From 28c5e3d706b36176834b9899b065d27f301898ce Mon Sep 17 00:00:00 2001 From: Kyle Douglass Date: Mon, 9 Oct 2023 15:41:40 +0200 Subject: [PATCH 1/5] Add section describing what the buffer does --- texts/smlm-lab-course/smlm-lab-manual.tex | 19 +++++++++++++++++++ 1 file changed, 19 insertions(+) diff --git a/texts/smlm-lab-course/smlm-lab-manual.tex b/texts/smlm-lab-course/smlm-lab-manual.tex index 8472926..fa76252 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 Monoethanolamine (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. Different fluorescent dyes perform better at different pH values, and there does not seem to be an easily discernible trend to predict the best pH for 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 of electrons. An 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 togethor 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} From 730f8182302881533534c5ae3ba016eb9df80816 Mon Sep 17 00:00:00 2001 From: Kyle Douglass Date: Mon, 9 Oct 2023 15:45:17 +0200 Subject: [PATCH 2/5] typos --- texts/smlm-lab-course/smlm-lab-manual.tex | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/texts/smlm-lab-course/smlm-lab-manual.tex b/texts/smlm-lab-course/smlm-lab-manual.tex index fa76252..4891dac 100644 --- a/texts/smlm-lab-course/smlm-lab-manual.tex +++ b/texts/smlm-lab-course/smlm-lab-manual.tex @@ -383,7 +383,7 @@ \section{The Role of the Buffer} These components serve three broad purposes. The first is to maintain the pH of the buffer at a constant value. Different fluorescent dyes perform better at different pH values, and there does not seem to be an easily discernible trend to predict the best pH for 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 of electrons. An 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 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 togethor 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. From 92c359ea18661ac6c679a291ef8167b335ce75d1 Mon Sep 17 00:00:00 2001 From: Kyle Douglass Date: Mon, 9 Oct 2023 15:46:38 +0200 Subject: [PATCH 3/5] more typos --- texts/smlm-lab-course/smlm-lab-manual.tex | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/texts/smlm-lab-course/smlm-lab-manual.tex b/texts/smlm-lab-course/smlm-lab-manual.tex index 4891dac..ad71b8b 100644 --- a/texts/smlm-lab-course/smlm-lab-manual.tex +++ b/texts/smlm-lab-course/smlm-lab-manual.tex @@ -385,7 +385,7 @@ \section{The Role 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 togethor 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. +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} From 3db9bd9e3aac24cb7acada1221cea4f0cb8d1c9a Mon Sep 17 00:00:00 2001 From: Kyle Douglass Date: Tue, 10 Oct 2023 09:27:07 +0200 Subject: [PATCH 4/5] Change comment about pH effects on dyes --- texts/smlm-lab-course/smlm-lab-manual.tex | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/texts/smlm-lab-course/smlm-lab-manual.tex b/texts/smlm-lab-course/smlm-lab-manual.tex index ad71b8b..f69490f 100644 --- a/texts/smlm-lab-course/smlm-lab-manual.tex +++ b/texts/smlm-lab-course/smlm-lab-manual.tex @@ -381,7 +381,7 @@ \section{The Role of the Buffer} \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. Different fluorescent dyes perform better at different pH values, and there does not seem to be an easily discernible trend to predict the best pH for 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. +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. From b1943cd46dc85f45bf3089231bb72eb271765160 Mon Sep 17 00:00:00 2001 From: Kyle Douglass Date: Tue, 10 Oct 2023 09:33:25 +0200 Subject: [PATCH 5/5] Change MEA common name --- texts/smlm-lab-course/smlm-lab-manual.tex | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/texts/smlm-lab-course/smlm-lab-manual.tex b/texts/smlm-lab-course/smlm-lab-manual.tex index f69490f..134cd04 100644 --- a/texts/smlm-lab-course/smlm-lab-manual.tex +++ b/texts/smlm-lab-course/smlm-lab-manual.tex @@ -376,7 +376,7 @@ \section{The Role of the Buffer} \item Trizma base \item NaCl \item Glucose - \item Monoethanolamine (commonly called MEA) + \item 2-Aminoethanol (commonly called MEA) \item Glucose oxidase \item Catalase diluted in glycerol \end{itemize}