A small bleach region of 0.4µm diameter was used at first to ensure mini- mal invasiveness and our imaging time was kept below one minute. The high rate of fluorescence depletion of mobile species is most evident for pre23k-GFP in panels (A-D)of figure3.4and all panels of TP-GFP in fig- ure3.6, as compared to the less mobile pre23k∆TPP-GFP in figure3.2and Hcf106-GFP in figure 3.8. It may be seen that the observational photo- bleaching rates of all constructs; figures 3.3, 3.5, 3.7and 3.9; were compa- rable although pre23k∆TPP-GFP appeared a little more resistant to photo- bleaching in this data series as can be seen from panel(B)in figure3.11.
We note the pointwise standard deviation bands for observational pho- tobleaching are especially tight around the pointwise averages for the ho- mogeneous(A)TP-GFP, less mobile(B)Hcf106-GFP and(C)pre23k∆TPP- GFP in figure3.10. There is greater variability for when spot photobleach-
evidence of a range of behaviours in figure3.10.
There are certain features in the graphs of figure 3.10 that we will be unable to address with the data. All fluorescence depletion graphs show comparable sharp initial drops and the time at which spot bleaching is stopped is marked by a rise in the fluorescence graph. The rise is most likely caused by movement of fluorescent protein into the confocal imaging slice from above and below the slice. The initial drop must be connected with the onset of imaging but we have no way of determining its cause without a significant detour from the main line of enquiry. It should be noted that the initial drop occurs in a very short interval of time even when compared to the short total duration of the experiment so assigning it a lower priority is not unjustified. Whatever its cause, it serves as a warning to experimentalists who would try to make measurements in tiny intervals of time very close to the start of bleaching and imaging.
The magnitude of the slopes of the linear portion of the graphs shown in figure3.10are plotted in panel(B)of figure3.11to compare and quan- tify the rates of whole-chloroplast fluorescence depletion caused by spot bleaching. Panel (A) of figure 3.11 shows a correction for the observa- tional photobleaching effect. Even from the confocal images themselves it was clear that TP-GFP is highly mobile which is characterized by the small bleach spots seen in figures 3.6, as compared to pre23k-GFP and pre23k∆TPP-GFP, and we will see this again with simulations in which we are able to control the various bleach and diffusion parameters.
Figure 3.2: Modified FLIP pre23k∆TPP-GFP (small bleach region). Rows of five experimental repetitions (A-E) are shown (different chloroplasts). Later frames in each row show slightly lower levels of fluorescence than earlier frames in the row. The scale bar is 5 µm in length. pre23k∆TPP- GFP has relatively low mobility yet the bleach region appears smeared in a horizontal direction in the first-bleach (column 3) and last-bleach (column 4) columns which shows redistribution between the forward bleach scan of a line and the immediate backward imaging scan of the same line.
Figure 3.3: Observational control: pre23k∆TPP-GFP.Rows of five exper- imental repetitions(A-E)are shown (different chloroplasts). Later frames in each row show very similar levels of fluorescence to earlier frames in the row. The scale bar is 5µm in length.
Figure 3.4: Modified FLIP: pre23k-GFP (small bleach region). Rows of five experimental repetitions (A-E) are shown (different chloroplasts). Later frames in each row show lower levels of fluorescence than earlier frames in the row. The scale bar is 5µm in length. The high mobility of pre23k-GFP is evidenced by the extent the bleach region leads to a widening depleted local region (column 3) and noticeably less fluorescence near the end of the sequence (columns 4 to 6).
Figure 3.5: Observational control: pre23k-GFP.Rows of five experimental repetitions(A-E)are shown (different chloroplasts). Later frames in each row show very similar levels of fluorescence to earlier frames in the row. The scale bar is 5µm in length.
Figure 3.6: Modified FLIP: TP-GFP (small bleach region). Rows of five experimental repetitions (A-E) are shown (different chloroplasts). Later frames in each row show lower levels of fluorescence than earlier frames in the row. The scale bar is 5 µm in length. TP-GFP is an example of how modelling and simulation is important for testing our interpretation of photobleaching analyses as here we see the most rapid depletion in the later stages (columns 4 to 6) but the depletion is fairly uniform across the chloroplast without the expanding bleach region seen for pre23k-GFP.
Figure 3.7: Observational control: TP-GFP. Rows of five experimental repetitions(A-E)are shown (different chloroplasts). Later frames in each row show very similar levels of fluorescence to earlier frames in the row. The scale bar is 5µm in length.
Figure 3.8: Modified FLIP: Hcf106-GFP (small bleach region). Rows of five experimental repetitions (A-E) are shown (different chloroplasts). Later frames in each row show very similar levels of fluorescence to earlier frames in the row. The scale bar is 5 µm in length. The bleach region is strikingly apparent in the first post-bleach images (column 5) and even at the last of twenty post-bleach acquisitions (column 6).
Figure 3.9: Observational control: Hcf106-GFP.Rows of five experimental repetitions(A-E)are shown (different chloroplasts). Later frames in each row show very similar levels of fluorescence to earlier frames in the row. The scale bar is 5µm in length.
Figure 3.10: Fluorescence depletion: small bleach region FLIP. Each quadrant shows the spot photobleaching effect (five repetitions with pointwise standard deviation, s.d., shown) as compared to the observa- tional photobleaching (five repetitions with pointwise standard deviation shown). The FLIP spot photobleaching has the most pronounced effect in
(A)and(D)suggesting greater mobility. The pointwise standard deviation is greatest in(D), for both observation and FLIP, which suggests a greater range of binding and dynamic processes are involved.
50 60 70 80 90 100 TP−GFP Obs. Obs. s.d. TP−GFP FLIP FLIP s.d. A Hcf106−GFP Obs. Obs. s.d. Hcf106−GFP FLIP FLIP s.d. B 0 10 20 30 40 50 60 50 60 70 80 90 100 p23k∆∆TPP−GFP Obs. Obs. s.d. p23k∆∆TPP−GFP FLIP FLIP s.d. C 0 10 20 30 40 50 60 p23k−GFP Obs. Obs. s.d. p23k−GFP FLIP FLIP s.d. D Percentage fluorescence Time / seconds
Figure 3.11: Correcting for observation: small bleach region. The flu- orescence profiles with observational photobleaching effect removed are shown in (A). TP-GFP shows the greatest rate of fluorescence depletion during photobleaching and Hcf106-GFP shows only a small rate of loss. A quantification of the fluorescence loss during spot photobleaching (the approximately linear portion after the initial drop and before the recovery) is shown in(B). The standard deviation is shown at the top of each bar.
0 10 20 30 40 50 60 80 90 100 TP−GFPHcf106−GFP p23k∆∆TPP−GFP p23k−GFP Percentage fluorescence Time / seconds TP−GFP p23k−GFP Hcf106−GFP p23k∆∆TPP−GFP FLIP photobleaching Obs. photobleaching 0.0 0.2 0.4 0.6 0.8
Percentage fluorescence loss per second