Longitudinal assessment of fluorescence stability shows fluorescence intensity decreases over time: implications for fluorescence microscopy studies
Fig 10
AF546 is the most resistant to fluorescence intensity changes over time and photobleaching does not underlie the observed changes in fluorescence intensity.
All sections were stained with anti-OMP primary antibody, followed by staining with AF488-, AF546- or AF647-conjugated secondary antibody. Control Experiment 2 (a-i) consisted of an initial imaging session followed by a second imaging session 7 weeks later, using widefield microscopy. A two-way repeated measures ANOVA showed significant effects of fluorophore (F(2,6)=317.3, p < 0.001) and time point (F(1,6)=747.5, p < 0.001), and a significant interaction between fluorophore and time point (F(2,6)=317.3, p < 0.001). Note that some data points have similar values and overlap (n = 3). (a,b) Images of AF488-conjugated secondary antibody-stained section at weeks 1 and 7. (c) No significant difference in fluorescence intensity after six weeks without imaging for AF488-conjugated secondary antibody-stained sections. (d,e) Images of AF546-conjugated secondary antibody-stained section at weeks 1 and 7. (f) No significant difference in fluorescence intensity after six weeks without imaging for AF546-conjugated secondary antibody-stained sections. (g,h) Images of AF647-conjugated secondary antibody-stained section at weeks 1 and 7. (i) No significant difference in fluorescence intensity after six weeks without imaging for AF647-conjugated secondary antibody-stained sections. Control Experiment 3 (j-u) consisted of widefield imaging every two minutes, collecting a total of 7 data points across 13 minutes. A two-way repeated measures ANOVA showed significant effects of fluorophore (F(2,6)=5.79, p = 0.040) and time (F(2.16,13.0)=12.63, p < 0.001) but no significant interaction between fluorophore and time (F(4.32,13.0)=2.95, p = 0.059). (j-l) Images of AF488-conjugated secondary antibody-stained section at minutes 1, 7 and 13. (m) No change in fluorescence intensity for AF488-conjugated secondary antibody-stained sections imaged every two minutes. (n-p) Images of AF546-conjugated secondary antibody-stained section at minutes 1, 7 and 13. (q) No change in fluorescence intensity for AF546-conjugated secondary antibody-stained sections imaged every two minutes. (r-t) Images of AF647-conjugated secondary antibody-stained section at minutes 1, 7 and 13. (u) No change in fluorescence intensity for AF647-conjugated secondary antibody-stained sections imaged every two minutes. Each line of data represents a single mouse.