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.

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.

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.

Time-encoded stimulated Raman scattering microscopy of tumorous human pharynx tissue in the fingerprint region from 1500-1800 cm-1.

Stimulated Raman scattering (SRS) microscopy for biomedical analysis can provide a molecular localization map to infer pathological tissue changes. Compared to spontaneous Raman, SRS achieves much faster imaging speeds at reduced spectral coverage. By targeting spectral features in the information dense fingerprint region, SRS allows fast and reliable imaging. We present time-encoded (TICO) SRS microscopy of unstained head-and-neck biopsies in the fingerprint region with molecular contrast. We combine a Fourier-domain mode-locked (FDML) laser with a master oscillator power amplifier (MOPA) to cover Raman transitions from 1500-1800cm-1. Both lasers are fiber-based and electronically programmable making this fingerprint TICO system robust and reliable. The results of our TICO approach were cross-checked with a spontaneous Raman micro-spectrometer and show good agreement, paving the way toward clinical applications.

Frequency-doubled FDML-MOPA laser in the visible.

Wavelength-swept lasers enable high-speed measurements in absorption spectroscopy, Raman spectroscopy, nonlinear Raman hyperspectral microscopy, rapid confocal microscopy, short impulse generation, and most importantly for high-speed optical coherence tomography, with speeds up to video-rate volumetric imaging. Recently, we introduced a pulsed wavelength-swept laser based on the Fourier domain mode-locked (FDML) laser principle combined with a master-oscillator power amplifier (MOPA) architecture. The high peak powers reached with this laser enabled rapid two-photon microscopy and two-photon fluorescence lifetime microscopy and high-speed light detection and ranging measurements. Here, we present the extension of this laser into the visible wavelength range by frequency doubling the output from 1064 nm to 532 nm via second harmonic generation in a deuterated potassium dihydrogen phosphate crystal. The result is a wavelength-swept laser source around 532 nm that outputs a pulse train of distinct, almost monochromatic wavelengths at an 88 MHz pulse repetition rate and 342 kHz sweep repetition rate. This swept-source laser in the visible can open up new research applications in spectroscopy, metrology, sensing, and high-speed imaging.

Fourier-domain mode-locked laser combined with a master-oscillator power amplifier architecture.

Originally introduced in 2005 for high-speed optical coherence tomography, the rapidly wavelength-swept Fourier-domain mode-locked (FDML) laser still, to this day, enables highest imaging speeds through a very high-speed spectral tuning capability. The FDML laser achieves a tuning bandwidth of over 1/10th of its center wavelength and can sweep this entire bandwidth in less than a microsecond. Interestingly, even though it covers a very broad spectral range, instantaneously it has a narrow spectral linewidth that puts it in a unique space compared to other high-speed broadband laser sources, e.g., mode-locked lasers or supercontinuum sources. Although it has been applied for nonlinear Raman spectroscopy and imaging, a current drawback of this continuous wave laser is the relatively low instantaneous power of 10-100 mW. Here, we report the combination of an FDML laser with a master oscillator power amplifier (MOPA) architecture to increase the instantaneous power of the FDML for nonlinear optical interactions. The output of an FDML laser around 1060 nm is modulated to short pulses by using an electro-optic amplitude modulator and subsequently amplified using ytterbium-doped fiber amplifiers (YDFAs). This generates a spectral rainbow of 65 picosecond pulses, where each pulse has a distinct, monochromatic wavelength. The instantaneous power can be adjusted by the YDFAs to reach nonlinear optical excitation regimes. This wavelength-swept FDML-MOPA laser will have a vast range of applications in, e.g., nonlinear optics, spectroscopy, imaging, and sensing.

Pulse-to-pulse wavelength switching of a nanosecond fiber laser by four-wave mixing seeded stimulated Raman amplification.

