Yoshihiko Wakazono

Fiber-coupled confocal microscope (FCM) for real time imaging of cellular signals in vivo

To study cellular morphology and functions in vivo in realtime, we developed a fiber-coupled confocal microscope (FCM), and observed fluorescently-labeled cells inside the body of anesthetized rat. We developed an imaging fiber bundle (IFB), which consisted of an objective lens and a multi-fiber assembly (unit fiber: NA > 0.4, 3 micron in diameter). By combining the IFB with a real-time confocal scanner, we detected intracellular signals of the molecular messenger, and the death signals in the form of fluorescence changes even from cells located deep (> 2 mm) inside the solid organs. The FCM we developed is very promising for detailed studies in both the cell-based researches and clinical researches.

Intracellular dynamics observed by mode switching of microscope with a light incidence to the interface at alternate angles through the ultra high NA objective

In order to study the dynamic change in the cell, we modified the evanescence microscope with an ultra high NA objective lens so as to modulate the penetration depth of the evanescent wave. We employed a galvanomirror to aim and switch the laser beam rapidly at the back focal plane near the periphery of 1.45 or 1.65 NA objectives. Under this microscope equipped with a 1.45 NA objective, images of the fluorescent bead were clearly distinguishable by the modulation of the penetration depth of the evanescent wave. Thus, translocation dynamics of protein kinase Cα (PKCα) upon cell activation were compared every 0.5 s between two modes using HeLa cells expressing PKCα fused with the green fluorescent protein (GFP). Stimulation of the cell with phorbol ester induced a transient increase in GFP fluorescence images illuminated by the thin evanescent field, but not in the image illuminated by the thick evanescent field. Later, a persistent increase in fluorescence appeared at cell borders in the both images. Using a 1.65 NA objective, trafficking of secretory vesicles was studied in MIN6 cells expressing insulin-GFP. Occasionally, the change in fluorescence of a vesicle observed under one illumination mode appeared very different from the other, allowing unique assignments of the fluorescence change to a certain combination of vesicle movement and a chemical response of fluorescent molecules. The ultra high NA lens provides a large window for evanescent illumination with a wide range of penetration depth, thus is useful for analyzing 3D events in the cell.

Dynamics of the Cell Membrane Observed under the Evanescent Wave Microscope and the Confocal Microscope

The objective lens of a microscope plays an essential role for high resolution imaging. Especially, the numerical aperture (NA) of the objective lens is the determining factor for gaining the resolving power. In 1994, a novel objective lens that had a numerical aperture of 1.65 was developed by Susumu Terakawa (the author) and Katsuyuki Abe (Olympus Optical Co.). Use of this ultra high NA lens opened a new field of light microscopy beneficial for observations of living cells at a molecular level and for studies of the cell membranes in their dynamic aspects.

Slit-scanning microscope with a high-NA objective lens for analysis of synaptic function

By employing the total internal reflection fluorescence (TIRF) microscope with an ultra high NA (1.65) objective lens, we demonstrated detailed dynamics of exocytosis in various types of secretory vesicles. However, the TIRF microscopy could be applied to observations only on the plasma membrane and its immediate vicinity. To observe the vesicles in the deeper region of cytoplasm, we modified the TIRF optics to project a slit beam thinner than 1 μm in width to the cell. The slit beam illumination spotted single secretory vesicles inside the cell better and their movement and exocytosis easier. By scanning the slit beam, a fluorescence microscopy was possible at a high signal-to-noise ratio useful for measurement and analysis of single exocytosis in neurons and endocrine cells.

Fluorescence micro-imaging of living cells and biomolecules with ultrahigh NA objectives

Optical imaging with a high detecting power is very instrumental to dynamic observations of bio-molecular objects in water. To pioneer this field of imaging researches further, we used novel objective lenses of 1.65 and 1.45 in NA comparatively with 1.35 NA lens. The lenses of ultra high NA have a high resolving power and very useful properties for observation of high contrast images of fluorescently labeled molecules and subcellular organelles including DNA and membrane fusion proteins.