Simple method to enhance the photostability of the fluorescence reporter R6G for prolonged single-molecule studies

Abstract

For fluorescence-based single-molecule studies, photobleaching of the dye reporter often limits the time window over which individual molecules can be followed. As such, many strategies, for example, using a cocktail of chemical reagents, have been developed to decrease the rate of photobleaching. Herein, we introduce a new and highly effective method to enhance the photostability of one of the commonly used fluorescent dyes, rhodamine 6G (R6G). We show that micrometer-sized polydimethylsiloxane (PDMS) wells, when the PDMS surface is properly treated, not only provide a confined environment for single-molecule detection but can also significantly increase the survival time of individual R6G molecules before photobleaching. Moreover, our results suggest, consistent with several previous studies, that R6G photobleaching involves a radical state.

Figure 1

A representative fluorescence intensity time trace of R6G molecules encapsulated in POPC GUVs.

Figure 2

Photobleaching statistics of R6G molecules encapsulated in POPC GUVs. The smooth line is the best fit of these data to the following equation: S(t) = A·exp(−t/τ)+B, with A = 32.7, τ = 85.6 s, and B = 1.6.

Figure 3

A fluorescence image of the PDMS wells. The unequal image brightness is caused by uneven laser illumination.

Figure 4

Comparison of FCS traces obtained in a BSA-coated PDMS well and on a BSA-covered glass slide, as indicated. In both cases, the concentration of the R6G solution used was 1 nM.

Figure 5

A representative fluorescence intensity time trace of single R6G molecules confined in BSA-coated PDMS wells.

Figure 6

Fluorescence intensity time traces of single R6G molecules confined in either BSA-coated (blue) or PEG-coated (red) PDMS wells. For ease of comparison, the original data have been offset. The variation in fluctuations among individual traces is resulting from different collection times (thus different bin time).

Figure 7

Compassion of high resolution FCS curves obtained in a BSA-coated PDMS well and on a BSA-covered glass slide, as indicated. In both cases, the concentration of the R6G solution used to prepare the respective samples was 1 nM.

Figure 8

A representative FCS curve (A) and a fluorescence intensity time trace (B) of single R6G molecules in untreated PDMS wells.

Figure 9

(A) Comparison of FCS curves of single R6G molecules in freshly oxygen plasma treated PDMS wells. As indicated, these data were obtained before and after 30 minutes of continuous laser (1.6 mW) illumination of the sample; and (B) the fluorescence intensity time trace obtained after the 30 minute photobleaching event.