Harmonic Imaging of Stem Cells in Whole Blood at GHz Pixel Rate.

The pre-clinical validation of cell therapies requires monitoring the biodistribution of transplanted cells in tissues of host organisms. Real-time detection of these cells in the circulatory system and identification of their aggregation state is a crucial piece of information, but necessitates deep penetration and fast imaging with high selectivity, subcellular resolution, and high throughput. In this study, multiphoton-based in-flow detection of human stem cells in whole, unfiltered blood is demonstrated in a microfluidic channel. The approach relies on a multiphoton microscope with diffractive scanning in the direction perpendicular to the flow via a rapidly wavelength-swept laser. Stem cells are labeled with metal oxide harmonic nanoparticles. Thanks to their strong and quasi-instantaneous second harmonic generation (SHG), an imaging rate in excess of 10 000 frames per second is achieved with pixel dwell times of 1 ns, a duration shorter than typical fluorescence lifetimes yet compatible with SHG. Through automated cell identification and segmentation, morphological features of each individual detected event are extracted and cell aggregates are distinguished from isolated cells. This combination of high-speed multiphoton microscopy and high-sensitivity SHG nanoparticle labeling in turbid media promises the detection of rare cells in the bloodstream for assessing novel cell-based therapies.

Pulsed swept-source FDML-MOPA laser with kilowatt picosecond pulses around 1550†nm.

Swept-source lasers are versatile light sources for spectroscopy, imaging, and microscopy. Swept-source-powered multiphoton microscopy can achieve high-speed, inertia-free point scanning with MHz line-scan rates. The recently introduced spectro-temporal laser imaging by diffractive excitation (SLIDE) technique employs swept-source lasers to achieve kilohertz imaging rates by using a swept-source laser in combination with a diffraction grating for point scanning. Multiphoton microscopy at a longer wavelength, especially in the shortwave infrared (SWIR) region, can have advantages in deep tissue penetration or applications in light detection and ranging (LiDAR). Here we present a swept-source laser around 1550 nm providing high-speed wavelength agility and high peak power pulses for nonlinear excitation. The swept-source laser is a Fourier-domain mode-locked (FDML) laser operating at 326 kHz sweep rate. For high peak powers, the continuous wave (cw) output is pulse modulated to short picosecond pulses and amplified using erbium-doped fiber amplifiers (EDFAs) to peak powers of several kilowatts. This FDML-master oscillator power amplifier (FDML-MOPA) setup uses reliable, low-cost fiber components. As proof-of-principle measurement, we show third-harmonic generation (THG) using harmonic nanoparticles at the 10 MHz pulse excitation rate. This new, to the best of our knowledge, laser source provides unique performance parameters for applications in nonlinear microscopy, spectroscopy, and ranging.

Four-wave mixing seeded by a rapid wavelength-sweeping FDML laser for nonlinear imaging at 900 nm and 1300 nm.

Four-wave mixing (FWM) enables the generation and amplification of light in spectral regions where suitable fiber gain media are unavailable. The 1300 nm and 900 nm regions are of especially high interest for time-encoded (TICO) stimulated Raman scattering microscopy and spectro-temporal laser imaging by diffracted excitation (SLIDE) two-photon microscopy. We present a new, to the best of our knowledge, FWM setup where we shift the power of a home-built fully fiber-based master oscillator power amplifier (MOPA) at 1064 nm to the 1300-nm region of a rapidly wavelength-sweeping Fourier domain mode-locked (FDML) laser in a photonic crystal fiber (PCF) creating pulses in the 900-nm region. The resulting 900-nm light can be wavelength swept over 54 nm and has up to 2.5 kW (0.2 µJ) peak power and a narrow instantaneous spectral linewidth of 70 pm. The arbitrary pulse patterns of the MOPA and the fast wavelength tuning of the FDML laser (419 kHz) allow it to rapidly tune the FWM light enabling new and faster TICO-Raman microscopy, SLIDE imaging, and other applications.