Here, we present a protocol that involves genetically coupled spectrally distinct photoactivatable and fluorescent proteins. These fluorescent protein chimeras permit quantification of the PA-FP fraction that is photoactivated to be fluorescent, i.e., the photoactivation efficiency. The protocol reveals that different modes of photoactivation yield different photoactivation efficiencies.
Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells and protein ensembles. Thus far, no method has been available to quantify in bulk and in live cells how many of the PA-FPs expressed are photoactivated to fluoresce.
Here, we present a protocol involving internal rulers, i.e., genetically coupled spectrally distinct (photoactivatable) fluorescent proteins, to ratiometrically quantify the fraction of all PA-FPs expressed in a cell that are switched on to be fluorescent. Using this protocol, we show that different modes of photoactivation yielded different photoactivation efficiencies. Short high-power photoactivation with a confocal laser scanning microscope (CLSM) resulted in up to four times lower photoactivation efficiency than hundreds of low-level exposures applied by CLSM or a short pulse applied by widefield illumination. While the protocol has been exemplified here for (PA-)GFP and (PA-)Cherry, it can in principle be applied to any spectrally distinct photoactivatable or photoconvertible fluorescent protein pair and any experimental set-up.
In 2002, the first broadly applicable photoactivatable (PA-GFP1) and photoconvertible (Kaede2) fluorescent proteins were described. These optical highlighter fluorescent proteins change their spectral properties upon irradiation with UV-light, i.e., they become bright (photoactivatable fluorescent proteins, i.e., PA-FPs), or change their color (photoconvertible FPs). To date, several reversible and irreversible photoactivatable and photoconvertible fluorescent proteins have been developed3,4. In ensemble or bulk studies, optical highlighters have been used to study the dynamics of entire cells or proteins, and the connectivity of subcellular compartments. Furthermore, optical highlighters enabled single-molecule based superresolution imaging techniques such as PALM5 and FPALM6.
Although the photochemical processes during photoactivation or -conversion have been described for many optical highlighters and even crystallographic structures before and after photoactivation/ -conversion have been made available7,8, the underlying photophysical mechanism of photoactivation and -conversion is not completely understood. Furthermore, thus far only crude estimates exist of the efficiency of photoactivation and -conversion, that is, the fraction of fluorescent proteins expressed that is actually photoconverted or photoactivated to be fluorescent. In vitro ensemble studies have been reported quantifying the shift in absorption spectra and the amount of native and activated protein in a gel9,10,11.
Here, we present a protocol involving fluorescent protein chimeras to assess the fraction of photoactivated fluorescent proteins in bulk and in live cells. Whenever working with genetically encoded fluorescent proteins, the absolute amount of protein expressed varies from cell to cell and is unknown. If one cell expressing a PA-FP shows a brighter signal after photoactivation than another cell, it cannot be differentiated if this brighter signal is due to higher expression of the PA-FP or a more efficient photoactivation of the PA-FP. To standardize the expression level in cells, we introduce internal rulers of genetically coupled spectrally distinct fluorescent proteins. By coupling the genetic information of a photoactivatable fluorescent protein to a spectrally distinct always-on fluorescent proteins, internal rulers are created that will still be expressed to an unknown total amount but in a fixed and known relative amount of 1:1. This strategy allows the quantitative characterization of different UV-light photoactivation schemes, i.e., the assessment of the relative amount of PA-FPs that can be photoactivated with different modes of photoactivation, and thereby permits to define photoactivation schemes that are more effective than others. Furthermore, this strategy allows in principle the assessment of the absolute quantification of the photoactivated PA-FP fraction. To this end, it is important to realize that the presented ensemble studies are intensity-based which makes the analysis more complex as laid out in this protocol. Parameters determining the measured fluorescent intensity, i.e., different molecular brightness, absorbance and emission spectra and FRET effects, need to be considered when comparing fluorescence intensities of different fluorescent proteins.
The presented ratiometric intensity-based quantification of photoactivation efficiency is exemplified for PA-GFP and PA-Cherry in live cells, but is in principle broadly applicable and can be used for any photoactivatable fluorescent protein under any experimental condition.
1. Plasmid Construction
2. Cell Culture and Transfection
3. Imaging and Photoactivation
4. Image Analysis and Algorithm for Ratiometric Intensity-based Quantification of Photoactivation Efficiency
The protocol presented here shows the ratiometric quantification of the fraction of fluorescent proteins that are photoactivated to be fluorescent (Figure 1). This fraction differs depending upon the mode of photoactivation.
A typical result using short time high-power photoactivation with a confocal laser scanning microscope (CLSM) is shown in Figure 2c. After titrating the laser power as measured at the objective lens by pixel dwell time and ATOF transmission, the maximum photoactivation efficiency for PA-GFP was about 8% and for PA-Cherry about 16%. The comparably low photoactivation efficiency of PA-GFP and PA-Cherry may be explained by simultaneous photoactivation and -destruction when exposed to a continuous deterministic stream of photons by CLSM. The shorter fluorescence lifetimes and the different, right-shifted absorption spectra of the red PA-FPs may contribute to the higher photoactivation efficiency of PA-Cherry compared to PA-GFP.
Using low laser power and hundreds of iterations, a higher photoactivation efficiency can be achieved. 450 iterations of UV-light delivered by a CLSM over a total of 4 min yielded a photoactivation efficiency of 29% for PA-GFP (Figure 3c). The higher photoactivation efficiency with repetitive exposure to UV-light photons may suggest a multi-step photoactivation process. Alternatively, the applied UV-light is strong enough to photoactivate but not enough to photodestruct which leads cumulative over time to a higher fraction of photoactivated fluorescent proteins.
