A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy

Eisuke Adachi, Yutaka Kazoe, Yohei Sato, Yuko Suzuki, Tetsumei Urano, Takehiko Ueyama, Naoaki Saito, Viacheslav O. Nikolaev, Martin J. Lohse, Makoto Tominaga, Hideo Mogami

Research output: Contribution to journalArticle

3 Citations (Scopus)


Physiological phenomena are regulated by multiple signal pathways upon receptor stimulation. Here, we have introduced a new technique with a combination of prism-based total internal reflection fluorescence microscopy (PBTIRFM) and epifluorescence microscopy (EPI) to simultaneously monitor multiple signal pathways. This instrumentation allows us to visualize three signal pathways, Ca2+, cyclic adenosine monophosphate (cAMP)/ protein kinase A (PKA), and diacylglycerol (DAG)/protein kinase C (PKC) signals in living cells. Three fluorescent indicators were employed for this purpose: (1) Fura-2 AM as a calcium sensor; (2) Epacl-camp, a cyan fluorescent protein-yellow fluorescent protein fluorescence resonance energy transfer-based cAMP indicator, as a cAMP sensor; and (3) C1-tagged monomeric red fluorescent protein, a tandem DAG-binding domain of PKC γ, as a DAG sensor or myristoylated alanine-rich C kinase substrate-tagged DsRed for the PKC activation pathway. The DAG signal was monitored by PBTIRFM, whereas the Ca 2+ and cAMP signals were monitored by EPI, Adenosine trisphosphate resulted in generation of all three second messengers in triple probe-loaded Cos-7 cells. The spectral overlap between these signal probes was evaluated by means of linear unmixing. Forskolin also evoked Ca2+, cAMP/PKA, and DAG/PKC signals whereas acetylcholine activated Ca2+ and DAG/PKC signals as well as inhibiting cAMP generation in triple probe-loaded insulin-secreting cells. Thus, the optical observation system combining PBTIRFM and EPI offers a great advance in analyzing interplay of multiple signaling pathways, such as these second messengers, upon G-proteincoupled receptor stimulation in living cells.

Original languageEnglish
Pages (from-to)227-234
Number of pages8
JournalPflugers Archiv European Journal of Physiology
Issue number1
Publication statusPublished - 2009 Nov 1



  • G-protein-coupled receptor
  • Live cell imaging
  • Prism-based total internal reflection fluorescence microscopy linear unmixing
  • Second messenger

ASJC Scopus subject areas

  • Physiology
  • Clinical Biochemistry
  • Physiology (medical)

Cite this