A wavelength agile 900 nm 2.5 kW peak power fiber laser is created by four-wave mixing (FWM) in a photonic crystal fiber (PCF), while amplifying a 1300 nm Fourier-domain mode-locked (FDML) laser. The FWM process is pumped by a home-built 1064 nm master oscillator power amplifier (MOPA) laser and seeded by a home-built 1300 nm FDML laser, generating high power pulses at wavelengths, where amplification by active fiber media is difficult. The 900 nm pulses have a spectral linewidth of 70 pm, are tunable over 54 nm and have electronic pulse-to-pulse tuning capability. These pulses can be used for nonlinear imaging like two-photon or coherent anti-Stokes Raman microscopy (CARS) microscopy including spectro-temporal laser imaging by diffracted excitation (SLIDE) and time-encoded (Tico) stimulated Raman microscopy.
We demonstrate a 3.3 MHz A-scan rate OCT for rapid scanning of large areas of human skin. The mosaicking performance and different OCT imaging modalities including intervolume speckle contrast are evaluated.
We present a combination of sub-nanosecond two-photon microscopy (TPM) and megahertz-rate optical coherence tomography (MHz-OCT) via a double ferrule for future endoscopic setups. The double ferrule combines a Hi1060 fiber of the OCT system and a double cladding (DC) fiber of the TPM setup. The use of sub-nanosecond pulses for TPM simplifies the setup substantially as no dispersion management is required. The inner cladding of the DC fiber collects the fluorescent light. The double ferrule was tested in a microscope setup. We characterized our system and collected first imaging data.
In Fourier domain mode locked (FDML) lasers, extremely precise and stable matching of the filter tuning period and light circulation time in the cavity is essential for ultra-low noise operation. During the operation of FDML lasers, the ultra-low noise mode can be lost due to temperature drifts of the already temperature stabilized cavity resulting in increased intensity noise. Until now, the filter frequency is continuously regulated to match the changing light circulation time. However, this causes the filter frequency to constantly change by a few mHz and leads to synchronization issues in cases where a fixed filter frequency is desired. We present an actively cavity length controlled FDML laser and a robust and high precision feedback loop algorithm for maintaining ultra-low noise operation. Instead of adapting the filter frequency, the cavity length is adjusted by a motorized free space beam path to match the fixed filter frequency. The closed-loop system achieves a stability of ~0.18 mHz at a sweep repetition rate of ~418 kHz which corresponds to a ratio of 4×10-10. We investigate the coherence properties during the active cavity length adjustments and observe no noise increase compared to fixed cavity length. The cavity length control is fully functional and for the first time, offers the possibility to operate an FDML laser in sweet spot mode at a fixed frequency or phase locked to an external clock. This opens new possibilities for system integration of FDML lasers.
Fourier domain mode locked (FDML) laser are fast swept light sources. Measuring the linewidth and coherence length of such light sources is not straightforward, but very important for a physical understanding of FDML lasers and their performance in optical coherence tomography (OCT). In order to characterize the dynamic (“instantaneous”) linewidth, we performed beat signal measurements between a stationary narrowband continuous wave laser and an FDML laser and detected the signals with a 63 GHz real time oscilloscope. The evaluation of the beat signals of consecutive FDML wavelength sweeps yields information about the phase evolution within one sweep and over several sweeps. These measurements suggest the existence of a distinct comb like mode structure of the FDML laser and help to determine the locking strength of individual modes (comb lines).
The routine pathology workflow relies on cutting tissue into single-cell layer thick slices using paraffin or frozen sectioning. We propose a fast method to obtain sections of equivalent quality optically using the strong sectioning capabilities of two-photon microscopy (TPM). Hematoxylin and eosin (HE) equivalent staining of the tissue is achieved using acridine orange and sulforhodamine 101. We improved our previously presented pulsed fiber laser to deliver adjustable pulse durations of ~30ps at repetition rates of up to 16MHz and kW peak power. We can now image up to one square centimeter of tissue with sub-micrometer resolution within 15 minutes.
