In the history of the development of optical imaging techniques, optical microscopy systems have been generally understood in a static manner, i.e., the optical properties of the microscopy systems can be fully analyzed in time-independent geometrical optics-based and/or physical optics-based models. In this thesis, we show that for multiphoton excitation fluorescence microscopy, there is still certain space for investigations and innovations in temporal aspects, and this space is not quite accessible if the dynamics of light pulses are over-simplified or even ignored as they usually were in previous studies. Specifically, we investigate the possibilities of using 0-order diffraction to generate time delays to reduce out-of-focus excitation in wide-field multiphoton excitation fluorescence mi- croscopy. We discover the fundamental limitation, i.e., the inter-channel light leakage issue, of such a methodology, and develop a physical optics-based simulation to optimize the optical systems under this limitation. Furthermore, we also invent a new method, fiber-bundle illumination, to ultimately resolve this limitation.
Our methods, especially the fiber-bundle illumination technique, can be the most powerful optical-sectioning techniques among the existing ones upon optimization. It provides conceptu- ally true scanningless illumination for high-frame-rate imaging, equivalent axial response to single- point-scanning multiphoton excitation fluorescence microscopy, extremely low (average) excitation intensity that is suitable for living-cell imaging, and at the same time its optical design is almost as simple as a conventional far-field optical microscope. Such simplicity makes the technique itself potentially low cost, easy to be used, maintained and even innovated by briefly trained persons.
We can therefore expect this technique to trigger more and more exciting inventions, explorations and discoveries in various research fields such as fiber optics, bio-imaging, and ultimately, biology. I believe that our techniques can greatly increase the availability and user-friendliness of diffraction- limited volumetric fluorescence imaging techniques, and thus broadly benefit bio-imaging-related researches in the near future.
Table 4.1: Features of wide-field optical-sectioning techniques. Ratio of in-focus to out-of-focus excitation is estimated assuming that the microscope objective is a 60X NA 1.42 oil-immersion lens;
the corresponding depth of field is ∼0.8 µm in the visible band. *Instrumentally achievable frame rates. **The width of field of view is assumed to be 100µm.
System Simplicity
Frame Rate*
Ratio of In-Focus to Out-of-Focus Excitation
Specific Drawbacks Multifocal
Confocal Microscopy
Moderate ∼500 fps
or less ∼3 Fixed pinhole size
(Time- Multiplexed) Multifocal Multiphoton Microscopy
Moderate ∼500 fps
or less ∼3
Structured Illumination Microscopy
High >1,000 fps ∼1/25 Degraded signal-to-noise ratio Selective
Plane Illumination Microscopy
Moderate >1,000 fps ∼1/2**
Trade-off between axial resolution and width of field of view
Inconvenient for sample handling Temporal
Focusing Microscopy
High >1,000 fps ∼1/12 [6] Wavelength-dependent optical path Requires ultrafast amplifiers Dense
Time- Multiplexed Multifocal Multiphoton Microscopy + SIM
High >1,000 fps ∼1/4 Requires ultrafast amplifiers
Fiber
Bundle-Based Time-
Multiplexed Multifocal Multiphoton Microscopy
High >1,000 fps ∼3 Requires ultrafast amplifiers
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