Takahiko Mizuno

Hadamard-transform fluorescence-lifetime imaging

We discuss a Hadamard-transform-based fluorescence-lifetime-imaging (HT-FLI) technique for fluorescence-lifetime-imaging microscopy (FLIM). The HT-FLI uses a Fourier-transform phase-modulation fluorometer (FT-PMF) for fluorescence-lifetime measurements, where the modulation frequency of the excitation light is swept linearly in frequency from zero to a specific maximum during a fixed duration of time. Thereafter, fluorescence lifetimes are derived through Fourier transforms for the fluorescence and reference waveforms. The FT-PMF enables the analysis of multi-component samples simultaneously. HT imaging uses electronic exchange of HT illumination mask patterns, and a high-speed, high-sensitivity photomultiplier, to eliminate frame-rate issues that accompany two-dimensional image detectors.

Measurement of absolute frequency of continuous-wave terahertz radiation in real time using a free-running, dual-wavelength mode-locked, erbium-doped fibre laser

A single, free-running, dual-wavelength mode-locked, erbium-doped fibre laser was exploited to measure the absolute frequency of continuous-wave terahertz (CW-THz) radiation in real time using dual THz combs of photo-carriers (dual PC-THz combs). Two independent mode-locked laser beams with different wavelengths and different repetition frequencies were generated from this laser and were used to generate dual PC-THz combs having different frequency spacings in photoconductive antennae. Based on the dual PC-THz combs, the absolute frequency of CW-THz radiation was determined with a relative precision of 1.2 × 10−9 and a relative accuracy of 1.4 × 10−9 at a sampling rate of 100 Hz. Real-time determination of the absolute frequency of CW-THz radiation varying over a few tens of GHz was also demonstrated. Use of a single dual-wavelength mode-locked fibre laser, in place of dual mode-locked lasers, greatly reduced the size, complexity, and cost of the measurement system while maintaining the real-time capability and high measurement precision.

Photon-counting 1.0 GHz-phase-modulation fluorometer

We have constructed an improved version of a photon-counting phase-modulation fluorometer (PC-PMF) with a maximum modulation frequency of 1.0 GHz, where a phase domain measurement is conducted with a time-correlated single-photon-counting electronics. While the basic concept of the PC-PMF has been reported previously by one of the authors, little attention has been paid to its significance, other than its weak fluorescence measurement capability. Recently, we have recognized the importance of the PC-PMF and its potential for fluorescence lifetime measurements. One important aspect of the PC-PMF is that it enables us to perform high-speed measurements that exceed the frequency bandwidths of the photomultiplier tubes that are commonly used as fluorescence detectors. We describe the advantages of the PC-PMF and demonstrate its usefulness based on fundamental performance tests. In our new version of the PC-PMF, we have used a laser diode (LD) as an excitation light source rather than the light-emitting diode that was used in the primary version. We have also designed a simple and stable LD driver to modulate the device. Additionally, we have obtained a sinusoidal histogram waveform that has multiple cycles within a time span to be measured, which is indispensable for precise phase measurements. With focus on the fluorescence intensity and the resolution time, we have compared the performance of the PC-PMF with that of a conventional PMF using the analogue light detection method.

Scan-less confocal phase imaging based on dual-comb microscopy

Confocal imaging and phase imaging are powerful tools in life science research and industrial inspection. To coherently link the two techniques with different depth resolutions, we introduce an optical frequency comb (OFC) to microscopy. Two-dimensional (2D) image pixels of a sample were encoded onto OFC modes via 2D spectral encoding, in which OFC acted as an optical carrier with a vast number of discrete frequency channels. Then, a scan-less full-field confocal image with a depth resolution of 62.4 um was decoded from a mode-resolved OFC amplitude spectrum obtained by dual-comb spectroscopy. Furthermore, a phase image with a depth resolution of 13.7 nm was decoded from a mode-resolved OFC phase spectrum under the above confocality. The phase wrapping ambiguity can be removed by the match between the confocal depth resolution and the phase wrapping period. The proposed hybrid microscopy approach will be a powerful tool for a variety of applications.Confocal laser microscopy (CLM) is a powerful tool in life science research and industrial inspection because it offers two-dimensional optical sectioning or three-dimensional imaging capability with micrometer depth selectivity. Furthermore, scan-less imaging modality enables rapid image acquisition and high robustness against surrounding external disturbances in CLM. However, the objects to be measured must be reflective, absorptive, scattering, or fluorescent because the image contrast is given by the optical intensity. If a new image contrast can be provided by the optical phase, scan-less CLM can be further applied for transparent non-fluorescent objects or reflective objects with nanometer unevenness by providing information on refractive index, optical thickness, or geometrical shape. Here, we report scan-less confocal dual-comb microscopy offering a phase image in addition to an amplitude image with depth selectivity by using an optical frequency comb as an optical carrier of amplitude and phase with discrete ultra-multichannels. Our technique encodes confocal amplitude and phase images of a sample onto a series of discrete modes in the optical frequency comb with well-defined amplitude and phase to establish a one-to-one correspondence between image pixels and comb modes. The technique then decodes these images from comb modes with amplitude and phase. We demonstrate confocal phase imaging with milliradian phase resolution under micrometer depth selectivity on the millisecond timescale. As a proof of concept, we demonstrate the quantitative phase imaging of standing culture fixed cells and the surface topography of nanometer-scale step structures. Our technique for confocal phase imaging will find applications in three-dimensional visualization of stacked living cells in culture and nanometer surface topography of semiconductor objects.

Video-rate confocal phase imaging by use of scan-less dual comb microscopy

We demonstrate the confocal phase imaging at a video rate by a combination of dual comb spectroscopy (DSC) with 2D spectral encoding (2D-SE). After the image pixels of the sample is encoded on an optical frequency comb (OFC) by 2DSE, DSC of the image-encoded OFC passing through the confocal pinhole gives the mode-resolved amplitude and phase spectra. Based on one-to-one correspondence between the image pixels and OFC modes, the confocal amplitude and phase images are decoded from the mode-resolved amplitude and phase spectra, respectively. The phase spectrum measurement without the need for mechanical scanning enables the video-rate confocal phase imaging.