Table II.

Protocol for quantitation of fluorescence intensity values

1. Acquire optical images

• Set up specimen and imaging system for optimal signal detection, low background, and low noise (Table I)

2. Acquire digital images

• Use software to monitor intensity values in the image to choose the best acquisition settingsa

• Use full dynamic range of the camera for fixed specimensa

• For live-cell work, it is often necessary to sacrifice SNR to minimize specimen exposure to light and maintain cell health and viabilitya

• Consider binning to increase SNRa

• Avoid high camera gain when a large dynamic range is neededa

• Avoid saturating pixels in the imagea

• Eliminate or minimize exposure of specimen to fluorescence excitation light prior to image acquisitiona

• Focus carefully, preferably with phase or DICb

3. Store images

• Always save the raw imagesc

• Use either no compression or lossless compressionc

4. Process images

• Use flat-field correction to correct for uneven illuminationd

• Be sure any other image processing used prior to quantitation preserves relative intensity valuesc,d

5. Analyze images

• Subtract local background value from intensity measurementse

• Do not measure intensity values on compressed or pseudo-colored imagesc

• Validate image segmentation and analysis methodf

• Calculate and report the error in your measurementsdg

^a(Waters, 2007)

^b(Inoué and Spring, 1997)

^c(Russ, 2007)

^d(Wolf et al., 2007)

^e(Hoffman et al., 2001)

^f(Dorn et al., 2008)

^g(Cumming et al., 2007)