Checklist for optimizing images for quantitation
| Increase signal: |
| ✓ Choose a bright (high quantum yield, high extinction coefficient) and photo-stable fluorophorea |
| ✓ Image through a clean No. 1.5 coverslipb |
| ✓ Mount specimen as close to the coverslip as possiblec |
| ✓ Use high NA clean objective lens with lowest acceptable magnificationd |
| ✓ Choose fluorescence filter sets that match fluorophore spectrad |
| ✓ Align arc lamp for Koehler illuminationd |
| ✓ For fixed specimens, use a glycerol-based mounting medium containing anti-photobleaching inhibitors3 |
| ✓ Remove DIC Wollaston prism and analyzer from light pathe |
| ✓ Use a cooled CCD camera with at least 60% quantum efficiencyd |
| ✓ Use camera binningd |
| Decrease noise: |
| ✓ Use a cooled CCD camera with less than 8 electrons readout noise and negligible dark noisef |
| ✓ Use amplification (e.g., EM-CCDs) only when signal is limitingf |
| ✓ Increase signal (see above) to reduce relative contribution of Poisson noisef |
| Decrease background: |
| ✓ Clean coverslips and opticse |
| ✓ Perfect fluorophore labeling protocol to minimize nonspecific labelingg |
| ✓ Mount specimens in minimally fluorescent medium (e.g., without phenol red)d |
| ✓ Use band-pass filter sets that block autofluorescenced |
| ✓ Turn off the room lightsd |
| ✓ Close down the field diaphragm to illuminate only the object of interestd |
| ✓ When out-of-focus fluorescence is high, consider using deconvolution, confocal, or TIRFh |
| Increase signal: |
| ✓ Choose a bright (high quantum yield, high extinction coefficient) and photo-stable fluorophorea |
| ✓ Image through a clean No. 1.5 coverslipb |
| ✓ Mount specimen as close to the coverslip as possiblec |
| ✓ Use high NA clean objective lens with lowest acceptable magnificationd |
| ✓ Choose fluorescence filter sets that match fluorophore spectrad |
| ✓ Align arc lamp for Koehler illuminationd |
| ✓ For fixed specimens, use a glycerol-based mounting medium containing anti-photobleaching inhibitors3 |
| ✓ Remove DIC Wollaston prism and analyzer from light pathe |
| ✓ Use a cooled CCD camera with at least 60% quantum efficiencyd |
| ✓ Use camera binningd |
| Decrease noise: |
| ✓ Use a cooled CCD camera with less than 8 electrons readout noise and negligible dark noisef |
| ✓ Use amplification (e.g., EM-CCDs) only when signal is limitingf |
| ✓ Increase signal (see above) to reduce relative contribution of Poisson noisef |
| Decrease background: |
| ✓ Clean coverslips and opticse |
| ✓ Perfect fluorophore labeling protocol to minimize nonspecific labelingg |
| ✓ Mount specimens in minimally fluorescent medium (e.g., without phenol red)d |
| ✓ Use band-pass filter sets that block autofluorescenced |
| ✓ Turn off the room lightsd |
| ✓ Close down the field diaphragm to illuminate only the object of interestd |
| ✓ When out-of-focus fluorescence is high, consider using deconvolution, confocal, or TIRFh |