In the past, several approaches have been developed to obtain quantitative readouts from fluorescent images (reviewed by 37 (link),38 (link)). With the possibility to express fluorescence proteins from their endogenous locus, these methods can now be used for proteomics studies in almost any eukaryotic cell. Typically, quantitative microscopy relies on the computation of a calibration function to convert fluorescence intensities to physical quantities. The calibration function can be computed using intra or extracellular fluorescent standards 39 (link),40 (link), or, as we present in this protocol, direct measurements of the concentrations within cells 41 (link)–43 (link). Protein numbers can also be directly measured by step-wise photobleaching 44 (link),45 , photon emission statistics 45 –47 (link), or, in compartments with freely diffusing components, by analyzing fluorescence fluctuations 5 (link),6 (link).
Extracellular fluorescent standards require purified FP, the same FP as used for tagging the POI. Depending on the application, solutions of known concentrations 48 (link) or diffraction limited complexes with known stoichiometry (e.g. Virus like particles 49 (link)) are measured along with the cells expressing the FP tagged POI. The method is relatively simple but requires additional sample preparation, knowledge of stoichiometry and concentration, and most importantly does not ensure whether the intra- and extracellular excitation and emission properties of the FP are the same 38 (link). An alternative is the measurement of total protein amounts with quantitative immunoblotting 37 (link),39 (link) or Mass-spectrometry 28 (link),36 (link). Using additional specialized equipment and reagents, both methods provide an estimate of the total protein amount that can be related to the total cell fluorescence. However, measurements are at the population level and the total amount of protein per cell needs to be extrapolated. Inaccuracies arise in the conversion of immuno reactivities to protein amount, estimate of total number of cells per immunoblot, and when the total protein level strongly differs between cells (e.g. cell cycle variations).
Intracellular calibration standards circumvent the issue of differences in fluorescence in an extracellular environment, but require an a priori knowledge of the stoichiometry or concentration. This method has been extensively used in yeast with proteins on the kinetochore as calibration standard 40 (link),48 (link),50 . In mammalian cells an intracellular calibration standard has not been agreed upon making the method not yet applicable.
Direct measurement of protein numbers using discrete photobleaching events is not suited for long time imaging. However, it can be used to define intracellular calibration standards 45 . Counting by photon statistics requires a specialized hardware with at least four detectors and bright fluorescent probes 46 ,47 (link). The possibility to tag proteins with organic dyes in live cells using endogenous expression of SNAP 51 (link), CLIP 52 (link) or Halo 53 (link) tagged proteins could make the method more accessible in the future.
In this protocol the calibration curve is computed from FCS concentration measurements in cells. Compared to methods that use calibration standards the method requires a specialized confocal setup that can then be used for further imaging. Data analysis requires parameter estimation and fitting. Thanks to the availability of software solutions (e.g. QuickFit3http://www.dkfz.de/Macromol/quickfit ; SimFCS, https://www.lfd.uci.edu/globals/ ; the Supplementary software in this protocol Supplementary Software 1 –4 ) the user can perform the analysis without a specialized knowledge of FCS.
Extracellular fluorescent standards require purified FP, the same FP as used for tagging the POI. Depending on the application, solutions of known concentrations 48 (link) or diffraction limited complexes with known stoichiometry (e.g. Virus like particles 49 (link)) are measured along with the cells expressing the FP tagged POI. The method is relatively simple but requires additional sample preparation, knowledge of stoichiometry and concentration, and most importantly does not ensure whether the intra- and extracellular excitation and emission properties of the FP are the same 38 (link). An alternative is the measurement of total protein amounts with quantitative immunoblotting 37 (link),39 (link) or Mass-spectrometry 28 (link),36 (link). Using additional specialized equipment and reagents, both methods provide an estimate of the total protein amount that can be related to the total cell fluorescence. However, measurements are at the population level and the total amount of protein per cell needs to be extrapolated. Inaccuracies arise in the conversion of immuno reactivities to protein amount, estimate of total number of cells per immunoblot, and when the total protein level strongly differs between cells (e.g. cell cycle variations).
Intracellular calibration standards circumvent the issue of differences in fluorescence in an extracellular environment, but require an a priori knowledge of the stoichiometry or concentration. This method has been extensively used in yeast with proteins on the kinetochore as calibration standard 40 (link),48 (link),50 . In mammalian cells an intracellular calibration standard has not been agreed upon making the method not yet applicable.
Direct measurement of protein numbers using discrete photobleaching events is not suited for long time imaging. However, it can be used to define intracellular calibration standards 45 . Counting by photon statistics requires a specialized hardware with at least four detectors and bright fluorescent probes 46 ,47 (link). The possibility to tag proteins with organic dyes in live cells using endogenous expression of SNAP 51 (link), CLIP 52 (link) or Halo 53 (link) tagged proteins could make the method more accessible in the future.
In this protocol the calibration curve is computed from FCS concentration measurements in cells. Compared to methods that use calibration standards the method requires a specialized confocal setup that can then be used for further imaging. Data analysis requires parameter estimation and fitting. Thanks to the availability of software solutions (e.g. QuickFit3