Optical coherence tomography (OCT) applications like ultra-widefield and full eye-length imaging are of high interest for various diagnostic purposes. In swept-source OCT these techniques require a swept light source, which is coherent over the whole imaging depth. We present a zero roll-off 1060 nm Fourier Domain Mode Locked-Laser (FDML-Laser) for retinal OCT imaging at 1.7 MHz A-scan rate and first long-range imaging results with it. Several steps such as improved dispersion compensation and frequency regulation were performed and will be discussed. Besides virtually no loss in OCT signal over the maximum depth range of 4.6 mm and very good dynamic range was observed. Roll-off measurements show no decrease of the point-spread function (PSF), while maintaining a high dynamic range.
Two-Photon Microscopy (TPM) can provide three-dimensional morphological and functional contrast in vivo. Through proper staining, TPM can be utilized to create virtual, HE equivalent images and thus can improve throughput in histology-based applications. We previously reported on a new light source for TPM that employs a compact and robust fiber-amplified, directly modulated laser. This laser is pulse-to-pulse wavelength switchable between 1064 nm, 1122 nm, and 1186 nm with an adjustable pulse duration from 50ps to 5ns and arbitrary repetition rates up to 1MHz at kW-peak powers. Despite the longer pulse duration, it can achieve similar average signal levels compared to fs-setups by lowering the repetition rate to achieve similar cw and peak power levels. The longer pulses lead to a larger number of photons per pulse, which yields single shot fluorescence lifetime measurements (FLIM) by applying a fast 4 GSamples/s digitizer. In the previous setup, the wavelengths were limited to 1064 nm and longer. Here, we use four wave mixing in a non-linear photonic crystal fiber to expand the wavelength range down to 940 nm. This wavelength is highly suitable for imaging green fluorescent proteins in neurosciences and stains such as acridine orange (AO), eosin yellow (EY) and sulforhodamine 101 (SR101) used for histology applications. In a more compact setup, we also show virtual HE histological imaging using a direct 1030 nm fiber MOPA.
We present a new design of a 1060nm Fourier Domain Mode Locked-Laser (FDML-Laser) that combines 1.67 MHz A-scan rate with a centimeter scale coherence length. The extended coherence length is achieved by synchronizing the cavity roundtrip time over the 75 nm sweep with a relative accuracy of 10-7. We will show that this requires careful combination of multiple fiber types in the cavity with a gradient heated chirped Fiber Bragg grating.
Newly developed microscopy methods have the goal to give researches in bio-molecular science a better understanding of processes ongoing on a cellular level. Especially two-photon excited fluorescence (TPEF) microscopy is a readily applied and widespread modality. Compared to one photon fluorescence imaging, it is possible to image not only the surface but also deeper lying structures. Together with fluorescence lifetime imaging (FLIM), which provides information on the chemical composition of a specimen, deeper insights on a molecular level can be gained. However, the need for elaborate light sources for TPEF and speed limitations for FLIM hinder an even wider application. In this contribution, we present a way to overcome this limitations by combining a robust and inexpensive fiber laser for nonlinear excitation with a fast analog digitization method for rapid FLIM imaging. The applied sub nanosecond pulsed laser source is perfectly suited for fiber delivery as typically limiting non-linear effects like self-phase or cross-phase modulation (SPM, XPM) are negligible. Furthermore, compared to the typically applied femtosecond pulses, our longer pulses produce much more fluorescence photons per single shot. In this paper, we show that this higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to not only use a single pulse per pixel for TPEF imaging but also to resolve the exponential time decay for FLIM. To evaluate our system, we acquired FLIM images of a dye solution with single exponential behavior to assess the accuracy of our lifetime determination and also FLIM images of a plant stem at a pixel rate of 1 MHz to show the speed performance of our single pulse two-photon FLIM (SP-FLIM) system.
