Date of download: 6/21/2016 Copyright © 2016 SPIE. All rights reserved. Schematic diagram of thermal destruction and photothermal treatment (PTT) study using real-time phase-contrast imaging system with live cell chamber. The live cell imaging chamber is designed to maintain the temperature of culture medium at 37°C with saturated moisture and a fixed atmosphere of air/ CO2. (a) The live cell imaging chamber with the cancer cell line on an indium tin oxide (ITO) heating plate is heated to calculate the heating-induced cellular destruction rate. (b) The light source for phase-contrast imaging is a 525-nm-wavelength LED, and the source for the PTT is a 960-nm-wavelength laser. A region containing both the cancer cell line (blue area) and the nanoshell-loaded macrophages (red area) is irradiated using a 960-nm-wavelength laser and estimated using real-time phase-contrast imaging system (dotted square). Figure Legend: From: Real-time phase-contrast imaging of photothermal treatment of head and neck squamous cell carcinoma: an in vitro study of macrophages as a vector for the delivery of gold nanoshells J. Biomed. Opt. 2012;17(12): doi: /1.JBO

Date of download: 6/21/2016 Copyright © 2016 SPIE. All rights reserved. Electron microscopy imaging and optical properties of the nanoshell. (a) Transmission electron microscopy (TEM) image of the nanoshells and uncoated silica particles (inset). Scale bar in the inset=160 nm. (b) Ultraviolet-visible-near infrared (UV-vis-NIR) spectrum of the nanoshells. An extinction maximum (λMAX) is obtained at 960 nm. Figure Legend: From: Real-time phase-contrast imaging of photothermal treatment of head and neck squamous cell carcinoma: an in vitro study of macrophages as a vector for the delivery of gold nanoshells J. Biomed. Opt. 2012;17(12): doi: /1.JBO

Date of download: 6/21/2016 Copyright © 2016 SPIE. All rights reserved. Cancer cell viability with respect to increasing temperature from ITO plate heating and laser irradiation. Each plot shows a single time-course measurement. (a) Relative change in the living cell area with heating time (the area at t=0 is normalized to 100%). An abrupt change in the cell area occurs at approximately 20 min. (b) Temperatures measured at the ITO heating plate and within the culture dish as a function of time. At 20 min, the temperature of the culture medium adjacent to cancer cells is >44°C (Video 1) (c) Cell death rate measured using the lactate dehydrogenase (LDH) assay during heating. The LDH level rises with increasing heating time. (d) Temperature change induced by irradiation with an NIR laser (2 W/cm2). The culture medium is continuously illuminated with an NIR laser for 30 min, and the nanoshell (NS)-mixed medium contains only NS at a concentration of 27.4 pM. The red dots with a solid line represent the measured temperature in the NS-mixed medium during irradiation, and the blue dots with a dashed line represent the measured temperature in the medium without NS during irradiation. The red arrow indicates the treatment time required to reach the target temperature (5 min, 44°C to 45°C) (Video 1, MOV, QuickTime, 4.3 MB) [URL: Figure Legend: From: Real-time phase-contrast imaging of photothermal treatment of head and neck squamous cell carcinoma: an in vitro study of macrophages as a vector for the delivery of gold nanoshells J. Biomed. Opt. 2012;17(12): doi: /1.JBO

Date of download: 6/21/2016 Copyright © 2016 SPIE. All rights reserved. Changes in the culture medium temperature and the macrophage endocytosis rate with respect to the NS concentration. (a) The medium temperature with changing NS concentration within the NS-mixed medium. The culture medium is continuously illuminated with an NIR laser for 30 min. The temperature of culture medium increases with increasing NS concentration, and the target temperature (>44°C to 45°C) is not achieved in low concentrations (<1.37 pM), irrespective of irradiation time. (b) Light transmittance of the NS-loaded macrophages. The light transmission decreases with increasing NS concentration that is co- incubated with the macrophages. Figure Legend: From: Real-time phase-contrast imaging of photothermal treatment of head and neck squamous cell carcinoma: an in vitro study of macrophages as a vector for the delivery of gold nanoshells J. Biomed. Opt. 2012;17(12): doi: /1.JBO

Date of download: 6/21/2016 Copyright © 2016 SPIE. All rights reserved. Photothermal treatment using NS-loaded macrophages. (a) Quantitative phase image (top) and differential interference contrast image (bottom) of a macrophage without NS. (b) Quantitative phase image (top) and differential interference contrast image (bottom) of a macrophage with small NS aggregates (red arrows). (c) Phase-contrast image of cancer cells without macrophages before (top) and after (bottom) 5 min of laser irradiation at 2 W (control). (d) Phase-contrast image of cancer cells with the NS- loaded macrophages before (top) and after (bottom) 5 min of laser irradiation. This image shows selective cell death near the NS- loaded macrophages (Video 2, MOV, QuickTime, 4.2 MB) [URL: Figure Legend: From: Real-time phase-contrast imaging of photothermal treatment of head and neck squamous cell carcinoma: an in vitro study of macrophages as a vector for the delivery of gold nanoshells J. Biomed. Opt. 2012;17(12): doi: /1.JBO