Hoseong Song

Two-photon microscopy using an Yb 3+ -doped fiber laser with variable pulse widths

Most of the two-photon fluorescence microscopes are based on femtosecond Ti:Sapphire laser sources near the 800 nm wavelength. Here, we introduce a new confocal two-photon microscope system using a mode-locked Yb(3+)-doped fiber laser. The mode-locked fiber laser produces 13 ps pulses with large positive chirping at a repetition rate of 36 MHz with an average power of 80 mW. By using an external grating pair pulse compressor, the pulse width and the frequency chirping of the laser output are controlled for optimum two-photon excitation. For a given objective lens, the optimum condition was obtained by monitoring the two-photon-induced-photocurrent in a GaAsP photodiode at the sample position. The performance of this pulse width optimized two-photon microscope system was demonstrated by imaging Vybrant DiI-stained dorsal root ganglion cells in 2 and 3 dimensions.

Self-starting passive mode-locked ytterbium fiber laser with variable pulse width

We report a scheme for controlling pulse width in a robust self-starting mode-locked ytterbium fiber laser using a semiconductor saturable absorber mirror (SESAM). We demonstrate that the pulse width in a mode-locked laser made of all-normal-dispersive fiber can be adjusted by changing ump power to the laser or by adjusting the axial position of the SESAM with respect to a focusing beam. We have obtained optical pulse width of 7.4 ps and the adjustable range was 2 ps without dispersion compensators in the all-normal-dispersive cavity and provides a high reliability of turn-key operation. We have explained that the principle of position dependent pulse width change in a mode-locked laser with a SESAM and verified with numerical simulations.

Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope

High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail.

A custom-built two-photon microscope based on a mode-locked Yb3+ doped fiber laser

Two-photon microscopy is a very attractive tool for the study of the three-dimensional (3D) and dynamic processes in cells and tissues. One of the feasible constructions of two-photon microscopy is the combination a confocal laser scanning microscope and a mode-locked Ti:sapphire laser. Even though this approach is the simplest and fastest implementation, this system is highly cost-intensive and considerably difficult in modification. Many researcher therefore decide to build a more cost-effective and flexible system with a self-developed software for operation and data acquisition. We present a custom-built two-photon microscope based on a mode-locked Yb3+ doped fiber laser and demonstrate two-photon fluorescence imaging of biological specimens. The mode-locked fiber laser at 1060 nm delivers 320 fs laser pulses at a frequency of 36 MHz up to average power of 80 mW. The excitation at 1060 nm can be more suitable in thick, turbid samples for 3D image construction as well as cell viability. The system can simply accomplish confocal and two-photon mode by an additional optical coupler that allows conventional laser source to transfer to the scanning head. The normal frame rate is 1 frames/s for 400 x 400 pixel images. The measured full width at half maximum resolutions were about 0.44 μm laterally and 1.34 μm axially. A multi-color stained convallaria, rat basophilic leukemia cells and a rat brain tissue were observed by two-photon fluorescence imaging in our system.

Ultra-high-speed phase-sensitive optical coherence reflectometer with a stretched pulse supercontinuum source

We demonstrate an ultra-high-speed phase-sensitive time-wavelength-domain optical coherence reflectometer with a stretched pulse supercontinuum source. A pulsed fiber laser operating at 10 MHz repetition rate was used to generate a pulsed supercontinuum of 30 ps pulse duration by using a nonlinear optical fiber. The supercontinuum pulses are stretched into 70 ns pulses with a highly dispersive fiber. With this stretched pulse source, we have built a phase-sensitive optical coherence reflectometer that measures the spectral interferogram of reflected light. By using the linear relation between the wavelength and the temporal position in a linearly chirped pulse, ultra-high-speed spectrum measurement can be obtained with this method in the time domain. We have demonstrated ultra-high-speed two-dimensional surface profiling for a standard image target and high-speed single-point monitoring for a fixed point under vibrational motion. It is shown that the measurement speed for the position of a single point can be as fast as 2.5 MHz, while the position accuracy can be better than 4.49 nm.

