Methodological considerations for global analysis of cellular FLIM/FRET measurements

Abstract

Abstract. Global algorithms can improve the analysis of fluorescence energy transfer (FRET) measurement based on fluorescence lifetime microscopy. However, global analysis of FRET data is also susceptible to experimental artifacts. This work examines several common artifacts and suggests remedial experimental protocols. Specifically, we examined the accuracy of different methods for instrument response extraction and propose an adaptive method based on the mean lifetime of fluorescent proteins. We further examined the effects of image segmentation and a priori constraints on the accuracy of lifetime extraction. Methods to test the applicability of global analysis on cellular data are proposed and demonstrated. The accuracy of global fitting degrades with lower photon count. By systematically tracking the effect of the minimum photon count on lifetime and FRET prefactors when carrying out global analysis, we demonstrate a correction procedure to recover the correct FRET parameters, allowing us to obtain protein interaction information even in dim cellular regions with photon counts as low as 100 per decay curve.

Quantifying intracellular protein binding thermodynamics during mechanotransduction based on FRET spectroscopy

“…One solution is to set an intensity threshold at a high level such that low intensity pixels are ignored but that often left us with few workable pixels per cells. We have developed an FR scaling method that can correct for this skew, allowing us to work with many more pixels at lower intensity, as discussed in our previous publication [43]. In addition to FR scaling, we also found that mean filtering is very effective in removing noise in our thermodynamic parameter calculations.…”

“…FR values can be skewed at low intensity pixels during global analysis [43]. One solution is to set an intensity threshold at a high level such that low intensity pixels are ignored but that often left us with few workable pixels per cells.…”

“…For lifetime imaging [41-43], the laser is tuned to 890nm with 40mW power to excite GFP paxillin before the objective, resulting in low power at the sample so as to minimize photobleaching and cell damage. In order to minimize contributions from scattered laser light and autofluorescence, the cell's fluorescence signal is selected with a short-pass Schott BG-39 filter and short-pass 700nm filter from Chroma (Rockingham, VT).…”

“…FLIM/FRET data were analyzed using the global analysis algorithm that was described extensively in our previous publication [43]. Briefly, from GPax transfected cells, the non-FRETting lifetime, τ D , of GFP is obtained using a single-exponential fit to lifetime decays.…”

“…The bound GPax will have shortened lifetime due to the presence of FRET to mCherry. Quantitative FRET imaging further allows a measure of the relative concentrations of bound, C FATmCh – GPax , and free, C GPax , paxillin [43]. Intensity measurements coupled with FCS calibration at the green and red emission channels quantify the number concentrations of the green ( C FATmCh – GPax and C GPax ) and red ( C FATmCh – GPax and C FATmCh ) fluorescent species, respectively.…”