21.2 STED measurements of spectral shifts
21.3.3 Implications of blueing for STED imaging
The shift of a fluorophore‘s emission spectrum towards the blue is in all likelihood accompanied by a similar shift of its absorption spectrum. An indication for this is that often a blue-shift coincided with an increased emission intensity and a red-shift with decreased total intensity. A blue-shift of the absorption spectrum will in turn lead to a lower absorption and therefore lower stimulated emission cross section at the far-red STED wavelength. This will reduce the STED efficiency at a given STED laser intensity and thereby enlarge the FWHM, as seen in table 21.4. Thus, the spatial resolution presumably deteriorates.
One can read from table 21.1 that with STED illumination, 61% of the measured molecules underwent at least one spectral shift during the observation period. Of these, 42% shifted towards the blue and 34% both towards blue and red consecutively, adding up to 76%. Taken together, 46% of all measured molecules underwent at least one blueing step. In table 21.4, one can see that of all selected time trace sections, 40%
comprised a small and 22% a large blue-shift. Since 46% of the measured molecules transitioned at least once to a blue-shifted state, of which 55% shifted far, in summary 25% of all molecules were at least temporarily in a state where their emission can compromise the spatial resolution of STED images.
21.3 Summary and discussion 137
However, the molecules were observed in a far blue-shifted state for approximately one quarter of the time period that the original state was observed before further photoconversion or complete photobleaching, as stated in table 21.4.
When a small, countable number of fluorophores is to be imaged, one could scan many short frames analogous to the measurements in the previous section. After acquisition, the spectral distribution could be screened and if a blue-shift is detected, these frames could be discarded before building up the final image. However, one would need to investigate how many fluorophores can be present at most in the observation volume in order to reliably detect individual spectral shifts and to discriminate them from blinking events.
In the graphs in Appendix E, pages 128-129, of [142], one can see that while a fixed cell stained with Star635P is imaged over several frames, the spectral distribution of the measured fluorophore ensemble on the four APDs 5-8 changes continuously towards the blue. Simultaneously, the total fluorescence intensity of the fluorophore population excited with red light decreases while the fluorescence intensity of the fluorophore population excited with green light increases, but with a lower rate. Together, this indicates that a dynamic equilibrium between photoconversion and photobleaching is established. The rate of the spectral change and intensity changes depends on the applied STED intensity. The same behaviour is shown for other red fluorophores.
However, the resolution could not be monitored simultaneously because the large nuclear pore complex had been labeled.
Analogous measurements of the fluorescence intensity and spectral distribution of a labeled target structure imaged over several frames could be performed to analyze the dynamic equilibrium of blueing and bleaching rates for a given fluorophore, STED intensity and other scan parameters. Ideally, a small target structure would be cho-sen which allows the simultaneous measurement of the spatial resolution. Then, an informed choice of scan settings and the number of frames to acquire could be made, which balances total fluorescence signal and blueing in order to optimize the spatial resolution.
138 Chapter 21 Spectral shifting of fluorophores
22
Effect of spectral shifts on the FRET efficiency
22.1 Overlap integral changes
Changes of the emission spectra of single fluorophores were observed in the measure-ments described in the previous chapter 21.1. In all likelihood, the absorption spectra of the molecules change in a similar fashion. An indication for this is that the total fluorescence intensity changed simultaneously, it often increased upon blueing and decreased upon redding.
The FRET efficiencyEFRETdepends on the spectral overlap integral J(λ)between the donor emission and acceptor absorption spectra as follows:
J(λ)= normalized to an area of one.
The overlap integral of Atto532 emission and Star635P absorption was calculated with the MATLAB code appended in supplementary section 29. Additionally, it was calculated after the spectra of one or both fluorophores had been shifted numerically
Atto532
Tab. 22.1: Spectral shifts change FRET efficiency.
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by the experimentally determined maximum and minimum amount stated in table 21.2 and table 21.3. After the overlap integral was computed, the Förster radiusR0 was calculated. For this, an orientation factorκ=2/3was assumed and the refractive indexn=1.33of water was used. It was further assumed that the shape of absorption and emission spectra remained unchanged and that the donor‘s QY and acceptor‘s absorption coefficient staid constant. The FRET efficiency was then computed fromR0
and a distance ofr=6.21nm between the fluorophores. The results are stated in table 22.1.
The FRET efficiency should be 50% by choice of r, but one can see in table 22.1 that it changes between 28% and 71% due to the changes of the overlap integral. Based on the more or less continuous distribution of spectral signatures in the scatter plots in Fig.
21.4 and Fig. 21.5, I expect that theoretically, all values ofEFRETin this range could be observed. Therefore, spectral changes would prevent a quantitative analysis ofEFRETin such single molecule experiments, even in confocal mode. One could only ascertain the presence or absence of FRET.