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We demonstrate dynamic imaging of molecular distribution in unstained living cells using Raman scattering. By combining slit-scanning detection and optimizing the excitation wavelength, we imaged the dynamic molecular distributions of cytochrome c, protein beta sheets, and lipids in unstained HeLa cells with a temporal resolution of 3 minutes. We found that(More)
We demonstrate the use of saturated excitation in confocal fluorescence microscopy to improve the spatial resolution. In the proposed technique, we modulate the excitation intensity temporally and detect the harmonic modulation of the fluorescence signal which is caused by the saturated excitation in the center of the laser focus. Theoretical and(More)
Raman microscopy is a promising technology for visualizing the distribution of molecules in cells. A challenge for live-cell imaging using Raman microscopy has been long imaging times owing to the weak Raman signal. Here we present a protocol for constructing and using a Raman microscope equipped with both a slit-scanning excitation and detection system and(More)
Metal nanoparticles have recently emerged as ubiquitous surface-enhanced Raman scattering (SERS) agents for nano-imaging and nano-analysis. These applications make use of the unique optical properties of metal nanoparticles to enhance the efficiency of Raman scattering, whereas the small size of the nanoparticles localizes the enhanced Raman scattering at(More)
We propose a multifocus scanning technique in second-harmonic-generation (SHG) microscopy in which the SHG detection efficiency and the image acquisition rate are improved by several tens of times compared with typical single-focus scanning techniques. Because of a microlens array scanner, the laser beam of a mode-locked Ti:sapphire laser is split into ~100(More)
Ischemic insult to the heart produces myocyte Ca2+ ([Ca2+]i) overload. However, little is known about spatiotemporal changes in [Ca2+]i within the ischemic heart in situ at the cellular level. Using real-time confocal microscopy, we successfully visualized [Ca2+]i dynamics at the border zone on the subepicardial myocardium of the heart 2 h after coronary(More)
The calcium ion (Ca2+) concentrations in a cell are responsible for the control of vital cellular functions and have been widely studied as a means to investigate and control cell activities. Here, we demonstrate Ca2+ wave generation in HeLa cells by femtosecond laser irradiation and show unexpected properties of the Ca2+ release and propagation. When the(More)
Noninvasive, straightforward methods to inactivate selected proteins in living cells with high spatiotemporal resolution are needed. Chromophore-assisted laser inactivation (CALI) can be used to photochemically inactivate proteins, but it has several drawbacks, such as procedural complexity and nonspecific photodamage. Here we show that by application of(More)
We performed label-free observation of molecular dynamics in apoptotic cells by Raman microscopy. Dynamic changes in cytochrome c distribution at the Raman band of 750 cm(-1) were observed after adding an apoptosis inducer to the cells. The comparison of mitochondria fluorescence images and Raman images of cytochrome c confirmed that changes in cytochrome c(More)