Phase-sensitive optical coherence reflectometer using a supercontinuum source

We report a high-speed phase-sensitive optical coherence reflectometer (OCR) with a stretched supercontinuum source. Firstly, supercontinuum source has been generated by injecting an amplified fiber laser pulses into a highly nonlinear optical fiber. The repetition rate and pulse duration of the generated supercontinuum source are 10 MHz and 30 ps respectively. The supercontinuum pulses are stretched into 70 ns pulses with a dispersion-compensating fiber (DCF). This pulse stretching technique enables us to measure the spectral information in the time domain. The relationship of time-wavelength has been measured by modified time-of-flight method. We have built a phase-sensitive OCR with this stretched pulse source and a two-dimensional (2D) scanning system. The displacement sensitivity of our proposed system has been investigated. We have demonstrated high-speed 2D imaging capability and single-point dynamics measurement performance of our proposed system.

Photobleaching Property of Confocal Laser Scanning Microscopy with Masked Illumination

Confocal laser scanning microscopy (CLSM) has become the tool of choice for high-contrast fluorescence imaging in the study of the three-dimensional and dynamic properties of biological system. However, the high cost and complexity of commercial CLSMs urges many researchers to individually develop low cost and flexible confocal microscopy systems. The high speed scanner is an influential factor in terms of cost and system complexity. Resonant galvo scanners at several kHz have been commonly used in custom-built CLSMs. However, during the repeated illumination for live cell imaging or 3D image formation, photobleaching and image distortion occurred at the edges of the scan field may be more serious than the center due to an inherent property (e.g. sinusoidal angular velocity) of the scan mirror. Usually, no data is acquired at the edges due to large image distortion but the excitation beam is still illuminated. Here, we present the photobleaching property of CLSM with masked illumination, a simple and low cost method, to exclude the unintended excitation illumination at the edges. The mask with a square hole in its center is disposed at the image plane between the scan lens and the tube lens in order to decrease photobleaching and image distortion at the edges. The excluded illumination section is used as the black level of the detected signals for a signal quantizing step. Finally, we demonstrated the reduced photobleaching at the edges on a single layer of fluorescent beads and real-time image acquisition without a standard composite video signal by using a frame grabber.

Coherence measurements of supercontinuum source based on a fiber laser and highly nonlinear dispersion shifted fiber

We constructed a passively mode-locked Er-doped fiber laser (PML-EDFL). It generates ~ 1.3 ps pulses at a repetition rate of 12 MHz with an average output power of 0.7 mW. These pulses are amplified in a short Er-doped fiber amplifier (EDFA) which is composed of low nonlinearity EDF. The average power of amplified pulse is about 15 mW. And its pulse width is about 880 fs. An all-fiber supercontinuum (SC) is generated by putting the amplified fiber laser pulse at the wavelength of 1. 56 &mgr;m into the highly nonlinear dispersion shifted fiber (HN-DSF) whose zero dispersion wavelength is 1.537 &mgr;m and nonlinear coefficient is about 10.5/W/km at the input wavelength. The polarization state of the generated SC spectra is well defined such that it can be properly controlled by the polarization controller. By using a delayed pulsed method, we report an experimental study of the coherence of SC spectra generated through a HN-DSF. In this paper, the strong dependence of the spectral coherence on the HN-DSF length is observed experimentally. And optimal conditions for obtaining wide SC with high coherence are investigated in detail. We believed that our proposed all-fiber laser based SC source with high coherence has many important applications in recently developed frequencydomain measurement techniques such as optical coherence tomography (OCT), optical frequency domain imaging (OFDI), optical frequency domain reflectometry (OFDR) and their instrumentation.

All-fiber supercontinuum generation based on a low noise femtosecond fiber laser and highly nonlinear dispersion shifted fiber

We report a wideband and almost flat spectrum supercontinuum (SC) generation by using a passive mode-locked Er-doped fiber laser (PML-EDFL) and a highly nonlinear dispersion shifted fiber (HN-DSF). The fiber laser pulses are characterized with a conventional second harmonic generation frequency resolved optical gating (SHG-FROG) method. The dependence of the SC spectral width on the HN-DSF length, the spectral stability of the generated SC spectrum, and conditions for obtaining smooth spectra without a fine structure are investigated experimentally. In this paper, wide spectrum covering 1100-1750 nm wavelength range is generated with only 8.5 cm long HN-DSF when laser is single pulse operation regime. In addition, the flat spectrum with ±1 dB uniformity was obtained at the wavelength region of 1130-1510 nm. And spectral stability is about ±0.2 dB at the uniformity wavelength region. We believe that our proposed all-fiber based SC source has many important applications in recently developed frequency-domain measurement techniques such as optical coherence tomography (OCT), Optical frequency domain imaging (OFDI), optical frequency domain reflectrometry (OFDR), and their instrumentation.