From: W. Trevor King Date: Thu, 13 Jun 2013 17:05:57 +0000 (-0400) Subject: future: Fill in conclusions/future chapter X-Git-Tag: v1.0~95 X-Git-Url: http://git.tremily.us/?a=commitdiff_plain;h=ba315ee98188c8a3e130a749e7285193119e6670;p=thesis.git future: Fill in conclusions/future chapter --- diff --git a/src/future/hardware.tex b/src/future/hardware.tex new file mode 100644 index 0000000..ef82147 --- /dev/null +++ b/src/future/hardware.tex @@ -0,0 +1,49 @@ +\section{Hardware} +\label{sec:future:hardware} + +Very few approach--bind--retract cycles actually pick up a protein and +produce a clean unfolding curve. This makes it hard to gather +sufficient unfolding statistics unless you can run experiments +continuously over a long time span. While our modified +MultiMode\citep{multimode} gets the job done, some hardware upgrades +would allow futher automation, increasing throughput through longer +run times. + +MultiModes measure the position of the cantilever my monitoring the +reflected laser with a four-segment photodiode. While the vertical +and horizontal signals are accessible in the cable connecting the +MultiMode to its controlling NanoScope, the total photodiode signal is +not. The loss of laser signal---which can occur when bubbles in the +fluid cell obstruct the laser---results in low voltage deflection that +is independent of piezo position. This flat-line deflection also +occurs when mechanical drift moves the surface out of range for the +piezo positioner. In the drift case, we would like to use the stepper +motor to reduce the tip--surface separation. In the loss-of-signal +case, we would like to \emph{increase} the tip--surface separation to +avoid accidentally crashing the tip into a surface we can no longer +detect. By exposing the total photodiode signal to the control +software, we could unambiguously distinguish these two cases. This +would allow for longer runs, aggressively using the stepper motor to +mitigate mechanical drift. + +We could also reduce deflection signal noise---which is especially +important for accurate cantilever calibration +(\cref{sec:calibcant})---by automating photodiode positioning. The +four-segment photodiode has the least signal noise when the deflected +laser lands near the point between all four sections. However, +mechanical drift in microscope alignment causes the spot location to +vary with time. We currently use manual thumbscrews to re-zero the +photodiode as needed, but unmonitored overnight runs would require +computer-controlled positioning. Similar automatic positioning would +be useful for automatically aligning the incoming laser with the +cantilever. While laser--cantilever alignment seems to be less +sensitive than cantilever--photodiode alignment, automatic laser +alignment would also open the door to automatic piezo calibration +through measurements of laser interference patterns\citep{jaschke95}. + +Finally, our current hardware does not address potential piezo +hysteresis, nonlinearity, or drift. Newer piezos often use capacitive +feedback, adjusting the driving voltage as needed to maintain the +target extension. Besides making existing distance measurements more +accurate, the increased stability opens the door to slower pulling +speeds needed to monitor proteins with less stable folded positions. diff --git a/src/future/main.bib b/src/future/main.bib new file mode 100644 index 0000000..03fe43b --- /dev/null +++ b/src/future/main.bib @@ -0,0 +1,13 @@ +@string{Bruker = "Bruker"} + +@misc{ multimode, + author = Bruker, + title = {{M}ulti{M}ode atomic force microscope with a NanoScope + controller}, + note = {This microscope was originally developed by + \href{http://www.di.com/}{Digital Instruments} (DI). DI was + aquired by \href{http://www.veeco.com/}{Veeco} in February of + 1998, and passed off to + \href{http://www.bruker-axs.com/multimode_8_scanning_probe_microscope.html}{Bruker} + in August 2010}, +} diff --git a/src/future/main.tex b/src/future/main.tex index 0a46023..4ee8a81 100644 --- a/src/future/main.tex +++ b/src/future/main.tex @@ -1,2 +1,7 @@ \chapter{Conclusions and future work} \label{sec:future} + +\input{future/overview} +\input{future/salt} +\input{future/hardware} +\input{future/software} diff --git a/src/future/overview.tex b/src/future/overview.tex new file mode 100644 index 0000000..e4da9ee --- /dev/null +++ b/src/future/overview.tex @@ -0,0 +1,16 @@ +Single molecule force spectroscopy (SMFS) provides an experimental +window the mechanics and kinetics of individual molecules and domains. +Single molecule measurements extend bulk measurements, by offering +insight into the variations within a population of molecules in +addition to insight into the aggregate behavior of bulk solutions. +They also bridge the gap between chemists experimenting at the bulk +level and theorists simulating at the amino-acid level. By providing +data for comparison, SMFS lays the ground work for improving and +validating all-atom protein simulations. These simulations can then +be used in predictive biological applications such as high throughput +drug screening. By developing open source experiment control and +analysis software, I have made it easier for new labs to get started +in this field and for existing labs to collaborate on critical tools. +I validate my experiment and analysis suite by carring out new +experiments showing that increased \CaCl\ concentration significantly +decreases the stability of folded I27. diff --git a/src/future/salt.tex b/src/future/salt.tex new file mode 100644 index 0000000..8bbe12f --- /dev/null +++ b/src/future/salt.tex @@ -0,0 +1,18 @@ +\section{Salt} +\label{sec:future:salt} + +As expected\citep{chauhan97,itkin11,zidar11}, increasing the ionic +strength of the buffer did significantly decrease the unfolding force +(folding stability) of I27. For labs with strong gene-splicing +capability, it would be interesting to replace the glutamic acids +involved in the major bonding (\cref{fig:I27:H-bonds}) with +alternative groups to gauge the specificity of the effect. + +While the statistics are strong for the two concentrations we tested +(standard PBS and PBS with an additional $0.5\U{M}$ \CaCl), it would +be useful to study destabilization scaling over a range of +concentrations. Carrying out these experiments over a range of +pulling speeds with additional force clamp