From 68ab7d36aba4b4c720e964fda6d669f57466e7cb Mon Sep 17 00:00:00 2001
From: Kyle Douglass <kyle.douglass@epfl.ch>
Date: Fri, 1 Sep 2023 14:12:59 +0200
Subject: [PATCH] Add epi-illumination strategies to SMLM course text

---
 figures/focal-volume/README.md                |   1 +
 figures/focal-volume/focal-volume.svg         | 313 ++++++++++++++++++
 .../smlm-lab-course/figures/focal-volume.png  | Bin 0 -> 41769 bytes
 texts/smlm-lab-course/refs.bib                |  40 +++
 texts/smlm-lab-course/smlm-lab-manual.tex     |  26 ++
 5 files changed, 380 insertions(+)
 create mode 100644 figures/focal-volume/README.md
 create mode 100644 figures/focal-volume/focal-volume.svg
 create mode 100644 texts/smlm-lab-course/figures/focal-volume.png

diff --git a/figures/focal-volume/README.md b/figures/focal-volume/README.md
new file mode 100644
index 0000000..b7564c6
--- /dev/null
+++ b/figures/focal-volume/README.md
@@ -0,0 +1 @@
+The focal volume of a microscope. This the volume within which a point source will appear in focus in an image. Point sources outside of this volume will appear out-of-focus. The axial extent of the focal volume is called the depth of focus.
diff --git a/figures/focal-volume/focal-volume.svg b/figures/focal-volume/focal-volume.svg
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diff --git a/texts/smlm-lab-course/refs.bib b/texts/smlm-lab-course/refs.bib
index 5a224b4..4bd7778 100644
--- a/texts/smlm-lab-course/refs.bib
+++ b/texts/smlm-lab-course/refs.bib
@@ -168,3 +168,43 @@ @article{douglass-naturephotonics-2016
 	keywords = {smlm-course},
 	pages = {705--708},
 }
+
+@article{tokunaga-naturemethods-2008,
+	title = {Highly inclined thin illumination enables clear single-molecule imaging in cells},
+	volume = {5},
+	copyright = {2008 Springer Nature America, Inc.},
+	issn = {1548-7105},
+	url = {https://www.nature.com/articles/nmeth1171},
+	doi = {10.1038/nmeth1171},
+	abstract = {We describe a simple illumination method of fluorescence microscopy for molecular imaging. Illumination by a highly inclined and thin beam increases image intensity and decreases background intensity, yielding a signal/background ratio about eightfold greater than that of epi-illumination. A high ratio yielded clear single-molecule images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify molecular dynamics, interactions and kinetics in cells for molecular systems biology.},
+	language = {en},
+	number = {2},
+	urldate = {2023-09-01},
+	journal = {Nature Methods},
+	author = {Tokunaga, Makio and Imamoto, Naoko and Sakata-Sogawa, Kumiko},
+	month = feb,
+	year = {2008},
+	note = {Number: 2
+Publisher: Nature Publishing Group},
+	keywords = {smlm-course},
+	pages = {159--161},
+}
+
+
+@article{axelrod-traffic-2001,
+	title = {Total {Internal} {Reflection} {Fluorescence} {Microscopy} in {Cell} {Biology}},
+	volume = {2},
+	issn = {1600-0854},
+	url = {https://onlinelibrary.wiley.com/doi/abs/10.1034/j.1600-0854.2001.21104.x},
+	doi = {10.1034/j.1600-0854.2001.21104.x},
+	abstract = {Key events in cellular trafficking occur at the cell surface, and it is desirable to visualize these events without interference from other regions deeper within. This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation. The technique has many other applications as well, most notably for studying biochemical kinetics and single biomolecule dynamics at surfaces. A brief summary of these applications is provided, followed by presentations of the physical basis for the technique and the various ways to implement total internal reflection fluorescence in a standard fluorescence microscope.},
+	language = {en},
+	number = {11},
+	urldate = {2023-09-01},
+	journal = {Traffic},
+	author = {Axelrod, Daniel},
+	year = {2001},
+	note = {\_eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1034/j.1600-0854.2001.21104.x},
+	keywords = {smlm-course},
+	pages = {764--774},
+}
diff --git a/texts/smlm-lab-course/smlm-lab-manual.tex b/texts/smlm-lab-course/smlm-lab-manual.tex
index 6940b35..1887062 100644
--- a/texts/smlm-lab-course/smlm-lab-manual.tex
+++ b/texts/smlm-lab-course/smlm-lab-manual.tex
@@ -118,6 +118,32 @@ \subsection{Image Formation in Linear Shift Invariant Systems}
 
 We can reduce the degree of blurring by increasing the numerical aperture of the objective, which has the effect of reducing the size of the PSF. Ultimately, however, diffraction prevents us from shrinking the PSF down to a point. As a result, a microscope image will never exactly reproduce an image of the object.
 
