Quantitative photo activated localization microscopy: unraveling the effects of photoblinking

Abstract

In this work we discuss how to use photophysical information for improved quantitative measurements using Photo Activated Localization Microscopy (PALM) imaging. We introduce a method that reliably estimates the number of photoblinking molecules present in a biological sample and gives a robust way to quantify proteins at the single-cell level from PALM images. We apply this method to determine the amount of β2 adrenergic receptor, a prototypical G Protein Coupled Receptor, expressed on the plasma membrane of HeLa cells.

Figure 1. Protein counting in vitro and in silico.

a) Experimental (markers) and simulated (blue line) total numbers of mEos2 molecules localized as a function of dark time t_d, where dark time t_d is time allowed for a molecule to go dark before being identified as a different molecule when fluorescence resumes. Simulated counts, out of total, ascribed respectively to missed counts (green), multiple counts (pink) and noise (gray). For all samples the duration of the acquisition is 20000×50 ms frames. Red curve shows best fit to data for dark time t_d values comprised between 0.05 s and 2 s. If no missed counts were to occur, the asymptote of the decaying curve of the observed counts would converge to the effective number of molecules present in the sample. Fitting to the equation (1) yields t_off = 0.2±0.1 s and <n_blinks> = 0.7±0.1 consistent with what reported respectively in b) and c) and Figure S2. N from the fit yields N = 121±6 molecules/µm² whereas the total density of the simulated sample is 135 molecules/µm² including noise counts, giving a 10% agreement. b) Histogram of the number of times an mEos2 molecule undergoes photoblinking before definitive photobleaching. Experimental values and single exponential best fits are shown, 1/e decay values is <n__blinks> = 1.05±0.11. c) Histogram of the measured off-times t_off-high = 0.1±0.01 s d) Left: single molecule kymographs of 6 blinking/reactivating molecules. The duration of the dark times spans from 1 to 4 units (0.05–0.15 s), resulting in the off-times histograms on the right. If now molecular localizations are performed considering a dark time t_d as 0.05 s, 0.1 s and 0.15 s the resulting number of counts are 13, 9, and 7 respectively. The difference between these values corresponds to the values of the off-time histogram e) Calibration curve for the concentrations extracted by fitting eq. 1 to PALM data. Blue circles: reconstructed concentration from PALM experiment (0.06 molecules/µm²/nM in TIRF) vs absorbance-derived concentration and best fit. Each point corresponds to the average of PALM experiments on three different samples. Red markers display the ratio between the concentration extracted directly from PALM at t_d = 0.05 s and the concentration obtained using our method.

Figure 2. Semi-empirical fit to simulated data.

Simulated PALM experiments on in-silico samples generated at three different densities while keeping the photophysical parameters constant. a) 10, b) 100 and c) and d) 1000 molecules/µm² . The simulated counts curves are fitted to equation (1), using only the first 5 points of the curve, up to a t_d = 50*10 ms = 0.5 s. a) 10 molecules/µm² we observe an error of 1 particle/µm² between the value extracted from the fit and the number of molecules present in the in-silico experiment, b) 100 molecules/µm² we observe an error of 2% that goes up to c) 20% at 1000/µm². However, upon reducing the photoactivation power in the densest sample d) this error reduces to approximately 5%. By reducing the photoactivation rate of a factor three, the fit can be extended (blue curve) up to 30 photoactivation cycles obtaining an error of 7%.

Figure 3. Comparison of counts vs t_d curve for three photoactivatable fluorescent proteins.

Normalized number of localized molecules vs t_d for three different photo-activatable fluorescent proteins, PA-GFP, Dronpa and mEos2.

Figure 4. Estimation of molecular density of β2-adrenergic receptor.

a) Red Markers - number of localized molecules as a function of dark time t_d with density estimation in the cell. Molecules localized in a 1 µm² region of the plasma membrane of the fixed HeLa cell expressing β2-mEos2 displayed in b). Blue line -fit using equation (1) limited to the overcounting regime yields an estimated density of approximately 60 molecules/µm², t_off = 0.36±0.05 s and n_blink = 0.63±0.07. b) TIRF-PALM image of a region of the cell expressing β2-mEos2 (t_d = 10 s, localization precision<35 nm) in c) its corresponding TIRF image d), e), f), and g) insets from a) displaying isolated localized receptors by a gaussian function for 4 different dark time values d) t_d = 0.5 s e) t_d = 1 s f) t_d = 5 s and g) t_d = 10 s.