Mr. Junichi Nishimura Profile

Junichi Nishimura

at Olympus Corp

SPIE Involvement:

Author

Publications (1)

This will count as one of your downloads.

You will have access to both the presentation and article (if available).

DOWNLOAD NOW

This content is available for download via your institution's subscription. To access this item, please sign in to your personal account.

Email or Username Forgot your username?

Password Forgot your password?

Show

Keep me signed in

No SPIE account? Create an account

Proceedings Article | 14 February 2007 Paper

Cross-talk-free fluorescence cross-correlation spectroscopy by the switching method

Junichi Nishimura, Yasuo Takahashi, Masataka Kinjo, Atsushi Miyawaki

Proceedings Volume 6444, 64440I (2007) https://doi.org/10.1117/12.699992

KEYWORDS: Switching, Signal detection, Proteins, Luminescence, Sensors, Molecular interactions, Cell death, Data acquisition, In vitro testing, Fluorescence spectroscopy

Read Abstract +

Fluorescence cross correlation spectroscopy (FCCS) is now widely used as a powerful technique to analyze molecular interactions in vitro and also in vivo. This method basically requires two laser excitations for two target molecules distinctly labeled with fluorophores. Coincidence of the two molecules can be analyzed at detectors, corresponding to each emission wavelength. However, due to spectral overlap of the two fluorophores, a cross-talk signal causes critical difficulties of data assessments by false positive cross correlations. To overcome this problem, we have developed a new cross-talk free FCCS system; the excitation laser pulses are switched alternatively by modulating of a conventional Acousto-optic tunable filter (AOTF) in the excitation laser unit. Alternatively, the specimen is illuminated with two different wavelengths, and cross correlation can be correctly calculated by this switching state, and therefore, we can eliminate spectra cross-talk. In this report, we show system outlines and demonstrate the feasibility of the switching FCCS for enzymatic cleavage of proteins in living cells. A fusion protein of two fluorophores (EGFP and mRFP) inserted with cleavage site of caspase3 was expressed in HeLa cells, and the cleaving process during apoptotic cell death was monitored. We measured a relative cross correlation amplitude obtained by FCCS with and without switching system. More significant changing of relative cross correlation amplitude by protein cleavage was detected with switching than that without switching. This result indicates that the switching FCCS measurement can exclude cross-talk signal. Hence, the switching FCCS enables a reliable analysis of molecular interaction in living cells with higher sensitivity than FCCS without switching.

My Library

You currently do not have any folders to save your paper to! Create a new folder below.

Folder Name

Folder Description

View contact details

UPDATE YOUR PROFILE

Is this your profile? Update it now.

Sign into your SPIE.org account

Don’t have a profile and want one?

Create an account on SPIE.org

Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks. You are receiving this notice because your organization may not have SPIE eBooks access.*

*Shibboleth/Open Athens users─please sign in to access your institution's subscriptions.

To obtain this item, you may purchase the complete book in print or electronic format on SPIE.org.

ORGANIZATIONAL
Sign in with credentials provided by your organization.

Organizational Username

Organizational Password

Show/Hide Password

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available

Members:

Non-members: ADD TO CART

Keywords/Phrases

Search In:

Publication Years