To investigate whether the size of fluorescent particles affects the modulation efficiency of ultrasound-modulated fluorescence (UMF), we measured UMF and DC (direct current) signals of the fluorescence emission from four different-sized fluorescent particles: (1) three carboxylate-modified fluorescent microspheres (FM) with diameters of 20？nm, 200？nm, and 1.0？μm and (2) streptavidin-conjugated Alexa Fluor 647 with a diameter of approximately 5？nm. The UMF and DC signals were simultaneously measured using a broadband lock-in amplifier and a narrowband amplifier, respectively. The ratio of the UMF strength to the DC signal strength is defined as the modulation efficiency. This modulation efficiency was then used to evaluate the effects of fluorophore size and concentration. Results show that the modulation efficiency was improved by approximately a factor of two when the size of the fluorescent particles is increased from 5？nm to 1？μm. In addition, the linear relationship between the UMF strength and ultrasound pressure (observed in our previous study) was maintained regardless of the fluorescent particle sizes. 1. Introduction Tissue fluorescence imaging has been well developed and widely used because of its high sensitivity and specificity [1, 2]. Fluorescence techniques can provide unique tissue physiological information when compared with other noninvasive imaging modalities (ultrasound, magnetic resonance imaging, computed tomography, etc.) and are sensitive to tissue microenvironments, such as tissue pH, temperature, and gas/ion concentrations. Also, they are relatively cost efficient, flexible in imaging probes selection (from organic dyes, to quantum dots, and to nanoparticles or microparticles), highly sensitive to imaging probes (fM-nM, 10？15–10？9？mole/liter), and nonionizing radiative [1–3]. Commonly used high-resolution fluorescence microscopy faces a fundamental challenge due to tissue’s strong optical scattering, which typically limits penetration depth to a few hundred micrometers [3]. Techniques used to image deep tissue at ranges of millimeters or centimeters, such as fluorescence diffuse optical tomography (FDOT) [4], take advantage of diffused photons that have been scattered many times before being detected. These diffused photons can penetrate biological tissue up to tens of millimeters at the red or near infrared (NIR) region [3] at the expense of spatial resolution (limited to ~1–5？mm) [4]. Ultrasound-modulated fluorescence (UMF) has been proposed to increase spatial resolution while maintaining imaging depth [5]. This is possible
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