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 |
| 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 |