Atomic Force Microscopy (AFM) plays important roles in nanobiology. We here review the recent development and application of AFM technology from the technical point of view. In the visualization mode, AFM produces not only static images at lower time-resolution in the seconds to minutes, but also motion images at higher time-resolution in the milliseconds. Furthermore, AFM combined with fluorescent techniques can now address varieties of biological questions from genes to proteins and from proteins to cells. In the force mode, the tip directly interacts with the molecules of interests to measure the inter- and/or intra-molecular physical properties.
Phase contrast microscopy is widely used in the field of cell biology. A weak point of this microscopy is a kind of optical noise, 'halo', which appears around the objects and reduces resolution of the images. To remove the halo, Otaki recently introduced apodization method to the phase contrast microscopy and improved quality of the images. Here, we review a mechanism of halo reduction by the apodized phase contrast microscopy.
Positron emission tomography (PET) is a powerful noninvasive method for molecular imaging in living systems, including the brain, heart, and other active tissues and organs. The need to develop new PET tracers has grown with the increase in use of this technique in biochemistry, medicine (diagnosis), and drug development. We here describe the overview of the recent advances and perspective in this interdisciplinary scientific area, focusing on current PET technology and new tracer synthesis particularly based on new methodologies for incorporating a short-lived 11C-nuclide into bioactive organic molecules through Pd(0)-mediated rapid methylation and carbonylations. The former method has been applied to the synthesis of 15R-[11C] TIC methyl ester, an efficient PET tracer for imaging a novel CNS-type prostacyclin receptor (IP2) in living human brain.
MRI (magnetic resonance imaging) is a non-destructive and non-invasive visualizing method, and has been widely used in clinical medicine and biological studies. Combining MRI with recent developments of molecular and cellular biology together with genetically modified animals is now opening up a new era of MRI, where various biological information, rather than only morphological shape, can be obtained in vivo at cellular and sub-cellular level by sophisticatedly designed MR reporter systems. This short review starts with a brief description of the basic concept of MRI and introduces recent topics.
We address biological questions concerning spatio-temporal patterns of signaling by EGF and astrocytic contact. First, the pattern of EGF signaling was visualized using fluorescent indicators for Ras activation and tyrosine phosphorylation in single live COS cells following local stimulation. Ligand-independent propagation of EGF signaling occured only when the receptor density on the plasma membrane was high, such as in carcinoma cells. Second, local contact with astrocytes via integrin receptors elicited PKC activation in individual dissociated neurons cultured in astrocyte-conditioned medium. The PKC activation, initially focal, soon spread throughout the entire neuron, leading to global neuronal maturation.
Electron cryotomography was introduced as the most promising method to visualize proteins and supramolecules inside cells in their intact form. Current spatial resolution with the method is about 5 nm and supramolecules like ribosomes or proteasomes were identified. To extend the resolution to 1 nm for the identification of proteins, electron-phase microscopy could be introduced, by which several supramolecules had been identified for unstained ice-embedded cyanobacteria.