Lab on a Chip Lab Chip, 2012, 12, 1591 COMMUNICATION a- Department of Physics, School of Advanced Science and Engineering,　Waseda University. b- Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University. c- Consolidated Research Institute for Advanced Science and Medical Care, Waseda University. d- Waseda Bioscience Research Institute in Singapore (WABIOS), Waseda University,

Structure of Fluorescent nanothermometer: Poly(allylamine hydrochloride) TA) Europium thenoyltrifluoroacetonate The core is observed by transmission electron microscopy. The diameter of the spherical core is 113±18nm. The diameter of nanothermometer is 211 ±84.9 nm

Fluorescent Property: In phosphate-buffered saline (PBS) The fluorescence intensity of nano-thermometers decreased in a linear fashion with increase in temperature change over the range of 20 to 60°C. The thermosensitivity of the nano-thermometers, defined as the decrease of fluorescence intensity was 2.2% per °C. The pH (4-10) and ionic strength (0-500mM) do not affect the fluorescent intensity.

Incorporated with Fluoroscein isothiocyanate: To observe the nanoparticles in confocal laser microscopy equipped with visible laser sources. HeLa cells To determine the internalization pathway, pH-rodo dye used as the marker of endocytosis.(pH inside endocytic vesicles is near to 6)

Directional motion of nanoparticles along the Microtubule: Directional motion of nanoparticle were not affected by the addition of Latrunculin B, an inhibitor of actin polymerization. Depolymerization of microtubules induced by Nocodazole completely inhibited the directional motion. These results indicate that the nanothermometers enter via endocytosis are actively transported along microtubules.

Temperature dependance on Motion: Temperature increased by focusing the laser beam outside the cells for 1 second and the majority of transported nanothermometers were accelerated. The avg temperature: Velocity before heating- 36.0 ± 0.5°C 0.32 μm/s during heating- 47.2 ±3.9 °C. 0.93 μm/s after heating - 0.32 μm/s The absolute value of the average velocity was one-fourth to one-fifth of that previously obtained in the in vitro measurements of the thermal activation of kinesin molecules (1.27 µm/s at 35˚ C). The Q10 temperature coefficient, the rate of change in velocity when the temperature increased by 10 C, was (0.93/0.32)= 2.6

The temperature may affect: 1- The interaction of motor proteins with microtubules. 2- The change in the concentration of Ca2+ in the cytosol which could results in changes in the enzymatic activities of motor proteins. 3- decrease in the cytoplasmic viscosity.

Conclusion: 1- Determined the position of moving acidic organelles, endosomes/lysosomes ,in nano meter resolution and the temperature change via “walking nano- thermometers” 2- Simultaneously measured both microscopic temperature and molecular activities within living cells. Thank You