Guide for Using Fluorescence Microscopy to Quantify the Spread Area of Fixed Cells
|Publication Date:||1 January 2014|
This guide describes several measurement and technical issues involved in quantifying the spread area of fixed cells. Cell spreading and the distribution of cell spread areas of a population of cells are the result of a biological response that is dependent on intracellular signaling mechanisms and the characteristics of cell adhesion to a surface. Cell spread area is a morphological feature that can be responsive to alteration in the metabolic state or the state of stress of the cells. Changes in cell spread area can also indicate an alteration in the adhesion substrate that may be due to differences in manufacturing of the substrate material or be in response to extracellular matrix secretions. High quality measurement of cell spread area can serve as a useful metric for benchmarking and detecting changes cell behavior under experimental conditions.
The measurement described in this document is based on the use of fluorescence microscopy imaging of fixed cells and the use of image analysis algorithms to extract relevant data from the images. To produce robust cell spread area measurements, technical details involved in sample preparation, cell staining, microscopy imaging, image analysis and statistical analysis should be considered. Several of these issues are discussed within this document.
This standard is meant to serve as a guide for developing methods to reliably measure the area to which cells spread at a surface. This surface can be conventional tissue culture polystyrene or sophisticated engineered biomaterial surfaces. An example of a detailed procedure to measure the spreading area of cells on a tissue culture polystyrene surface is provided in the appendix section.
Cell morphology features such as cell spreading area and perimeter are generally reported in units of length. For example, spreading area per cell (that is, cell spread area) is likely reported in units of μm2. A spatial calibration standard is required to convert between numbers of pixels in a CCD camera image to μm2 as an SI unit.
This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
Sodium azide is used as a anti-bacterial reagent in the slide mounting media. This preserves the integrity of the mounting media. The toxicity of this reagent (for example, MSDS) should be considered before use of this reagent in large scale slide mounting procedures.