Single Molecule Imaging

Various proteins in a living cell are working with corporation as bio-element nano-machine. The cell constructs the molecule system for signal transudation by these proteins, and does a complex and flexible behaviors. Our purpose is to reveal the molecular mechanism of signal transudation in an immune reaction.　 It is thought that the information in the cell via the proteins transmits with the participation of few molecules. Furthermore, neither the structure nor the environment in the cell is uniform. To achieve our goal, we have developed new microscopic techniques to observe directly the tempo-spatial dynamics in living cells. Here, we would like to introduce the microscopic techniques we developed.

1)　Single molecular imaging by using a total internal reflection florescent microscope (TIRFM)
We have developed Single molecular imaging by using a TIRFM. The illumination by TIRF can excite the fluorescent dye nearby about 200 nm far from the glass surface by inserting the laser into the objective as do the total reflection on the boundary between the buffer and the glass. The background noise is reduced to 1/2000 by using this irradiation technique compared with a current fluorescence microscope. Therefore, a fluorescent image can be acquired with very good contrast, and we can detect the image of individual single molecule of fluorescent dyes or fluorescent proteins.

Single molecular imaging in living cell is enumerated as one of examples for bio- and/or medical applications using TIREM. We can observe the basic-process of signal transmits of the binding of ligand to the membrane, the dimerization, the complex formation, the phosphorylation, the diffusion of membrane proteins and so on. Thus, original behavior of the proteins can be observed by single molecular imaging in living cells by using TIRFM, while an electro microscope permits us to observe those in only fixed cells.

We observe PTEM, which is involved in Chemotaxis of cell, using TIRFM. The red spots are individual molecules of PTEM recruited onto the cell membrane.

2)　Nanometry of the proteins in living cells
Nanometry of the proteins in living cells We can peruse the movement of the protein by calculating the centroid of the fluorescent spot. If the fluorescent intensity is enough high, the accuracy became to be about 2 nm. We labeled HER2 (Human EGF Receptor 2), which is one of membrane proteins, with CdSe fluorescent dye (Qdot) via the antidoby, and tracked the movements of the individual fluorescent spots (Fig 2A). The membrane proteins was known to diffuse on the membrane previously, we found the diffusional movement was reflected the structures of somewhat on the membrane such as actin network, caveola or something (Fig 2B).

The nanometry in the cell enables us to observe "Life" movement of the protein that cannot be observed so far.

3)　3D single particle tracking
We constructed a new optics that can obtain the two distinct focal images with a CCD camera (DIO, Dual focus Imaging Optics). The principle of 3D tracking by using DIO is simple. The image of the fluorescent particle between on two focus planes is projected onto both focus images, and fluorescent intensity and the blurring of both the images correlate to the position of the vertical direction of the particle. We completed the algorithm that calculated the position of the vertical direction of the single particle by the comparing the fluorescent intensities obtained in two different focuses. XY position is determined by fitting the image with 2D Gaussian function.

We tracked the vesicle transport by dynein which is the motor protein associated with microtubule (Fig 3A). As the traveling direction of the vesicle is a coordinate axis, successive 8 nm steps were clearly detected, which indicating the small repeated structure of microtubule (Fig 3B). As the vesicle transport was viewed from the front, the vesicle was transported ahead with shaking right and left on the cylindrical column. The radius was 109 nm (Fig 3C), which consisted with the theoretical length from the center of the microtubule to the vesicle.

If the above-mentioned optics and the analysis method are used, the movement of the protein that moves in the cell can be observed as it is.

4) Inguinal skin window for chrocnic two-photo microscopic observation
Two-photon microscopy is a form of laser-scanning microscopy that uses localized nonliner optics to excite fluorescence by using near simultaneous absorption of two long wavelength (>800nm) photons. The long wavelengths used for two-photon microscopy are less damaging and penetrate more deeply into tissue than those used in other light microscopy. This distinct advantage makes it possible to perform real-time analysis of cell dynamics within their normal tissue environments in a living animal .

While two-photon microscopy to date is the only modality that can unveil cell dynamics in living animals, it is far less convenient than other imaging modalities like ultrasound and magnetic resonanse. One reason is that it is invasive, as the tissue to be observed needs to be exposed surgically by removing obstructive tissue like skin and fat. In lymphoid tissue, for example, the result is an imaging depth of a few hundreds micrometers with two-photon microscopy. Furthermore, the invasive surgical preparation makes it difficult to use one animal on different days and to follow the development of disease over a period of time.

To overcome the drawback of two-photon microscopy, we have developed an implantable observation window system for long-term observation of the mouse inguinal lymph node. To demonstrat the system's deasibility, we observed T cell migration and proliferation during homeostaic proliferation for two weeks.