Keisuke Isobe

Femtosecond laser disruption of subcellular organelles in a living cell

Subcellular organelles in living cells were inactivated by tightly focusing femtosecond laser pulses inside the cells. Photodisruption of a mitochondrion in living cells was experimentally confirmed by stacking three-dimensional confocal images and by restaining of organelles. The viability of the cells after femtosecond laser irradiation was ascertained by impermeability of propidium iodide as well as by the presence of cytoplasmic streaming.

Intracellular disruption of mitochondria in a living HeLa cell with a 76-MHz femtosecond laser oscillator

Femtosecond laser pulses can be used to selectively disrupt and dissect intracellular organelles. We report on disruption of mitochondria in living HeLa cells using a femtosecond laser oscillator with a repetition rate of 76 MHz. We studied the laser parameters used for disruption. The longterm viability of the cells after disruption of a single mitochondrion was confirmed by the observation of cell division, indicating that intracellular disruption of organelles using a femtosecond laser oscillator can be performed without compromising the long-term cell viability.

Multi-Spectral Two-Photon Excited Fluorescence Microscopy Using Supercontinuum Light Source

We report on a novel technique of multi-spectral two-photon-excited fluorescence microscopy by use of broadband supercontinuum light. A supercontinuum in the near-infrared region is generated in a 4.5-mm-long photonic crystal fiber using a Ti:sapphire femtosecond oscillator. The supercontinuum is used as a multi-spectral excitation light source in a two-photon excited fluorescence microscope. We demonstrate that three-color fluorescence images of organelles in a cell can be simultaneously acquired.

Single-organelle tracking by two-photon conversion

Spatial and temporal information about intracellular objects and their dynamics within a living cell are essential for dynamic analysis of such objects in cell biology. A specific intracellular object can be discriminated by photoactivatable fluorescent proteins that exhibit pronounced light-induced spectral changes. Here, we report on selective labeling and tracking of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. We performed selective labeling of a single mitochondrion in a living tobacco BY-2 cell using two-photon photoconversion of Kaede. Using this technique, we demonstrated that, in plants, the directed movement of individual mitochondria along the cytoskeletons was mediated by actin filaments, whereas microtubules were not required for the movement of mitochondria. This single-organelle labeling technique enabled us to track the dynamics of a single organelle, revealing the mechanisms involved in organelle dynamics. The technique has potential application in direct tracking of selective cellular and intracellular structures.

Stimulated parametric emission microscopy.

We propose a novel microscopy technique based on the four-wave mixing (FWM) process that is enhanced by two-photon electronic resonance induced by a pump pulse along with stimulated emission induced by a dump pulse. A Ti:sapphire laser and an optical parametric oscillator are used as light sources for the pump and dump pulses, respectively. We demonstrate that our proposed FWM technique can be used to obtain a one-dimensional image of ethanol-thinned Coumarin 120 solution sandwiched between a hole-slide glass and a cover slip, and a two-dimensional image of a leaf of Camellia sinensis.

Multifarious control of two-photon excitation of multiple fluorophores achieved by phase modulation of ultra-broadband laser pulses

We propose two-photon excited fluorescence (TPEF) microscopy employing a novel phase modulation technique of ultra-broadband laser pulses, which allows the relative excitation of an individual fluorophore with respect to other fluorophores. This technique is based on the generation of multi-wavelength pulse train, which independently interacts with each fluorophore. Our technique is applied to dual-color imaging of cells expressing two types of fluorescent proteins. We achieve the selective excitation of one over the other and vice versa. The product of the maximum contrast ratios exceeds 100. We also demonstrate yielded equal excitation rates in the simultaneous excitation. By the selective excitation of a donor fluorescent protein, fluorescence resonance energy transfer imaging is also achieved.

Single-pulse coherent anti-Stokes Raman scattering microscopy employing an octave spanning pulse

We demonstrate two complementary types of microscopy using an identical setup for single-pulse coherent anti-Stokes Raman scattering (CARS) imaging, which employs an ultrabroadband laser pulse with a spectral bandwidth of 4800 cm(-1) and enables the suppression of nonresonant CARS signals. One is a novel type of microscopy that uses spectral phase modulation for the selective excitation of a single Raman mode. The selective excitation is achieved by the modulated pulse focusing its difference-frequency spectrum into a narrow spectral region. Another type is Fourier-transform CARS (FT-CARS) microspectroscopy based on the measurement of the CARS spectrum obtained from the Fourier-transform of the interferometric autocorrelation (IAC) signal. Vibrational spectral imaging of chemical and biological samples is demonstrated using the two types of microscopy.

Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination

We demonstrate super-resolution imaging with background fluorescence rejection by interferometric temporal focusing microscopy, in which temporal focusing is combined with structured illumination. The lateral resolution and the optical sectioning capability are simultaneously improved by factors of 1.6 and 1.4, respectively, compared to conventional temporal focusing microscopy. Fluorescent beads (200 nm diameter) that are difficult to distinguish from the background fluorescence in conventional temporal focusing microscopy, are clearly visualized by interferometric temporal focusing microscopy.

Background-free deep imaging by spatial overlap modulation nonlinear optical microscopy

We demonstrate how the resolution and imaging depth limitations of nonlinear optical microscopy can be overcome by modulating the spatial overlap between two-color pulses. We suppress out-of-focus signals, which limit the imaging depth, by a factor of 100, and enhance the lateral and axial resolution by factors of 1.6 and 1.4–1.8 respectively. Using spatial overlap modulation, we demonstrate background-free three-dimensional imaging of fixed mouse brain tissue at depths for which the signals of the conventional technique are swamped by background noise from out-of-focus regions.

Measurement of two-photon excitation spectra of fluorescent proteins with nonlinear Fourier-transform spectroscopy

We present measurements of two-photon excitation (TPE) spectra of various fluorescent proteins with nonlinear Fourier-transform spectroscopy. By using an ultrabroadband laser pulse with a spectrum ranging from 700 to 1100 nm, the absolute TPE spectra of six typical fluorescent proteins (SeBFP, Sapphire, eGFP, eCFP, Venus, DsRed) were measured with high spectral resolution.