diff --git a/benchtop_scanning.tex b/benchtop_scanning.tex new file mode 100644 index 0000000..1db7f5a --- /dev/null +++ b/benchtop_scanning.tex @@ -0,0 +1,172 @@ +%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% +%%%% Load the document class and packages %%%% +%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% +\documentclass[a4paper]{report} +\usepackage{epsfig} % to insert PostScript figures +\graphicspath{ + {figures/} +} + +%Change figure names +\renewcommand{\figurename}{Fig} + +\usepackage[bf,footnotesize]{caption} % make captions small and label bold + + +\addtocounter{chapter}{1} %Because starting at zero is silly +\makeatletter +\renewcommand{\thesection}{\@arabic\c@section} +\renewcommand{\thefigure}{\@arabic\c@figure} +\makeatother + +\usepackage[a4paper,margin=2.7cm,tmargin=2.5cm,bmargin=2.5cm]{geometry} +\usepackage{textcomp} % To make nice degree symbols and others\usepackage[bf,footnotesize]{caption} % make captions small and label bold +\usepackage{wrapfig} +%to produce the clickable references along the left in Acroread. This +%package must be included last. +\usepackage[ps2pdf,bookmarks=TRUE]{hyperref} + + + +%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% +%%%% Hypertext references for Acrobat %%%% +%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% +\hypersetup{ +pdfauthor = {TENSS}, +pdftitle = {Benchtop Scanning}, +pdfkeywords = {optics, lenses, refraction, reflection, dispersion, + telescope, microscope}, +pdfcreator = {LaTeX with hyperref}, +pdfproducer = {dvips + ps2pdf} + } + + +\begin{document} + + + + +%set the number of sectioning levels +\setcounter{secnumdepth}{2} + +\begin{center} +\textbf{\Large{Benchtop Scanning}} +\end{center} + +\section{Introduction} + +\subsection{Scanning microscopy} +Widefield imaging uses lenses to form a real image of the sample which is magnified and imaged onto a CCD chip or viewed by eye. +In this scenario the sample is conjugate with the image plane. +In widefield microscopy the entire field of view is illuminated so a great deal of scattered light from outside of the focal plane ends up in the image plane. +Recall the definition of an image-forming condition: \textbf{light rays leaving one point (or region) of the object arrive at some other defined point (or region)}. +Scattered photons are so defined because they arrive at the image plane at a location \textit{not conjugate} with where they originally came from. +Thus, scattered photons fail to satisfy the image-forming condition. +Scattering is due to the inhomogeneous refractive index of the sample. +Photons that are not scattered are known ballistic photons. + +As you saw previously, image quality can be improved by an well set up K\"{o}hler illumination system. +However, even K\"{o}hler illumination does not fix the fundamental problems with wide-field imaging and it can only be used with thin samples where transmitted light illumination is possible. + +Fluorescence-based scanning microscopy greatly mitigates the problem of scattering. +A laser beam is scanned across the sample and excites a fluorophore. +Emitted fluorescence is collected via the objective and detected with a photomultiplier tube (PMT). +No image is formed on the PMT (it's a `single pixel'), instead the image is constructed \textit{post-hoc} on a computer from the time-series PMT data. +Indeed, in many scanning microscopes the object and the PMT are not even at conjugate planes. +The reason scattered light is less problematic in scanning microscopy depends on the design of the microscope: +With confocal, the scattered light is rejected by the pinhole. +With 2-photon, the region of excitation is highly restricted so the origin of all collected photons is known. + + + + +\subsection{Building a transmission scanning microscope} +Today you will build a transmitted light scanning microscope using a set of scanners, three lenses, two mirrors, and a laser pointer. +For detection you will use a photodiode located after the sample. +This setup of course provides none of the advantages of a scanning microscope, since it's not fluorescence based and there is no pinhole. +However, the arrangement of the excitation path optics will be identical to that found in the 2-photon microscope you will go on to build. + +\section{Instructions} + +\subsection{Set up the scanners and align the beam} +Set up the rail and scanners as follows: +\begin{itemize} +\setlength\itemsep{0.15em} +\item Bolt the optical rail to the breadboard using a clamp on each end. Do not over-tighten. +\item Place two irises on the ends of the rail and set them to the same height. +Locating