Over the last 20 years, optical coherence tomography (OCT) has become a valuable diagnostic tool in ophthalmology with several 10,000 devices sold today. Other applications, like intravascular OCT in cardiology and gastro-intestinal imaging will follow. OCT provides 3-dimensional image data with microscopic resolution of biological tissue in vivo. In most applications, off-line processing of the acquired OCT-data is sufficient. However, for OCT applications like OCT aided surgical microscopes, for functional OCT imaging of tissue after a stimulus, or for interactive endoscopy an OCT engine capable of acquiring, processing and displaying large and high quality 3D OCT data sets at video rate is highly desired.
We developed such a prototype OCT engine and demonstrate live OCT with 25 volumes per second at a size of 320x320x320 pixels. The computer processing load of more than 1.5 TFLOPS was handled by a GTX 690 graphics processing unit with more than 3000 stream processors operating in parallel. In the talk, we will describe the optics and electronics hardware as well as the software of the system in detail and analyze current limitations. The talk also focuses on new OCT applications, where such a system improves diagnosis and monitoring of medical procedures. The additional acquisition of hyperspectral stimulated Raman signals with the system will be discussed.
In order to realize fast OCT-systems with adjustable line rate, we investigate averaging of image data from an FDML based
MHz-OCT-system. The line rate can be reduced in software and traded in for increased system sensitivity and image
quality. We compare coherent and incoherent averaging to effectively scale down the system speed of a 3.2 MHz FDML
OCT system to around 100 kHz in postprocessing. We demonstrate that coherent averaging is possible with MHz systems
without special interferometer designs or digital phase stabilisation. We show OCT images of a human finger knuckle joint
in vivo with very high quality and deep penetration.
We present a new design of a Fourier Domain Mode Locked laser (FDML laser), which provides a new record in sweep
range at ~1μm center wavelength: At the fundamental sweep rate of 2x417 kHz we reach 143nm bandwidth and 120nm
with 4x buffering at 1.67MHz sweep rate. The latter configuration of our system is characterized: The FWHM of the
point spread function (PSF) of a mirror is 5.6μm (in tissue). Human in vivo retinal imaging is performed with the MHz
laser showing more details in vascular structures. Here we could measure an axial resolution of 6.0μm by determining
the FWHM of specular reflex in the image. Additionally, challenges related to such a high sweep bandwidth such as
water absorption are investigated.
We present full volumetric high speed OCT imaging of the retina with multiple settings varying in volume size and volume rate. The volume size ranges from 255x255 A-scans to 160x40 A-scans with 450 samples per depth scan with volume rates varying between 20.8 V/s for the largest volumes to 195.2 V/s for the smallest. The system is based on a 1060nm Fourier domain mode locked (FDML) laser with 1.6MHz line rate. Scanning along the fast axis is performed with a 2.7 kHz or 4.3 kHz resonant scanner operated in bidirectional scanning mode, while a standard galvo scanner is used for the slow axis. The performance is analyzed with respect to various potential applications, like intraoperative OCT.
OCT as a coherent imaging technique inherently suffers from speckle. We present a new dual parametric compounding approach to reduce speckle. The approach is to acquire several OCT volumes with different numerical apertures (NAs). Then in post processing, a first spatial compounding step is performed by averaging of adjacent B-frames. In a second step data from the different volume is averaged. Retinal imaging data comparing this idea with standard spatial compounding is presented and analyzed and necessary parameters such as the required variation of the NA and number of different NAs are discussed
Full volumetric high speed OCT imaging of the retina with multiple settings varying in volume size and volume rate is
presented. The volume size ranges from 255x255 A-scans to 160x40 A-scans with 450 samples per depth scan. The
volume rates vary between 20.8 V/s for the largest volumes to 195.2 V/s for the smallest. The system is based on a
1060nm Fourier domain mode locked (FDML) laser with 1.6MHz line rate. Scanning along the fast axis is performed
with a 2.7 kHz or 4.3 kHz resonant scanner operated in bidirectional scanning mode, while a standard galvo scanner is
used for the slow axis. The performance is analyzed with respect to various potential applications, like intraoperative
OCT.
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