Development of high-throughput Real-time single-cell secretion assay systems

• Project/Area Number: 25440074
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Biophysics
• Research Institution: The University of Tokyo (2014-2015); Institute of Physical and Chemical Research (2013)
• Principal Investigator: Shirasaki Yoshitaka 東京大学, 理学(系)研究科(研究院), 助教 (70469948)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2015: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2014: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2013: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: １細胞生物学 / サイトカイン分泌 / 全反射顕微鏡 / 導波路照明 / １細胞計測 / 細胞分泌 / 全反射照明 / 単一細胞解析 / 分泌活性測定 / 全反射蛍光顕微鏡 / サイトカイン / イムノアッセイ

Outline of Final Research Achievements

This project focused on developing the techniques for the single-cell secretion dynamics measurement, which will contribute to understanding of the detailed mechanism of Immune systems reguration at single-cell resolution. During this project, I succeeded in developing a novel micro fabricated waveguide chip for wide field total internal reflection fluorescence microscopy (TIRFM).
This chip has the optical refraction index matching layer on both side of quartz light wave guide slide, and nano-litter well array made by PDMS on top side.Using this chip with the wave-guide illuminator, we measured single-cell cytokine secretion dynamics from hundreds of single-cells every minute for over 2hours.
This optical wave-guide based TIRFM system has great potential for revealing single-cell secretion dynamics and will contribute to develop the measurement technique for analyzing cell-cell interaction via humoral factors.

Research Products

• [Journal Article] 1細胞分泌実時間イメージング法 (2015)
  • Author(s): 白崎善隆、山岸舞、小原收、上村想太郎
  • Journal Title: 細胞工学 Volume: 34(3) Pages: 304-309
  • NAID: 40020377011
• [Journal Article] Single-Cell Imaging of Caspase-1 Dynamics Reveals an All-or-None Inflammasome Signaling Response. (2014)
  • Author(s): Liu, T., Yamaguchi, Y., Shirasaki, Y., Shikada, K., Yamagishi, M., Hoshino, K., Kaisho, T., Takemoto, K., Suzuki, T., Kuranaga, E., Ohara, O., Miura, M.
  • Journal Title: Cell Reports Volume: 8 Issue: 4 Pages: 974-982
  • DOI: 10.1016/j.celrep.2014.07.012
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Real-time single-cell imaging of protein secretion (2014)
  • Author(s): Yamagishi, M. Suzuki, N. Izawa, K. Nakahara, A. Mizuno, J. Shoji, S. Heike, T. Harada, Y. Nishikomori, R. Ohara, O.
  • Journal Title: Sci Rep Volume: 4 Issue: 1 Pages: 4736-4736
  • DOI: 10.1038/srep04736
  • Peer Reviewed / Open Access
• [Presentation] Real-time single-cell secretion imaging revealed heterogeneity of the cell secretion (2015)
  • Author(s): Yoshitaka Shirasaki, Ting Liu, Yoshifumi Yamaguchi, Mai Yamagishi, Nobutake Suzuki, Masayuki Miura, Osamu Ohara, Sotaro Uemura
  • Organizer: The 53rd Annual Meeting of the Biophysical Society of Japan
  • Place of Presentation: 金沢大学 角間キャンパス （石川県金沢市）
  • Year and Date: 2015-09-13
• [Presentation] HIGH-THROUGHPUT SINGLE-CELL SECRETION MESUREMENT ON AN OPTICAL WAVEGUDE CHIP (2014)
  • Author(s): Yoshitaka Shirasaki, Nobutake Suzuki, Mai Yamagishi, Asahi Nakahara, Shuichi Shoji and Osamu Ohara
  • Organizer: The 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences
  • Place of Presentation: テキサス、サンアントニオ
  • Year and Date: 2014-10-26 – 2014-10-30
• [Presentation] １細胞分泌実時間測定によるIL-1β非古典的分泌機序の解明 (2014)
  • Author(s): 白崎 善隆、劉 霆、山口 良文、山岸 舞、鈴木 信勇、井澤 和司、水野 潤、庄子 習一、原田 慶恵、西小森 隆太、平家 俊男、三浦 正幸、小原 收
  • Organizer: 第52回日本生物物理学会年会
  • Place of Presentation: 北海道、札幌コンベンションセンター
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] Time-resolved live-cell FluoroSpot assay on Total Internal Reflection Fluorescence Microscopy (2014)
  • Author(s): Yoshitaka Shirasaki, Nobutake Suzuki, Mai Yamagishi, Osamu Ohara
  • Organizer: EMBL Conference Series Microfluidics 2014
  • Place of Presentation: ドイツ、ハイデルベルグ
  • Year and Date: 2014-07-23 – 2014-07-25
• [Presentation] 炎症性細胞死に伴うIL-１βサイトカイン分泌の１細胞イメージング (2014)
  • Author(s): 白崎 善隆、劉 霆、山口 良文、山岸 舞、鈴木 信勇、三浦 正幸、小原 收
  • Organizer: 第23回日本Cell Death学会学術集会
  • Place of Presentation: 東京都、東京医科歯科大学
  • Year and Date: 2014-07-18 – 2014-07-19
• [Presentation] リアルタイム分泌イメージング法を用いたインフラマソーム活性化に伴うIL-1β分泌機序の解析 (2014)
  • Author(s): 白崎 善隆, 山岸 舞, 鈴木 信勇, 劉 霆, 山口 良文, 改正 恒康, 星野 克明, 三浦 正幸, 小原 收
  • Organizer: 第66回日本細胞生物学会大会
  • Place of Presentation: 奈良県、新公会堂
  • Year and Date: 2014-06-11 – 2014-06-13
• [Presentation] Real-time single-cell imaging of IL-1β secretion by Inflammasomes (2014)
  • Author(s): Yoshitaka Shirasaki, Mai Yamagishi, Kazushi Izawa, Hirotsugu Oda, Toshio Heike, Ryuta Nishikomori, Osamu Ohara
  • Organizer: CSH 2014 meeting on Gene expression & signaling in the immune system
  • Place of Presentation: ニューヨーク、コールドスプリングハーバーラボラトリー
  • Year and Date: 2014-04-22 – 2014-04-26
• [Presentation] Real-time secretion analysis 64 revealed correlation of IL-1beta release and loss of cell membrane integrity (2013)
  • Author(s): Shirasaki Y, Yamagishi M, Izawa K, Nakagawa K, Nakahara A, Suzuki N, Mizuno J, Sekiguchi T, Heike T, Nishikomori R, Shoji S, Ohara O
  • Organizer: MicroTAS2013
  • Place of Presentation: Freiburg, Germany