them on fully lowered $75~mm$ posts is suitable. +\item Place the the scanners at one end of the rail and power them (ask for assistance). +The scanners need to be powered for the mirrors to be square with respect to each other. +\item Loosely bolt the scanners into place. +\item You may end up needing to the move the rail or the scanners during the alignment procedure. +\end{itemize} + +\vspace{1.5em} + +You're now ready to position and align the laser. +You will use two mirrors to align the beam going into the scanners. +Your goal is to have the beam hit the middle of the scan mirrors then go straight down the rail, parallel with the table. +This will be easiest if you position one mirror, angled at 45 degrees, close to the scanners. +The next mirror should be some distance from the first one and square with respect to it. +The laser pointer is placed at 45 degrees with respect to this second mirror. +Do the initial coarse alignment by moving and rotating the mirrors and the laser pointer. +Then bolt down the components and use the fine-adjustment screws on the mirror mounts. +Use the irises to ensure the beam runs straight down the rail. +You will likely find that the scanners aren't aligned with the irises and that either they or the rail need to be moved. +Expect to do the alignment procedure several times. +It's OK for our purposes if the alignment isn't \textit{perfect}. +Getting it to within a couple of beam diameters should be adequate. + + +\subsection{Add the optics} +You will use the 4X objective to image the scan pattern onto the sample using the objective's full NA. +Two things need to be achieved in order to make this possible. +\begin{enumerate} +\setlength\itemsep{0.1em} +\item The scan mirrors will need to be in a conjugate plane to the back aperture of the objective. +\item The beam will need to be expanded to fill the back aperture of the objective. +\end{enumerate} + +Both of these things can be satisfied by building a beam expander between the objective and the scanners. +Place the objective mid-way down the rail and align it with the beam. Use an iris to guide you. +Choose suitable lenses for your beam expander and locate them in the correct places +(think about what distance each element should be with respect to the others). +You can move the irises to help you position the lenses. +The lens nearer the scanners is called the \textbf{scan lens}. +The lens nearer the objective is called the \textbf{tube lens}. + +\subsection{Verifying the scan pattern} +\begin{itemize} +\item Connect the scan control cables to the analog outputs of your acquisition card. +The $x$ mirror is responsible for the `fast' axis and goes to AO0. The $y$ mirror is responsible for the slow axis and goes to to AO1. +Ask if you are unsure how to do this. +\item Start NI MAX, go to the test panels for your DAQ device and select analog output. +\item Play a $10~Hz$ sine wave of amplitude $3~V$ through each channel in turn. +\item Use a card or piece of paper to observe the beam motion through your optical system. +What do you see at $1f$ from the scan lens? +What do you see at the working distance of the objective? +What do you see on the back of the objective? +What about in front of the objective? +Satisfy yourself that all those things make sense. +\end{itemize} + + +\subsection{Obtaining images} +\begin{itemize} +\item Place a slide containing an EM grid at the working distance of the objective. +\item Place a photodiode close to the back of the slide and hook it up to AI0 on your DAQ. +\item You may get a better image with a collection lens in front of the detector, but this isn't critical\footnote{Or even K\"{o}hler, for that matter.}. +\item If you have been introduced to the scan software, start the `minimal' version to begin scanning and acquisition. +The brave can write their own scan software from scratch. +You can do it with MATLAB's Data Acquisition Toolbox in about 150 lines of code. +\item You will likely need to focus carefully to get an image: the depth of field is quite narrow. +\item Your image will look distorted. +Where does the distortion come from? +\item Modify the acquisition software to either (\textbf{a}) get rid of the artifact or (\textbf{b}) implement faster bidirectional scanning and correct the artifacts you see. You choose. +\item Modify the software to save the image stream to disk. Hint: \textbf{help imwrite} +\item Acquire images of the grid at three different scan amplitudes and calculate the number of microns per pixel. +\item Image a biological specimen, save the image, add a scale bar. +\end{itemize} + + +\end{document}