Innovations of wide-field optical-sectioning fluorescence microscopy : toward high-speed volumetric bio-imaging with simplicity

Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere.

The concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths.

Among existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often result in certain degrees of photo-damages because of the high focal intensity at the scanning point. In order to overcome such an issue, several wide-field optical-sectioning techniques have been proposed and demonstrated, although not without introducing new limitations and compromises such as low signal-to-background ratios and reduced axial resolutions. As a result, single-point-scanning optical-sectioning techniques remain the most widely used instrumentations for volumetric imaging of living biological systems to date.

In order to develop wide-field optical-sectioning techniques that has equivalent optical performance as single-point-scanning ones, this thesis first introduces the mechanisms and limitations of existing wide-field optical-sectioning techniques, and then brings in our innovations that aim to overcome these limitations. We demonstrate, theoretically and experimentally, that our proposed wide-field optical-sectioning techniques can achieve diffraction-limited optical sectioning, low out-of-focus excitation and high-frame-rate imaging in living biological systems. In addition to such imaging capabilities, our proposed techniques can be instrumentally simple and economic, and are straightforward for implementation on conventional wide-field microscopes. These advantages together show the potential of our innovations to be widely used for high-speed, volumetric fluorescence imaging of living biological systems.

A Q-swicthed all-solid-state single-longitudinal-mode laser with adjustable pulse-width and high repetition rate

A single-longitudinal-mode (SLM) laser-diode pumped Nd: YAG laser with adjustable pulse width is developed by using the techniques of pre-lasing and changing polarization of birefingent crystal. The Q-switching voltage is triggered by the peak of the pre-lasing pulse to achieve the higher stability of output pulse energy. The output energy of more than 1 mJ is obtained with output energy stability of 3% (rms) at 100 Hz. The pulse-width can be adjusted from 30 ns to 300 ns by changing the Q-switching voltage. The probability of putting out single-longitudinal-mode pulses is almost 100%. The laser can be run over four hours continually without mode hopping.

20-W average power, high repetition rate, nanosecond pulse with diffraction limit from an all-fiber MOPA system

In this article, we report an all-fiber master oscillator power amplifier (MOPA) system, which can provide high repetition rate and nanosecond pulse with diffraction-limit. The system was constructed using a (2 + 1) X 1 multimode combiner. The Q-Switched, LD pumped Nd:YVO4 solid-state laser wets used (is master oscillator. The 976-nm fiber-coupled module is used as pump source. A 10-m long China-made Yb3+-doped D-shape double-clad large-mode-area fiber was used as amplifier fiber. The MOPA produced as much as 20-W average power with nanosecond pulse and near diffraction limited. The pulse duration is maintained at about 15 its during 50-175 kHz. The system employs a simple and compact architecture and is therefore suitable for the use in practical applications such as scientific and military airborne LIDAR and imaging. Based oil this system. the amplification performances of. the all fiber amplifier is investigated. (C) 2008 Wiley Periodicals, Inc.

High-repetition-rate MHz acoustooptic Q-switched fiber laser

A simple actively Q-switched double-clad fiber laser combining an amplifying cavity is reported by using a dynamic acoustooptic Q-switching as a beam splitter. Sub-100-ns. pulses independence of the repetition rate of acoustooptic modulator are almost changeless with repetition rate varied from 50 kHz to 1.5 MHz. With 4.5-W absorbed power, 9.4-W peak-power pulses at 1.5-MHz repetition rate with 75-ns pulse duration are generated.

High power short pulse generation at high repetition rate from InGaN violet laser diodes

The generation of 22 ps pulses with peak powers of 0.74 W by a gain-switched InGaN violet laser diode is reported. Significant pulse width dependence on repetition rate is observed. © 2011 OSA.