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

High-speed, FPGA-based photon-counting fluorometer with high data-gathering efficiency

We have developed a low-cost, high-efficiency fluorometer using a field-programmable gate array and simultaneous detection of photoelectron pulse trains. The fluorometer covers a time span of 64 ns with a resolution of 1.0 ns/channel. Depending on the number of channels, the signal-gathering efficiency was improved by a factor of 100 relative to that of conventional time-correlated single-photon-counting. This is assuming that the fluorescence intensity is moderately high but still requires photon counting. The dead time for building a histogram has been reduced to zero, which means that the upper limit of the repetitive excitation frequency could exceed that determined by the time span. We describe instrumental details and demonstrate the basic performance

Phase-Modulation Fluorometer Using a Phase-Modulated Excitation Light Source

We propose a phase-modulation fluorometer (PMF) with a light-emitting diode (LED) or a laser diode (LD) used as an excitation light source (ELS) that is driven in the phase-modulation (PM) mode. The PM-ELS generates many frequency sidebands that spread in the vicinity of carrier frequency fc with the interval of modulation frequency fm depending on the maximum phase deviation Δφ . The scheme enables us to derive fluorescence lifetime values of a multicomponent sample at one time. We show a typical numerical simulation result for explaining the principle of operation. To demonstrate the effectiveness of the proposed PMF, we have measured fluorescence lifetimes of three kinds of inorganic fluorescent glasses and that of a mixture solution of 1×10−6M rhodamine 6G and 1×10−6M coumarin 152 in ethanol with a volume ratio of 1 :1

Scan-less full-field fluorescence-lifetime dual-comb microscopy using two-dimensional spectral mapping and frequency multiplexing of dual-optical-comb beats

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for quantitative fluorescence imaging because fluorescence lifetime is independent of concentration of fluorescent molecules or excitation/detection efficiency and is robust to photobleaching. However, since FLIM is based on point-to-point measurements, mechanical scanning of a focal spot is needed for forming an image, which hampers rapid imaging. In this article, we demonstrate scan-less full-field FLIM based on a one-to-one correspondence between two-dimensional (2D) image pixels and frequency-multiplexed RF signals. A vast number of dual-optical-comb beats between dual optical frequency combs is effectively adopted for 2D spectral mapping and high-density frequency multiplexing in radio-frequency region. Bimodal images of fluorescence amplitude and lifetime are obtained with high quantitativeness from amplitude and phase spectra of fluorescence RF comb modes without the need for mechanical scanning. The proposed method will be useful for rapid quantitative fluorescence imaging in life science.Comment: 38 pages, 8 figures, 1 tabl

Full-field fluorescence lifetime dual-comb microscopy using spectral mapping and frequency multiplexing of dual-comb optical beats

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for quantitative fluorescence imaging because fluorescence lifetime is independent of concentration of fluorescent molecules or excitation/detection efficiency and is robust to photobleaching. However, since most FLIMs are based on point-to-point measurements, mechanical scanning of a focal spot is needed for forming an image, which hampers rapid imaging. Here, we demonstrate scan-less full-field FLIM based on a one-to-one correspondence between two-dimensional (2D) image pixels and frequency-multiplexed radio frequency (RF) signals. A vast number of dual-comb optical beats between dual optical frequency combs are effectively adopted for 2D spectral mapping and high-density frequency multiplexing in the RF region. Bimodal images of fluorescence amplitude and lifetime are obtained with high quantitativeness from amplitude and phase spectra of fluorescence RF comb modes without the need for mechanical scanning. The parallelized FLIM will be useful for rapid quantitative fluorescence imaging in life science

Multicascade-linked synthetic wavelength digital holography using an optical-comb-referenced frequency synthesizer

Digital holography (DH) is a promising method for non-contact surface topography because the reconstructed phase image can visualize the nanometer unevenness in a sample. However, the axial range of this method is limited to the range of the optical wavelength due to the phase wrapping ambiguity. Although the use of two different wavelengths of light and the resulting synthetic wavelength, i.e., synthetic wavelength DH, can expand the axial range up to a few tens of microns, this method is still insufficient for practical applications. In this article, a tunable external cavity laser diode phase-locked to an optical frequency comb, namely, an optical-comb-referenced frequency synthesizer, is effectively used for multiple synthetic wavelengths within the range of 32 um to 1.20 m. A multiple cascade link of the phase images among an optical wavelength (= 1.520 um) and 5 different synthetic wavelengths (= 32.39 um, 99.98 um, 400.0 um, 1003 um, and 4021 um) enables the shape measurement of a reflective millimeter-sized stepped surface with the axial resolution of 34 nm. The axial dynamic range, defined as the ratio of the maximum axial range (= 0.60 m) to the axial resolution (= 34 nm), achieves 1.7*10^8, which is much larger than that of previous synthetic wavelength DH. Such a wide axial dynamic range capability will further expand the application field of DH for large objects with meter dimensions.Comment: 19 pages, 7 figure

Expression of ICAM-I on M cells covering isolated lymphoid follicles of the human colon.

To clarify the immunological function of 'M' (microfold or membranous) cells in the large intestine, we examined the expression of intercellular adhesion molecule-1 (ICAM-1) and HLA-class II antigens immunohistochemically in M cells and follicle-associated epithelia (FAE) covering isolated lymphoid follicles of the human colon in comparison with their expression in Peyer's patches of the small intestine. In Peyer's patches of the small intestine, ICAM-1 was not expressed on the epithelial cells covering the lymphoid follicles, but their cell surfaces were stained positively for HLA-DR. In contrast, colonic M cells expressed ICAM-1 on their cell surfaces but were negative for HLA class II antigens. By immunoelectron microscopy, ICAM-1 was seen to be distributed on the surface of microfolds, on the membranes of apical vesicles and on part of the basolateral plasma membranes of M cells, but was not expressed on adjacent FAE. These findings imply that the M cells in the colon and in Peyer's patches have different immunological roles. In addition, identification of ICAM-1 expression on the colonic M cells should help elucidate the pathogenesis of some inflammatory colonic diseases which appear to start in the lymphoid follicles of the colonic mucosa.</p