We report on a multi-color fiber laser based on four-wave mixing (FWM) and stimulated Raman scattering (SRS), delivering rapidly wavelength switchable narrowband output at 1064, 1122, and 1186 nm. High-power pulses from a nanosecond pulsed fiber master oscillator power amplifier at 1064 nm are combined with 1122 nm of seed light for Raman amplification at the first Stokes order in a standard single-mode fiber. With increasing power, we observe a narrowband spectral component at 1186 nm, without any additional seed or resonator at this wavelength. We analyze this occurrence of a narrowband second Stokes order both experimentally and theoretically and suggest it is a result of FWM seeding of the SRS amplification in the fiber. We demonstrate that the wavelength shifting can be controlled electronically within microseconds for very rapid and even pulse-to-pulse wavelength changes. This wavelength conversion method can extend the spectral coverage of single-wavelength fiber lasers for biomedical imaging.

Flexible A-scan rate MHz-OCT: efficient computational downscaling by coherent averaging.

In order to realize adjustable A-scan rates of fast optical coherence tomography (OCT) systems, we investigate averaging of OCT image data acquired with a MHz-OCT system based on a Fourier Domain Mode Locked (FDML) laser. Increased system sensitivity and image quality can be achieved with the same system at the cost of lower imaging speed. Effectively, the A-scan rate can be reduced in software by a freely selectable factor. We demonstrate a detailed technical layout of the strategies necessary to achieve efficient coherent averaging. Since there are many new challenges specific to coherent averaging in swept source MHz-OCT, we analyze them point by point and describe the appropriate solutions. We prove that coherent averaging is possible at MHz OCT-speed without special interferometer designs or digital phase stabilization. We find, that in our system up to ∼100x coherent averaging is possible while achieving a sensitivity increase close to the ideal values. This corresponds to a speed reduction from 3.3 MHz to 33 kHz and a sensitivity gain of 20 dB. We show an imaging comparison between coherent and magnitude averaging of a human finger knuckle joint in vivo with 121 dB sensitivity for the coherent case. Further, the benefits of computational downscaling in low sensitivity MHz-OCT systems are analyzed.

Spectro-temporal encoded multiphoton microscopy and fluorescence lifetime imaging at kilohertz frame-rates.

Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration. In terms of imaging speeds, however, mechanical scanners still limit the acquisition rates to typically 10-100 frames per second. Here we present a high-speed non-linear microscope achieving kilohertz frame rates by employing pulse-modulated, rapidly wavelength-swept lasers and inertia-free beam steering through angular dispersion. In combination with a high bandwidth, single-photon sensitive detector, this enables recording of fluorescent lifetimes at speeds of 88 million pixels per second. We show high resolution, multi-modal - two-photon fluorescence and fluorescence lifetime (FLIM) - microscopy and imaging flow cytometry with a digitally reconfigurable laser, imaging system and data acquisition system. These high speeds should enable high-speed and high-throughput image-assisted cell sorting.

Wavelength agile multi-photon microscopy with a fiber amplified diode laser.

Multi-photon microscopy is a powerful tool in biomolecular research. Less complex and more cost effective excitation light sources will make this technique accessible to a broader community. Semiconductor diode seeded fiber lasers have proven to be especially robust, low cost and easy to use. However, their wavelength tuning range is often limited, so only a limited number of fluorophores can be accessed. Therefore, different approaches have been proposed to extend the spectral coverage of these lasers. Recently, we showed that four-wave mixing (FWM) assisted stimulated Raman scattering (SRS) can be harnessed to red-shift high power pulses from 1064 nm to a narrowband output at 1122 nm and 1186 nm and therefore extend the number of accessible fluorophores. In this contribution, we show the applicability of all three wavelengths for multi-photon microscopy and analyze the performance.

Single pulse two photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate.

Two-photon-excited fluorescence lifetime imaging microscopy (FLIM) is a chemically specific 3-D sensing modality providing valuable information about the microstructure, composition and function of a sample. However, a more widespread application of this technique is hindered by the need for a sophisticated ultra-short pulse laser source and by speed limitations of current FLIM detection systems. To overcome these limitations, we combined a robust sub-nanosecond fiber laser as the excitation source with high analog bandwidth detection. Due to the long pulse length in our configuration, more fluorescence photons are generated per pulse, which allows us to derive the lifetime with a single excitation pulse only. In this paper, we show high quality FLIM images acquired at a pixel rate of 1 MHz. This approach is a promising candidate for an easy-to-use and benchtop FLIM system to make this technique available to a wider research community.

