Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy

Abstract

Finding the right combination of a fluorescent dye and a mounting medium is crucial for optimal microscopy of fixed samples. It was recently shown that Vectashield, one of the most commonly used mounting media for conventional microscopy, can also be applied to super-resolution direct stochastic optical reconstruction microscopy (dSTORM). dSTORM utilizes conventional dyes and starts with samples in a fluorescent "ON" state. This helps in identifying structures of interest. Subsequently, labelled samples are induced into blinking, which is necessary for determining the position of single molecules and reconstruction of super-resolution images. This is only possible with certain fluorescent dyes and imaging buffers. One of the most widely used dyes for dSTORM, Alexa Fluor 647 (AF647), blinks in Vectashield. However, after preparing immunocytochemical samples in Vectashield, we noticed that the fluorescence intensity of AF647 is quenched. This is particularly evident for dimmer immunostainings, such as stainings of some components of neuronal cytoskeleton and axonal initial segment. Because structures of interest cannot be identified in quenched samples, loss of fluorescence intensity hinders imaging of AF647 in Vectashield. This has consequences for both conventional and dSTORM imaging. To overcome this, we provide: 1) a quantitative analysis of AF647 intensity in different imaging media, 2) a quantitative analysis of the suitability of Vectashield for dSTORM imaging of high and low-abundance AF647-labelled targets. Furthermore, for the first time, we quantitatively analyse the performance of Alexa Fluor Plus 647, a new variant of AF647-conjugated antibody, in dSTORM imaging.Super-resolution microscopy (SRM) provides unique insights into the subcellular organisation of cells and tissues. Several techniques have been developed to make nanoscale imaging possible 1-4 . Single-molecule localisation techniques, such as photoactivated localisation microscopy (PALM) 5 , fluorescence photoactivation localisation microscopy (FPALM) 6 , stochastic optical reconstruction microscopy (STORM) 7 and direct stochastic optical reconstruction microscopy (dSTORM) 8 rely on temporal separation of fluorescence emission. Temporal separation is achieved by blinking, which separates individual fluorescent molecules not only in time, but also in space. The resulting spatiotemporal separation of fluorescence emission enables precise determination of the location of fluorescent molecules, which is necessary to reconstruct SRM images 7,9 . Whereas PALM and FPALM utilize genetically engineered fluorescent proteins, dSTORM relies on synthetic fluorophores (dyes). To achieve blinking in dSTORM, dye molecules are switched to a dark "OFF" state from which they stochastically and spontaneously recover to a fluorescent "ON" state, multiple times before bleaching. Blinking can be induced in certain dyes by using strong lasers and particular imaging buffers, such as thiol-containing reducing agents (e.g. β-mercaptoethanol) with or without enzymatic ox...

Sodium sulfite as a switching agent for single molecule based super-resolution optical microscopy

“…The mechanism of switching, and glycerol’s role thereof, is not known. Another unknown is the cause of the stark reduction in brightness of the fluorophore AF647 in the sulfite switching buffer, similar to what has recently been reported of the Vectashield dSTORM buffer 12 . Once the mechanism behind this behavior is established, it might be possible to find a switching buffer that switches the fluorophore emission as well as sulfite, while maintaining the brightness observed with the primary thiols.…”

Chemical Analysis of Single Cells and Organelles

“…Arsić et al showed the effect of Vectashield, one of the most commonly used mounting media, causing the loss of fluorescence intensity of Alexa Fluor 647 (AF647) via fluorescence quenching . As AF647 blinks in Vectashield, this dye-mounting media combination is often used for dSTORM.…”

“…Arsić et al showed the effect of Vectashield, one of the most commonly used mounting media, causing the loss of fluorescence intensity of Alexa Fluor 647 (AF647) via fluorescence quenching. 98 As AF647 blinks in Vectashield, this dye-mounting media combination is often used for dSTORM. However, the authors observed that the fluorescence intensity of AF647 is quenched after mounting immunolabeled samples in Vectashield.…”

The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks

“…The following secondary antibodies were from Thermo Fisher Scientific and diluted 1/1000: goat anti-rabbit IgG Alexa Fluor 488 (A11034), donkey anti-mouse IgG Alexa Fluor 568 (A10037), donkey anti-rabbit IgG Alexa Fluor 568 (A10042) and goat anti-mouse IgG1 Alexa Fluor 647 (A21240). Cells in glass bottom dishes were imaged directly in PBS, whereas cells on coverslips were washed once in water and mounted in VECTASHIELD (unless imaging samples with Alexa Fluor 647, in which case SlowFade Diamond Antifade Mountant (Thermo Fisher Scientific) was used to avoid fluorescence quenching 114 ).…”