Two-photon excitation imaging of pancreatic islets with various fluorescent probes.
Various fluorescent probes were assessed for investigating intact islets of Langerhans using two-photon excitation imaging. Polar fluorescent tracers applied on the outside rapidly (within 3 min) penetrated deep into the islets via microvessels. Likewise, an adenovirus carrying a Ca(2+)-sensitive green fluorescent protein mutant gene, yellow cameleon 2.1, was successfully transfected and enabled ratiometric cytosolic Ca(2+) measurement of cells in the deep layers of the islets. Interestingly, FM1-43, which is lipophilic and does not permeate the plasma membrane, also rapidly reached deep cell layers of the islets. In contrast, lipophilic fluorescent probes that permeate the plasma membrane (for example, fura-2-acetoxymethyl and BODIPY-forskolin) accumulated in the superficial cell layers of the islets, even 30 min after application. Thus, two-photon excitation imaging of pancreatic islets is a promising method for clarifying signaling mechanisms of islet cells, particularly when it is combined with membrane-impermeable probes. In addition, our data suggest that membrane-permeable antagonists may affect only the superficial cell layers of islets, and so their negative effects should be interpreted with caution.
Takahashi
Noriko
N
Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Japan. norikomd@nips.ac.jp
Nemoto
Tomomi
T
Kimura
Ryoichi
R
Tachikawa
Akira
A
Miwa
Akiko
A
Okado
Haruo
H
Miyashita
Yasushi
Y
Iino
Masamitsu
M
Kadowaki
Takashi
T
Kasai
Haruo
H
D000818:Animals; D001896:Boron Compounds / Q000493:pharmacokinetics; D005576:Colforsin / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D016257:Fura-2 / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D007515:Islets of Langerhans / Q000502:physiology*; D007546:Isoquinolines / Q000493:pharmacokinetics; D008055:Lipids; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D017785:Photons; D011726:Pyridinium Compounds / Q000493:pharmacokinetics; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics; D014867:Water
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; C073804:FM1 43; D005456:Fluorescent Dyes; D007546:Isoquinolines; D008055:Lipids; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D014867:Water; D005576:Colforsin; C017475:lucifer yellow; D016257:Fura-2