Fiber-optic fluorescence imaging

Abstract

Optical fibers guide light between separate locations and enable new types of fluorescence imaging. Fiber-optic fluorescence imaging systems include portable handheld microscopes, flexible endoscopes well suited for imaging within hollow tissue cavities and microendoscopes that allow minimally invasive high-resolution imaging deep within tissue. A challenge in the creation of such devices is the design and integration of miniaturized optical and mechanical components. Until recently, fiber-based fluorescence imaging was mainly limited to epifluorescence and scanning confocal modalities. Two new classes of photonic crystal fiber facilitate ultrashort pulse delivery for fiber-optic two-photon fluorescence imaging. An upcoming generation of fluorescence imaging devices will be based on microfabricated device components.

Figure 1

Photographs of photonic crystal fibers. (a) Hollow-core PBF. Wavelengths within the transmission band are localized to the air core, virtually eliminating SPM. (b) Large-mode area photonic crystal fiber (LMA-PCF). Light guidance in the silica core is ‘endlessly single mode’ and the large mode area reduces SPM. (c) Double-clad photonic crystal fiber. A silica LMA core and a surrounding airsilica inner cladding comprise an inner lower-NA fiber that can guide ultrashort pulses with reduced SPM. An outer cladding composed nearly exclusively of air creates a higher-NA fiber that can collect fluorescence efficiently. Acrylate structural support surrounds the outer cladding and is not fully shown. (d) Highly nonlinear photonic crystal fiber. The small silica core can be used to harness SPM for broadening the spectrum of ultrashort excitation pulses. Scale bars, 20 μm. Photographs kindly provided by R. Kristiansen (Crystal Fibre A/S).

Figure 2

Scanning mechanisms. (a) Proximal scanning. Cascaded galvanometer-mounted mirrors scan the excitation beam across the proximal end of a fiber bundle. (b) Proximal line-scanning. A cylindrical lens focuses the illumination to a line that is scanned across the face of a fiber bundle in one dimension. (c) Proximal scanning with a spatial light modulator, which can illuminate pixels sequentially without sweeping the beam. (d) Distal 2D mirror scanning. A piezoelectric driven tip-tilt mirror or a miniaturized MEMS mirror pivots in two angular dimensions. (e) Distal fiber tip scanning. The tip of the excitation delivery fiber is vibrated at resonance by an actuator (not shown). (f) Distal fiber-objective scanning. Both the fiber and the objective lens are mounted together on a cantilever (not shown) that is vibrated at resonance by an actuator.

Figure 3

An MEMS scanner. (a) Scanning electron micrograph of 2D MEMS vertical comb scanner. Electrostatically driven angular vertical comb actuators rotate the 1-mm-diameter mirror about the torsion beams. Gimbal mounting of the mirror and actuators allows 2D scanning. Scale bar, 500 μm. (b) Scanning electron micrograph of vertical comb actuators. Scale bar, 100 μm. Material is this figure is based on work by Piyawattanametha et al.

Figure 4

Fiber-optic fluorescence imaging embodiments. (a) One-photon FME using a GRIN microendoscope probe. Visible illumination is coupled into the probe, which focuses the light onto the sample. Fluorescence returns through the probe. (b) One-photon epifluorescence imaging using a fiber bundle. Illumination travels through the fiber bundle to a small objective or GRIN lens, which focuses the light onto the sample. Fluorescence returns through the bundle. (c) Confocal imaging using SMF. The SMF delivers illumination to lenses that collimate and focus the light onto the specimen. The SMF core also serves as a pinhole for collecting in focus but rejecting out-of-focus fluorescence emissions. For microscopy, this embodiment generally relies on galvanometer scanning mirrors, which would be located between the fiber and lenses. For flexible endoscopy, the miniaturized distal scanning mechanisms are appropriate. (d) Dual-axis confocal microscopy. One SMF delivers excitation light and a second SMF, mounted at an angle with respect to the first, collects fluorescence from the overlapping region of the two fiber apertures. (e) Fiber bundle confocal imaging. Visible excitation light is scanned across a fiber bundle. A miniaturized objective or GRIN lens focuses the light onto the sample. Fluorescence returns through the probe and is routed to a pinhole detector. (f) Two-photon FME with a GRIN microendoscope probe. Ultrashort pulses are coupled into the probe, which focuses the light onto the sample. Fluorescence returns back through the probe and is routed to a photodetector. (g) Single-fiber two-photon imaging. Ultrashort pulses exiting a single fiber are scanned in 2D before entering a miniaturized objective or GRIN lens, which focuses the near-infrared excitation pulses onto the sample. A coated microprism can serve as a dichroic mirror. A large-core multimode fiber collects fluorescence. (h) Fiber bundle two-photon imaging. Ultrashort pulses are scanned across the proximal end of a fiber bundle. A miniature objective or GRIN lens focuses the light onto the sample. Fluorescence returns through the bundle to a detector. (i) Multifocal two-photon imaging. A single fiber delivers the excitation light to a collimating lens. A micro-lens array divides the beam into multiple ‘beamlets’, which are scanned in 2D and focused onto the sample. In all panels, blue represents visible fluorescence excitation light, red represents ultrashort-pulsed near-infrared light for two-photon excitation, and green represents visible fluorescence emission. Arrows show the directions of light propagation. Detectors and cameras are omitted. Scanners and dichroic mirrors are not shown except where explicitly labeled.