Mr. Nelson L. Sivers Profile

Nelson L. Sivers

Graduate Student

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 | 24 March 2005 Paper

Fluorescence confocal microscopy with spectrally tuned, cryogenically cooled diode lasers

Nelson Sivers, Brian Van de Workeen, Susanne Lee

Proceedings Volume 5701, (2005) https://doi.org/10.1117/12.589635

KEYWORDS: Luminescence, Semiconductor lasers, Confocal microscopy, Signal to noise ratio, Diodes, Nitrogen, Interference (communication), Liquids, Optical filters, Mirrors

Read Abstract +

In confocal microscopy, the laser excitation must be near the fluorochrome absorption peak to efficiently excite the fluorochrome. However, most fluorochromes emitting in the red to infrared have absorption and emission spectra with overlapping wavelength ranges, i.e. small Stokes shifts. As a result, the laser excitation extends into the fluorescence wavelengths hindering separation of the reflected laser signal from the information-containing fluorescence signal. Therefore, compromises are necessary: (1) the entire reflected laser spectrum is filtered, eliminating some of the fluorescence signal; or (2) the entire fluorescence signal is recorded, including unwanted reflected laser light, increasing the background noise. These compromises must be addressed, even with fluorochromes - like Cy5 - having a relatively large Stokes shift. Cryogenically cooled diode lasers eliminate the need for these compromises by tuning the output wavelength away from the fluorochrome emission. By separating the excitation and emission spectra, the confocal fluorescence signal-to-noise ratio can be increased by filtering more of the reflected laser emission, without losing valuable fluorescence information. However, this results in slightly less efficient excitation of the fluorochrome. We will present spectrophotometric analyses of fluorochrome absorption, fluorescence, and diode laser emission as a function of diode operating temperature. We will show that as the diode lasers are cooled their output power increases and more than compensates for the lower fluorochrome excitation, resulting in significantly more intense fluorescence. Thus, by tuning the diode laser, more fluorescence information and less reflected laser light reach the detector, creating images with greater intensity and less background noise.

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