+\section{Types of Illuminations}
+
+Imaging fluorescent molecules is challenging because of their weak signals. Any background light, such as autofluorescence from the sample buffer or out-of-focus fluorescence from the sample, will reduce the signal-to-noise ratio of the image. As a result, it is important to minimize the amount of background light that reaches the camera.
+
+Out-of-focus fluorescence can be mitigated by a careful choice of illumination strategy. Any microscope will have a finite focal volume, as illustrated in \autoref{fig:depth-of-focus}. Sources of light from within this volume will appear in focus in the image, whereas sources outside of this volume will appear out-of-focus. Sources that lie outside the axial extent of this volume (called the depth of focus) contribute to background fluorescence.
+
+\begin{figure}[ht]
+    \centering
+    \includegraphics{focal-volume.png}
+    \caption{The focal volume of a microscope. This is the volume within which a point source will appear in focus in an image. Point sources outside of this volume will appear out-of-focus. The axial extent of the focal volume is called the depth of focus.}
+    \label{fig:depth-of-focus}
+\end{figure}
+
+Epi-illumination, as shown in \autoref{fig:epi-modes}, illuminates the entire sample, both within and outside of the focal volume. As a result, epi-illumination often produces images with high background fluorescence. However, there are other illumination modes available, two of which are also illustrated in \autoref{fig:epi-modes}. Highly inclined and laminated optical sheet (HILO) microscopy illuminates the sample with a thin sheet of light that is highly inclined with respect to the optical axis of the microscope \cite{tokunaga-naturemethods-2008}. Only regions of the sample within the light sheet will be illuminated. If the lightsheet is thin enough to fit within the focal volume, then out-of-focus fluorescence will be minimized because no fluorophores outside the focal volume will be excited.
+
+Total internal reflection fluorescence (TIRF) microscopy illuminates the sample with light that is totally internally reflected at the coverslip \cite{axelrod-traffic-2001}. Only regions of the sample within one wavelength of the coverslip will be illuminated.
+
+Why would one choose epi-illumination if the background is higher than the other methods? For one, epi-illumination often produces smooth illumination patterns on the sample, whereas HILO and TIRF can produce stripe-like artifacts due to the directionality of the illumination and scattering from the sample. It's also the simplest of all the techniques to implement and is good enough in many situations. HILO would be appropriate for targets deep inside thick cells, and TIRF is ideal for targets that are very close to the coverslip, such as the cell membrane.
+
+\begin{figure}[ht]
+    \centering
+    \includegraphics[width=1.0\textwidth]{epi-illumination-modalities.png}
+    \caption{Different illumination modalities for epi-fluorescence imaging. Epi-illumination illuminates the whole sample at once so that out-of-focus fluorescence contributes to the background. HILO illuminates the sample with a thin light sheet. Only regions of the sample within the light sheet will fluoresce. Finally, in TIRF, the illumination is totally internally reflected at the coverslip so that only regions of the sample within one wavelength of the coverslip are illuminated.}
+    \label{fig:epi-modes}
+\end{figure}
+
 \section{Digital Image Formation}
 
 The image described by \autoref{eq:image-formation} is a function of continuous spatial variables $x$ and $y$ and represents the optical power per area at a point $\left(x, y\right)$ in the image plane.\footnote{Equivalently, it is proportional to the photon flux at the image plane in units of $\text{photons} / s / m^2$.} A camera, which is used to record the image, is comprised of regularly-spaced, discrete, photo-sensitive elements called pixels. Each pixel converts the optical signal integrated over the time of exposure into a digital number called an analog-to-digital unit, or ADU. A well-designed camera produces images with pixel values (in units of ADUs) that are proportional to the number of photons that were incident on each pixel during an exposure.