experiments would also +increase confidence in the kinetic models used to summarize the data. +SMFS is a low-throughput technique, so such an exponential increase in +assembled data would be much easier with more reliable hardware. diff --git a/src/future/software.tex b/src/future/software.tex new file mode 100644 index 0000000..2c48245 --- /dev/null +++ b/src/future/software.tex @@ -0,0 +1,74 @@ +\section{Software} +\label{sec:future:software} + +Open source experiment control is possible! Even for a small lab, +with a single novice developer\footnote{ + I started this project a bit of LabVIEW and Matlab experience, but + only a few days of Python from the physics department's ``Welcome to + Drexel'' boot camp. I stumbled across version control on my own, + after a year of maintaining a directory full of version-stampted + tarballs. + +}, building reasonable software on top of existing pieces is possible. +After a significant investment in developing +\sawsim\ (\cref{sec:sawsim}), the \pyafm\ stack +(\cref{sec:pyafm,sec:calibcant}), and \Hooke\ (\cref{sec:hooke}), we +have a complete experiment control and analysis suite for single +molecule force spectroscopy. All of the software in the +stack---including the existing libraries and systems layers that I've +built on---is open source, so other labs are free to use, improve, and +republish it as they see fit. + +As the body of existing science increases, new researchers must become +at the same time more specialized and more interdisciplinary than +their fore-bearers. With a relatively fixed undergraduate curriculum, +new researchers cannot afford to spend time becoming experts in every +field that bears on their research project. By pooling resources +between labs, individual researchers can reduce time spent on generic +tooling and increase time spent on their particular project. +Experiment control, analysis, and simulation software is particularly +amenable to community development, because the cost of sharing +software between labs is minimal. + +Besides the low cost of transferring the data itself, the rise of +distributed version control systems such as \citetalias{git} have +reduced the administrative overhead of maintaining a project with many +far-flung contributors. Researchers can automatically fetch and merge +changes made by other groups, incorperating remote improvements. They +can also commit and push local improvements, which are then available +for remote researchers to incorperate. The version control systems +and workflows that facilitate this cooperation scale well, from small +projects with a single user, to huge projects like the Linux kernel +with thousands of developers contributing to each release. + +Once the software used in a lab has been published, it is also easier +to audit by others who may be skeptical of the summary published in a +journal article. For example, resolving the confusion about the +``Lorentzian'' (\cref{sec:calibcant:lorentzian}) would be trivial if +\citet{florin95} had also published their explicit procudure for +generating their figure. Do you think I'm not calibrating my +cantilevers correctly? Feel free to dig through my code. Let me know +if you find something wrong (or fix it and send me a patch!). Science +is built on reproducible experiments and analysis, and open sourcce +software allows you to explicitly specify your methods. With well +organized code, the specification should be clear from a high-level, +experiment-design choices down through low-level bit manipulation. + +Many researchers have not received formal training in software +development best practices, so how do we bootstrap this transition to +open source science? There is a wealth of documentation available +online for self-teaching, and scientists have lots of experience +reading technical writing in their own field. For those who are +overwhelmed by the amount of available resources, organizations such +as \href{http://http://software-carpentry.org/}{Software Carpentry} +are actively reaching out to scientists with short boot camps to lay +the ground work. Mastery of any subject takes a significant +investment, but gaining a working level of knowledge in distributed +version control should only take a few days\footnote{ + Software Carpentry allocates half a day to take students from ``What + is version control?'' to being functioning \citetalias{git} users. +}. The difficulty for the uninitiated is often not mastering the new +tool or workflow, but learning that it exists at all. There are a +number of papers highlighting best practices and tools that are good +surveys for guiding future +learing\citep{wilson06a,wilson06b,vandewalle09,aruliah12}. diff --git a/src/root.bib b/src/root.bib index 75c1cb7..e6187c9 100644 --- a/src/root.bib +++ b/src/root.bib @@ -2144,6 +2144,32 @@ project = "Cantilever Calibration" } +@article{ jaschke95, + author = MJaschke #" and "# HJButt, + title = {Height calibration of optical lever atomic force + microscopes by simple laser interferometry}, + journal = RSI, + year = 1995, + volume = 66, + number = 2, + pages = {1258--1259}, + publisher = AIP, + url = {http://rsi.aip.org/resource/1/rsinak/v66/i2/p1258_s1}, + doi = {10.1063/1.1146018}, + issn = {0034-6748}, + keywords = {atomic force microscopy;calibration;interferometry;laser + beam applications;mirrors;spatial resolution}, + abstract = {A new and simple interferometric method for height + calibration of AFM piezo scanners is presented. Except for a small + mirror no additional equipment is required since the fixed + wavelength of the laser diode is used as a calibration + standard. The calibration is appliable in the range between + several ten nm and several μm. Besides vertical calibration many + problems of piezo elements like hysteresis, nonlinearity, creep, + derating, etc. and their dependence on scan parameters or + temperature can be investigated.}, +} + @article { cao07, author = YCao #" and "# MBalamurali #" and "# DSharma #" and "# HLi, title = "A functional single-molecule binding assay via force spectroscopy", diff --git a/src/root.tex b/src/root.tex index 7c2bcc7..5aecfa7 100644 --- a/src/root.tex +++ b/src/root.tex @@ -82,6 +82,7 @@ defaultpen(fontsize(10)); // match drexel-thesis's default 10pt font size calibcant/main,% hooke/main,% salt/main,% + future/main,% packaging/main,% figures/main,% root}