diff --git a/texts/smlm-lab-course/smlm-lab-manual.tex b/texts/smlm-lab-course/smlm-lab-manual.tex index 8472926..134cd04 100644 --- a/texts/smlm-lab-course/smlm-lab-manual.tex +++ b/texts/smlm-lab-course/smlm-lab-manual.tex @@ -368,6 +368,25 @@ \subsection{Photoswitching vs Photoactivation} \label{table:photophysics-comparison} \end{table} +\section{The Role of the Buffer} + +The STORM buffer used in this course contains the following components. + +\begin{itemize} + \item Trizma base + \item NaCl + \item Glucose + \item 2-Aminoethanol (commonly called MEA) + \item Glucose oxidase + \item Catalase diluted in glycerol +\end{itemize} + +These components serve three broad purposes. The first is to maintain the pH of the buffer at a constant value. The photoswitching behavior of a given fluorophore depends on the pH, and there does not seem to be an easily discernible rule to predict the effects of pH on a given dye. The pH of each buffer must therefore be tuned to a specific dye through experimentation. Trizma and NaCl together help maintain the correct pH of the buffer. + +The second purpose of the buffer is to open the energetic pathway to the long-lived dark state. As mentioned above, this pathway is enabled by the reduction\footnote{A reduction occurs when a molecule gains electrons. Oxidation is the opposite process whereby a molecule losses electrons.} of the flourophore when it is in the triplet state. MEA is the reducing agent in the buffer. + +The third purpose of the buffer is to remove triplet-state oxygen from the aqueous environment of the sample. This is important because triplet-state oxygen quenches the triplet and reduced states of the dye, therefore serving as a precursor to more reactive oxygen species that make photobleaching more likely. Glucose oxidase (GLOX) and catalase together form an oxygen scavenging system, with glucose providing the fuel for the reaction. Glucose oxidase converts glucose and triplet oxygen to hydrogen peroxide, and catalase converts hydrogen peroxide to water and oxygen. + \chapter{Structural Biology of the Cell} \section{Microtubules}