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 high-speed image sensing technique with adjustable frame rate based on an ordinary CCD

In the sinusoidal phase modulating interferometer technique, the high-speed CCD is necessary to detect the interference signals. The reason of ordinary CCD's low frame rate was analyzed, and a novel high-speed image sensing technique with adjustable frame rate based on ail ordinary CCD was proposed. And the principle of the image sensor was analyzed. When the maximum frequency and channel bandwidth were constant, a custom high-speed sensor was designed by using the ordinary CCD under the control of the special driving circuit. The frame rate of the ordinary CCD has been enhanced by controlling the number of pixels of every frame; therefore, the ordinary of CCD can be used as the high frame rate image sensor with small amount of pixels. The multi-output high-speed image sensor has the deficiencies of low accuracy, and high cost, while the high-speed image senor with small number of pixels by using this technique can overcome theses faults. The light intensity varying with time was measured by using the image sensor. The frame rate was LIP to 1600 frame per second (f/s), and the size of every frame and the frame rate were adjustable. The correlation coefficient between the measurement result and the standard values were higher than 0.98026, and the relative error was lower than 0.53%. The experimental results show that this sensor is fit to the measurements of sinusoidal phase modulating interferometer technique. (c) 2007 Elsevier GmbH. All rights reserved.