Automated image analysis to quantify the subnuclear organization of transcriptional coregulatory protein complexes in living cell populations

Ty C. Voss; Ignacio A. Demarco; Cynthia F. Booker; Richard N. Day

doi:10.1117/12.529679

14 June 2004 Automated image analysis to quantify the subnuclear organization of transcriptional coregulatory protein complexes in living cell populations

Ty C. Voss, Ignacio A. Demarco, Cynthia F. Booker, Richard N. Day

Author Affiliations +

Proceedings Volume 5329, Genetically Engineered and Optical Probes for Biomedical Applications II; (2004) https://doi.org/10.1117/12.529679
Event: Biomedical Optics 2004, 2004, San Jose, CA, United States

Abstract

Regulated gene transcription is dependent on the steady-state concentration of DNA-binding and coregulatory proteins assembled in distinct regions of the cell nucleus. For example, several different transcriptional coactivator proteins, such as the Glucocorticoid Receptor Interacting Protein (GRIP), localize to distinct spherical intranuclear bodies that vary from approximately 0.2-1 micron in diameter. We are using multi-spectral wide-field microscopy of cells expressing coregulatory proteins labeled with the fluorescent proteins (FP) to study the mechanisms that control the assembly and distribution of these structures in living cells. However, variability between cells in the population makes an unbiased and consistent approach to this image analysis absolutely critical. To address this challenge, we developed a protocol for rigorous quantification of subnuclear organization in cell populations. Cells transiently co-expressing a green FP (GFP)-GRIP and the monomeric red FP (mRFP) are selected for imaging based only on the signal in the red channel, eliminating bias due to knowledge of coregulator organization. The impartially selected images of the GFP-coregulatory protein are then analyzed using an automated algorithm to objectively identify and measure the intranuclear bodies. By integrating all these features, this combination of unbiased image acquisition and automated analysis facilitates the precise and consistent measurement of thousands of protein bodies from hundreds of individual living cells that represent the population.

Citation Download Citation

Ty C. Voss, Ignacio A. Demarco, Cynthia F. Booker, and Richard N. Day "Automated image analysis to quantify the subnuclear organization of transcriptional coregulatory protein complexes in living cell populations", Proc. SPIE 5329, Genetically Engineered and Optical Probes for Biomedical Applications II, (14 June 2004); https://doi.org/10.1117/12.529679

ACCESS THE FULL ARTICLE

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: $17.00

Non-members: $21.00 ADD TO CART

PROCEEDINGS
8 PAGES

DOWNLOAD PAPER SAVE TO MY LIBRARY

GET CITATION

RIGHTS & PERMISSIONS

Get copyright permission Get copyright permission on Copyright Marketplace

KEYWORDS

Proteins

Image analysis

Luminescence

Computer programming

Spherical lenses

Algorithm development

Green fluorescent protein

Show All Keywords

Keywords/Phrases

Search In:

Publication Years