in the fluorescence intensity image (c).
• Figure 5: Ex-vivo endoscopic endogenous FLIM images of ex-vivo murine submandibular gland tissue at different depth z .