With widefield illumination, the fluorophores are stochastically and repetitively exposed to 405-nm photons. Here, exposure for only 250 ms yielded a 29% photoactivation efficiency for PA-GFP.
Figure 1: Concept of how to determine photoactivation efficiency in bulk and in live cells. By coupling spectrally distinct fluorescent proteins, internal rulers are created which allow for the ratiometric intensity-based assessment of photoactivation efficiency. Measured intensities of PA-Cherry and PA-GFP were related to expected intensities. Expected intensities were derived from determining the fluorescence intensity of the always-on fluorescent proteins in the GFP-Cherry, GFP-PA-Cherry or PA-GFP-Cherry chimeras prior to photoactivation. Figure modified from Renz and Wunder 201717. Please click here to view a larger version of this figure.
Figure 2: Bulk photoactivation of PA-GFP-Cherry (a) and GFP-PA-Cherry (b) as instantaneously and completely as possible using a confocal laser-scanning microscope in live cells. 8% of PA-GFP expressed was photoactivated with a pixel dwell time of 2 μs and an AOTF transmission of 38% which result in laser power of 90 μW, as measured at the objective lens and 3 iterations (c). Increasing the 405-nm laser power did not increase photoactivation efficiency. Figure modified from Renz and Wunder 201717. Please click here to view a larger version of this figure.
Figure 3: Iterative low-power photoactivation with a confocal laser-scanning microscope (a) and short high-power widefield illumination (b) yield higher photoactivation efficiencies. 29% of PA-GFP was photoactivated with a pixel dwell time of 2 μs and an AOTF transmission of 6%, which result in laser power of 40 μW, as measured at the objective lens and 450 iterations (c). Figure modified from Renz and Wunder 201717. Please click here to view a larger version of this figure.
So far, no method existed to determine in bulk the fraction of PA-FPs expressed in live cells that is photoactivated to be fluorescent. The presented protocol can be used for any spectrally distinct fluorescent protein pair. While exemplified here for the irreversible PA-FPs PA-GFP and PA-Cherry, this approach is in principle applicable to photoconvertible proteins as well. The spectrally distinct fluorescent protein, however, must be selected carefully to minimize spectral overlap given that photoconvertible fluorescent proteins shift their absorbance and emission spectra, e.g. from green to red fluorescence.
As outlined above, it is important to state that the presented approach is ratiometric and intensity-based. It can be used to standardize the unknown expression level in cells and define relative differences in photoactivation efficiency by different modes of photoactivation. The protocol can also be used to assess the absolute fraction of photoactivated PA-FPs. Then, different spectral properties of different FPs need to be taken into account.
The molecular brightness (MB) is the product of quantum yield (QY), extinction coefficient (EC) and percent absorbance at the given excitation wavelength relative to the absorbance peak. For Cherry12 and PA-Cherry13, respective values of QY and EC have been published. The percent absorbance at the given excitation wavelength of 543 nm relative to absorbance peak is 0.5 and 0.7, respectively.
MBCherry = 0.22 * 72,000 * 0.5 = 7,920
MBPA-Cherry = 0.46 * 18,000 * 0.7 = 5,796
Thus, the lower molecular brightness of PA-Cherry compared to Cherry can be taken into account by dividing IRed_expected by 1.37 (derived from 7,920/5,796).
However, it is unknown under which photoactivation conditions the published molecular brightness of PA-Cherry has been determined. This is important, since we show here that the mode of photoactivation changes the measured fraction of photoactivated PA-FPs. Furthermore, for the monomeric versions comprising the A206K mutation. i.e., mEGFP and PA-mEGFP, no molecular brightness has been published.
In this ratiometric intensity-based approach, the molecular brightness of the PA-FPs and the always-on FP counterparts in a first approximation have been considered identical. We decided on this approach, since (i) for some FPs no molecular brightness has been reported, and (ii) it is thus far unclear in how far different modes of photoactivation may affect the molecular brightness of the PA-FPs reported in the literature. Furthermore, (iii) for a comparative analysis the knowledge of the molecular brightness is not necessary; it is only needed for the intensity-based determination of the absolute fraction of photoactivated PA-FPs which can be calculated as shown above.
Our approach involving fluorescent protein chimeras as internal rulers shows that different exposure to UV-light yields different photoactivation efficiencies. Thereby, it defines options as how to photoactivate a larger PA-FP fraction and achieve a better signal-to-noise ratio. Furthermore, it opens up opportunities to differentially photoactivate different PA-FPs in the same cell given their differential response to UV-light or to differentially photoactivate the same PA-FP in different subcellular compartments by exposing it differently to UV-light. In summary, our protocol will help further the quantitative understanding of cellular processes using PA-FPs in live-cell microscopy.
The authors have nothing to disclose.
We would like to thank the Dorigo laboratory and the Neuroscience Imaging Service at Stanford University School of Medicine for providing equipment and space for this project.
pEGFP-N1 mammalian cell expression vector | Clontech | ||
DMEM w/o phenol red | Thermo Fisher Scientific | 11054020 | |
Trypsin w/o phenol red | Thermo Fisher Scientific | 15400054 | |
L-Glutamine (200 mM) | Thermo Fisher Scientific | 25030081 | |
HEPES | Thermo Fisher Scientific | 15630080 | |
LabTek 8-well chambers #1.0 | Thermo Fisher Scientific | 12565470 | |
Fugene 6 | Promega | E2691 |