Johanna Trägårdh

Multimode optical fiber (MMF) endoscopes have recently gained widespread attention as a novel tool for imaging deep within tissue using light microscopy. We here present a method for particle tracking through the MMF, which overcomes the lack of a fast enough wide-field fluorescence imaging modality for this type of endoscope, namely a discrete implementation of orbital particle tracking. We achieve biologically relevant tracking speeds (up to 1.2 μm/s) despite using a slow SLM for the wavefront shaping. We demonstrate a tracking accuracy of λ/50 for a 0.3 NA fiber and show tracking of a pinhole moving to mimic Brownian motion with diffusion rates of up to 0.3 μm²/s.

Multimode fibres show great potential for use as miniature endoscopes for imaging deep in tissue with minimal damage. When used for coherent anti-Stokes Raman scattering (CARS) microscopy with femtosecond excitation sources, a high band-width probe is required to efficiently focus the broadband laser pulses at the sample plane. Although graded-index (GRIN) fibres have a large bandwidth, it is accompanied by a strong background signal from four-wave mixing and other non-linear processes occurring inside the fibre. We demonstrate that using a composite probe consisting of a GRIN fibre with a spliced on step-index fibre reduces the intensity of the non-linear background by more than one order of magnitude without significantly decreasing the focusing performance of the probe. Using this composite probe we acquire CARS images of biologically relevant tissue such as myelinated axons in the brain with good contrast.

The interferometric acquisition of the transmission matrix (TM) of a multimode optical fibre (MMF), which is at the heart of multimode fibre-based endoscopic imaging methods, requires using a reference beam. Attempts to use an internal reference, that is to provide the reference in a common pathway geometry through the MMF itself, lead to a speckled reference intensity and consequential occurrence of “blind spots”—locations where insufficient optical power in the reference wave inflicts strong measurement errors. Here we show that combining a relatively small number of TMs, which are measured using different internal references, facilitates a complete elimination of blind spots, and thereby a significant enhancement of the imaging quality.

Multimode fiber-based endoscopes have recently emerged as a tool for minimally invasive endoscopy in tissue, at depths well beyond the reach of multiphoton imaging. Here, we demonstrate label-free second-harmonic generation (SHG) microscopy through such a fiber endoscope. We simultaneously fully control the excitation polarization state and the spatial distribution of the light at the fiber tip, and we use this to implement polarization-resolved SHG imaging, which allows imaging and identification of structural proteins such as collagen and myosin. We image mouse tail tendon and heart tissue, employing the endoscope at depths up to 1 mm, demonstrating that we can differentiate these structural proteins. This method has the potential for enabling instant and in situ diagnosis of tumors and fibrotic conditions in sensitive tissue with minimal damage.

We demonstrate a composite probe, based on a GRIN multimode fiber, where the non-linear fiber background, apparent in non-linear Raman imaging, is suppressed, while the GRIN fiber bandwidth is retained.

Advanced wavefront-shaping methods can be used to transform a simple multimode fiber into an ultra-thin laser scanning microscope. Here, we extend this technique to label-free non-linear microscopy with chemical contrast using coherent anti-Stokes Raman scattering (CARS) through a multimode fiber endoscope, which opens up new avenues for instant and in-situ diagnosis of potentially malignant tissue. We use a commercial, 125 μm diameter, 0.29 NA, GRIN fiber as the endoscopic probe. Wavefront shaping on a spatial light modulator is used to create a focus, where the 1-2 ps long pump and Stokes pulses are overlapped in time, which is scanned behind the fiber facet across the sample. The chemical selectivity is demonstrated by imaging 2 μm polystyrene and 2.5 μm PMMA beads with per pixel integration time as low as 1 ms for epi-detection. Epi-detection through the fiber is possible despite the fact that the CARS signal is emitted mainly in the forward direction, away from the fiber facet. Detecting the back-scattered signal from the underlying tissue, requires a large detector aperture to be efficient. By detecting through both the core and the cladding of the fiber, we obtain sufficient detection efficiency.

A GRIN multimode fiber (125 μm diameter, 0.29 NA) is transformed into a laser scanning microscope using wave-front shaping on an SLM. We demonstrate label-free non-linear microscopy, namely coherent anti-Stokes Raman scattering (CARS) and SHG.

Multimode fibres have recently been employed as high-resolution ultra-thin endoscopes, capable of imaging biological structures deep inside tissue in vivo. Here, we extend this technique to label-free non-linear microscopy with chemical contrast using coherent anti-Stokes Raman scattering (CARS) through a multimode fibre endoscope, which opens up new avenues for instant and in-situ diagnosis of potentially malignant tissue. We use a commercial 125 µm diameter, 0.29 NA GRIN fibre, and wavefront shaping on an SLM is used to create foci that are scanned behind the fibre facet across the sample. The chemical selectivity is demonstrated by imaging 2 µm polystyrene and 2.5 µm PMMA beads with per pixel integration time as low as 1 ms for epi-detection.

Multimode fibres have recently shown promise as miniature endoscopic probes. When used for non-linear microscopy, the bandwidth of the imaging system limits the ability to focus light from broadband pulsed lasers as well as the possibility of wavelength tuning during the imaging. We demonstrate that the bandwidth is limited by the dispersion of the off-axis hologram displayed on the SLM, which can be corrected for, and by the limited bandwidth of the fibre itself. The selection of the fibre is therefore crucial for these experiments. In addition, we show that a standard prism pulse compressor is sufficient for material dispersion compensation for multi-photon imaging with a fibre endoscope.

We demonstrate a femtosecond single pass Raman generator based on an YVO₄ crystal pumped by a high energy fiber laser at a wavelength of 1064 nm and a repetition rate of 1 MHz. The Raman generator shifts the pump wavelength to 1175 nm, in a broadband spectrum, making it suitable for multi-photon microscopy. We use the Raman generator for third harmonic generation imaging of live plant specimens as well as for two-photon fluorescence imaging of red fluorescent protein expressing HeLa cells. We demonstrate that the photo-damage to a live specimen is low.