Two-photon microscopy using fiber-based nanosecond excitation.

Two-photon excitation fluorescence (TPEF) microscopy is a powerful technique for sensitive tissue imaging at depths of up to 1000 micrometers. However, due to the shallow penetration, for in vivo imaging of internal organs in patients beam delivery by an endoscope is crucial. Until today, this is hindered by linear and non-linear pulse broadening of the femtosecond pulses in the optical fibers of the endoscopes. Here we present an endoscope-ready, fiber-based TPEF microscope, using nanosecond pulses at low repetition rates instead of femtosecond pulses. These nanosecond pulses lack most of the problems connected with femtosecond pulses but are equally suited for TPEF imaging. We derive and demonstrate that at given cw-power the TPEF signal only depends on the duty cycle of the laser source. Due to the higher pulse energy at the same peak power we can also demonstrate single shot two-photon fluorescence lifetime measurements.

A Time-Encoded Technique for fibre-based hyperspectral broadband stimulated Raman microscopy.

Raman sensing and microscopy are among the most specific optical technologies to identify the chemical compounds of unknown samples, and to enable label-free biomedical imaging. Here we present a method for stimulated Raman scattering spectroscopy and imaging with a time-encoded (TICO) Raman concept. We use continuous wave, rapidly wavelength-swept probe lasers and combine them with a short-duty-cycle actively modulated pump laser. Hence, we achieve high stimulated Raman gain signal levels, while still benefitting from the narrow linewidth and low noise of continuous wave operation. Our all-fibre TICO-Raman setup uses a Fourier domain mode-locked laser source to achieve a unique combination of high speed, broad spectral coverage (750-3,150 cm(-1)) and high resolution (0.5 cm(-1)). The Raman information is directly encoded and acquired in time. We demonstrate quantitative chemical analysis of a solvent mixture and hyperspectral Raman microscopy with molecular contrast of plant cells.

High definition live 3D-OCT in vivo: design and evaluation of a 4D OCT engine with 1 GVoxel/s.

We present a 1300 nm OCT system for volumetric real-time live OCT acquisition and visualization at 1 billion volume elements per second. All technological challenges and problems associated with such high scanning speed are discussed in detail as well as the solutions. In one configuration, the system acquires, processes and visualizes 26 volumes per second where each volume consists of 320 x 320 depth scans and each depth scan has 400 usable pixels. This is the fastest real-time OCT to date in terms of voxel rate. A 51 Hz volume rate is realized with half the frame number. In both configurations the speed can be sustained indefinitely. The OCT system uses a 1310 nm Fourier domain mode locked (FDML) laser operated at 3.2 MHz sweep rate. Data acquisition is performed with two dedicated digitizer cards, each running at 2.5 GS/s, hosted in a single desktop computer. Live real-time data processing and visualization are realized with custom developed software on an NVidia GTX 690 dual graphics processing unit (GPU) card. To evaluate potential future applications of such a system, we present volumetric videos captured at 26 and 51 Hz of planktonic crustaceans and skin.

Extended coherence length megahertz FDML and its application for anterior segment imaging.

We present a 1300 nm Fourier domain mode locked (FDML) laser for optical coherence tomography (OCT) that combines both, a high 1.6 MHz wavelength sweep rate and an ultra-long instantaneous coherence length for rapid volumetric deep field imaging. By reducing the dispersion in the fiber delay line of the FDML laser, the instantaneous coherence length and hence the available imaging range is approximately quadrupled compared to previously published MHz-FDML setups, the imaging speed is increased by a factor of 16 compared to previous extended coherence length results. We present a detailed characterization of the FDML laser performance. We demonstrate for the first time MHz-OCT imaging of the anterior segment of the human eye. The OCT system provides enough imaging depth to cover the whole range from the top surface of the cornea down to the crystalline lens.