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Biomedical Optics & Medical Imaging

Fluorescence tomography of biological tissue using ultrasonic tagging

Ultrasound-induced modulation permits the collection of fluorescence images from deep within light-scattering media.

Ultrasound-induced modulation permits the collection of fluorescence images from deep within light-scattering media.
3 October 2007, SPIE Newsroom. DOI: 10.1117/2.1200709.0870

Fluorescence labeling traces specific molecules that play physiological roles in living organisms, revealing the mysteries of life. Recently developed fluorescent probes with excellent optical performance are expected to extend fluorescence techniques to clinical diagnosis. However, living tissues scatter light strongly, restricting applications to the microscopic level or to the body surface. To permit noninvasive optical measurement for tomographic imaging of the human body, we have explored a technique to visualize fluorescence at a depth of tens of centimeters, with resolution of the order of millimeters.

Myriad efforts have been made over many years to develop fluorescence and other optical imaging techniques that work in light-scattering media. Time- and frequency-domain techniques1 for analyzing diffuse light and the extraction of forward-scattering light by coherent gating2,3 are the major approaches to optical tomography. The distribution of optical absorption has been measured using ‘ultrasonic tagging,’ which focuses more weakly scattered ultrasound to extract spatial information about optical properties.4–7 We have extended focused ultrasound to visualize fluorescence in dense scattering media.

Figure 1. (a) Tomographic map of ultrasound-modulated fluorescence from porcine muscle tissue that contains a localized fluorescent region. The X and Y axes are parallel to the laser and ultrasound beams, respectively. (b) Signal intensity profile along the X direction, sectioned at the center of Y-axis of the image, which includes the fluorescent region. (c) The tissue inserted in a measuring holder.

Previously, ultrasonic tagging was thought to be unsuitable for fluorescence imaging because speckle modulation in multiply-scattering media occurs only for coherent optical processes. However, even for fluorescence, density variation of the medium in a focused sound field might induce intensity modulation. By exploiting the spectral separation between fluorescence and excitation using an appropriate optical arrangement with a large-scale, highly efficient detector, an ultrasound field offers a detectable modulation of the fluorescence signal.8,9

For this demonstration study, we show tomographic imaging of fluorescence in dense scattering media using porcine muscle tissue. The tissue on the scanning plane is 40×40mm. A fluorescent region is formed by embedding an encapsulated fluorescent material (755nm emission wavelength) that is molded with columnar agarose gel (5mm long, 3mm in diameter) in the center of the tissue, at a depth of 20mm. The tissue is immersed in a water tank that has a focused-ultrasound transducer (38mm focal length, 3mm focal diameter) on one side wall. An excitation laser beam (726nm, 40mW) traverses the tissue through the water tank, perpendicular to the ultrasound beam, and excites the fluorescent material embedded in the tissue. The modulated component of the fluorescence signal is detected using a spectrum analyzer via a photomultiplier tube. Bi-axial scanning of the ultrasound focus in the tissue yields a two-dimensional tomographic image.

Figure 1(a) presents a tomographic image of fluorescence observed by scanning a 20×20mm area. A localized area of fluorescence is detected near the center of the tissue. The area of higher signal corresponds to the embedded fluorescent material. The profile of the signal intensity along the X direction (parallel to the laser beam), sectioned at the center of the Y-axis of the image (parallel to the ultrasound beam) is shown in Figure 1(b). The peak width almost matches the fluorescent region's diameter, considering the size of ultrasound focus. The signal-to-noise ratio in this experiment, together with the near-infrared scattering coefficient of living tissues, suggests that this technique could image fluorescence in biological tissues at a depth of roughly 30mm.

Nanomaterials, referred to as quantum-dot materials, have recently been considered as novel fluorescent probes in biological investigations. In the near future, quantum dots that label physiological substances will illuminate sites of dysfunction or disease, including cancer, within living bodies.

The current study demonstrates the potential to visualize fluorescence deep within biological tissue with the assistance of ultrasound. The results suggest that further progress in scanning techniques, such as arrayed ultrasound transducers, can produce a practical new bio-imaging system. This technique is expected to facilitate the expansion of fluorescent probes' use for biological and clinical applications. Fluorescent visualization of living tissues will be a crucial technology contributing to human health and welfare.

Masaki Kobayashi
Department of Electronics,
Tohoku Institute of Technology
Sendai, Japan

Masaki Kobayashi received a PhD in engineering from Tohoku University in 1993. From 1993 to 2000, he was a senior researcher at the Biophotonics Information Laboratories. He is currently a Professor of Electronics at Tohoku Institute of Technology. His interests are optical tomography and ultra-weak photon imaging for biomedical measurement.

Motohiro Takeda
Graduate School of Medicine
Tohoku University
Sendai, Japan