Nobuhiro Tomatsu

Simple PCR-Based DNA Microarray System To Identify Human Pathogenic Fungi in Skin

ABSTRACT Fungal diseases in immunocompromised hosts pose significant threats to their prognoses. An accurate diagnosis and identification of the fungal pathogens causing the infection are critical to determine the proper therapeutic interventions, but these are often not achieved, due to difficulties with isolation and morphological identification. In an effort to ultimately carry out the simultaneous detection of all human pathogenic microbes, we developed a simple system to identify 26 clinically important fungi by using a combination of PCR amplification and DNA microarray assay (designated PCR-DM), in which PCR-amplified DNA from the internal transcribed spacer region of the rRNA gene was hybridized to a DNA microarray fabricated with species-specific probes sets using the Bubble Jet technology. PCR-DM reliably identified all 26 reference strains; hence, we applied it to cases of onychomycosis, taking advantage of the accessibility of tissue from skin. PCR-DM detected fungal DNA and identified pathogens in 92% of 106 microscopy-confirmed onychomycosis specimens. In contrast, culture was successful for only 36 specimens (34%), 3 of which had results inconsistent with the results of PCR-DM, but sequence analysis of the isolates proved that the PCR-DM result was correct. Thus, PCR-DM provides a powerful method to identify pathogenic fungi with high sensitivity and speed directly from tissue specimens, and this concept could be applied to other fungal or nonfungal infectious human diseases in less accessible anatomical sites.

Motion artifact and speckle noise reduction in polarization sensitive optical coherence tomography by retinal tracking

We present a novel polarization sensitive optical coherence tomography (PS-OCT) system with an integrated retinal tracker. The tracking operates at up to 60 Hz, correcting PS-OCT scanning positions during the acquisition to avoid artifacts caused by eye motion. To demonstrate the practical performance of the system, we imaged several healthy volunteers and patients with AMD both with B-scan repetitions for frame averaging and with 3D raster scans. Under large retinal motions with up to 1 mm amplitude at 0.5 ~a few Hz frequency range, motion artifact suppression in the PS-OCT images as well as standard deviation noise reduction in the frame averaged retardation images are presented.

Analysis of optimum conditions of depolarization imaging by polarization-sensitive optical coherence tomography in the human retina.

Abstract. Measurement and imaging of depolarization by polarization-sensitive optical coherence tomography (PS-OCT) requires averaging of Stokes vector elements within two- or three-dimensional (3-D) evaluation windows to obtain the degree of polarization uniformity (DOPU). By use of a PS-OCT system with an integrated retinal tracker, we analyze optimum conditions for depolarization imaging, data processing, and segmentation of depolarizing tissue in the human retina. The trade-offs between figures of merit like DOPU imaging sensitivity, efficiency, and susceptibility are evaluated in terms of 3-D resolution. The results are used for a new, detailed interpretation of PS-OCT high-resolution images of the human retinal pigment epithelium and Bruch’s membrane.

Three-beam spectral-domain optical coherence tomography for retinal imaging

Abstract. A three-beam spectral domain optical coherence tomography system (OCT) whose center wavelength is 840 nm was developed. The three beams focus on fundus 3.1 mm apart from each other and are detected by a single line sensor. The distance between the beams is fixed and the beams scan a total area of 10×10  mm2 while keeping this separation during three-dimensional (3-D) measurement. The line rate of the sensor is 70 kHz, therefore the total speed is equivalent to 210k A-scans per second in this system. A 1000(x)×500(z)×250(y) voxel volumetric 3D OCT data set can be acquired within 2 s. Images of a model eye, a healthy human eye and a diseased eye taken by this system are shown and evaluated. The image quality of one B-Scan is as good as an image from a single-beam OCT. Adjustment among the beams is solved by additional signal processing using a model eye. A multi-beam OCT has the potential not only for high speed imaging but also functional imaging although problems such as compensation among the beams and motion artifacts must be solved.