We present three novel reproducible methodologies for the quantification of changes in actin organization from microscope images. Striation and integrative analysis were devised for the investigation of trans-cellular filaments and F-actin localization, respectively, in response to physiological or mechanical actin-modulatory conditions. Additionally, the Parker-Qusous (PQ) formula was developed as a measure of total quantity of F-actin, independent of cell volume changes, whereby fluorescence intensity was divided by the cube root of cell volume, squared. Values obtained were quantified in Mauricean Units (Mu; pixel/mu m3). Upon isolation, there was a 49% decrease in total F-actin fluorescence from 1.91 +/- 0.16 pixel/mu m3 (Mu) to 0.95 +/- 0.55 Mu, whereas upon culture, an apparent increase in total fluorescence was deemed insignificant due to an increase in average cell volume, with a rise, however, in striation units (StU) from 1 +/- 1 to 5 +/- 1 StU/cell, and a decrease in percentage cortical fluorescence to 30.45% +/- 1.52% (P = 7.8 x 10(-5)). Freshly isolated chondrocytes exhibited a decrease in total F-actin fluorescence to 0.61 +/- 0.05 Mu and 0.32 +/- 0.02 Mu, 10 min posthypertonic and hypotonic challenges, respectively. Regulatory volume decrease was inhibited in the presence of REV5901 with maintenance of actin levels at 1.15 Mu. Following mechanical impact in situ, there was a reduction in total F-actin fluorescence to 0.95 +/- 0.08 Mu and 0.74 +/- 0.06 Mu under isotonic and hypotonic conditions, respectively, but not under hypertonic conditions. We report simple methodologies for quantification of changes in actin organization, which will further our understanding of the role of actin in various cellular stress responses. These techniques can be applied to better quantify changes in localization of various proteins using fluorescent labeling. Microsc. Res. Tech. 2012. (C) 2012 Wiley Periodicals, Inc.