LEB-EPFL · kmdouglass · Dec 5, 2025 · Dec 4, 2025 · Dec 4, 2025
diff --git a/book/src/SUMMARY.md b/book/src/SUMMARY.md
@@ -4,7 +4,7 @@
   - [Learning Outcomes](./chapter_intro/learning_outcomes.md)
 - [Live Cell Fluorescence Imaging of Mitochondria](./chapter_live_cell_mito/README.md)
   - [The Epifluorescence Microscope](./chapter_live_cell_mito/microscope_components.md)
-  - [Materials](./chapter_live_cell_mito/materials.md)
+  - [Microscope Startup](./chapter_live_cell_mito/startup/README.md)
 - [Microscope Optics and Image Acquisition](./chapter_image_acquisition/README.md)
   - [Set up the Camera](./chapter_image_acquisition/camera.md)
   - [Align the Camera and Tube Lens](./chapter_image_acquisition/camera_tube_lens_alignment.md)

diff --git a/book/src/chapter_intro/learning_outcomes.md b/book/src/chapter_intro/learning_outcomes.md
@@ -20,12 +20,6 @@ At the end of this training, a student should be able to:
 - The students should know how to perform background subtraction and identify when it is necessary to do so
 - The students should be able to estimate how much brighter the signal is in one fluorescence channel vs. another
 
-### Measure the degree of fluorescence bleedthrough
-
-- The students should be able to define what fluorescence bleedthrough is and explain why it is problematic
-- The students should be able to perform the necessary measurements to assess bleedthrough
-- The students should be able to explain what steps one might take to alleviate the effects of bleedthrough
-
 ## Optics Module
 
 The purpose of the alignment module is to provide hands-on experience to students and post-docs who will build laboratory setups for fluorescence microscopy.

diff --git a/book/src/chapter_live_cell_mito/README.md b/book/src/chapter_live_cell_mito/README.md
@@ -1 +1,3 @@
 # Live Cell Fluorescence Imaging of Mitochondria
+
+In this section we will use a Zeiss Axio Observer 7 widefield fluorescence microscope to perform live cell imaging of mitochondria in U2OS cells. The cells have been modified to express [StayGold](https://www.fpbase.org/protein/staygold/) fluorescent protein that is targeting the mitochondrial matrix.
diff --git a/...cquisition/epifluorescence-microscope.svg → ..._cell_mito/epifluorescence-microscope.svg b/...cquisition/epifluorescence-microscope.svg → ..._cell_mito/epifluorescence-microscope.svg
diff --git a/book/src/chapter_live_cell_mito/materials.md b/book/src/chapter_live_cell_mito/materials.md
diff --git a/book/src/chapter_live_cell_mito/startup/README.md b/book/src/chapter_live_cell_mito/startup/README.md
@@ -0,0 +1,77 @@
+# Microscope Startup
+
+We will begin by starting up the microscope and its components.
+
+## Instructions
+
+### Step 0
+
+Turn on the air pump and the environmental controls for the incubator.
+
+It is good practice to turn these on 10 minutes or more before any experiment so that the environmental controls have a chance to stabilize.
+
+![](./step_0.jpg)
+![](./step_1.jpg)
+
+### Step 1
+
+Turn on the focus controller for the Zeiss's Definite Focus system.
+
+In addition, turn on the Zeiss's Power Supply 232 and SMC 2009 controllers.
+
+![](./step_2.jpg)
+
+### Step 2
+
+Turn on the CoolLED unit.
+
+![](./step_3.jpg)
+
+### Step 3
+
+Turn on the camera. You need not wait for the initialization to finish before turning on the control PC because the camera is connected via USB.
+
+![](./step_4.jpg)
+
+### Step 4
+
+Wait until the Definite Focus controller's initialization has finished. (You can tell whether it has finished because it will no longer display a message indicating that it is initializing on its LCD display screen.)
+
+Turn on the microscope.
+
+![](./step_5.jpg)
+
+### Step 5
+
+Log into the computer with your EPFL username.
+
+Find the shortcut to Micro-Manager on the desktop and double click it.
+
+*2025-12-05*: This shortcut should point to `C:\Program Files\Micro-Manager-2.0\ImageJ.exe`. It is a slightly out-of-date version of Micro-Manager that is intended to support the CoolLED light source.
+
+![](./step_7.jpg)
+![](./step_6.jpg)
+
+### Step 6
+
+After a few seconds or less You will see the Micro-Manager startup screen. Ensure that the `231031_ZeissAxioObserver7.cfg` configuration file is selected. We have placed it inside the Micro-Manager folder in case you must select it yourself.
+
+Click `OK` to continue.
+
+![](./step_8.jpg)
+
+### Step 7
+
+After a few more seconds, you will see the Micro-Manager control panel. If you do not see all the configuration settings at first, then expand the window by dragging its border.
+
+![](./step_9.jpg)
+
+### Step 8
+
+You can set the channel and objective by selecting the corresponding configuration settings in the control panel.
+
+![](./step_10.jpg)
+
+### Step 9
+
+Click the `Live` button. If you see a noisy image stream appear, then you are ready to proceed to the next steps.
diff --git a/book/src/chapter_live_cell_mito/startup/step_0.jpg b/book/src/chapter_live_cell_mito/startup/step_0.jpg
diff --git a/book/src/chapter_live_cell_mito/startup/step_1.jpg b/book/src/chapter_live_cell_mito/startup/step_1.jpg
diff --git a/book/src/chapter_live_cell_mito/startup/step_10.jpg b/book/src/chapter_live_cell_mito/startup/step_10.jpg
diff --git a/book/src/chapter_live_cell_mito/startup/step_2.jpg b/book/src/chapter_live_cell_mito/startup/step_2.jpg
diff --git a/book/src/chapter_live_cell_mito/startup/step_3.jpg b/book/src/chapter_live_cell_mito/startup/step_3.jpg
diff --git a/book/src/chapter_live_cell_mito/startup/step_4.jpg b/book/src/chapter_live_cell_mito/startup/step_4.jpg
diff --git a/book/src/chapter_live_cell_mito/startup/step_5.jpg b/book/src/chapter_live_cell_mito/startup/step_5.jpg
diff --git a/book/src/chapter_live_cell_mito/startup/step_6.jpg b/book/src/chapter_live_cell_mito/startup/step_6.jpg
diff --git a/book/src/chapter_live_cell_mito/startup/step_7.jpg b/book/src/chapter_live_cell_mito/startup/step_7.jpg
diff --git a/book/src/chapter_live_cell_mito/startup/step_8.jpg b/book/src/chapter_live_cell_mito/startup/step_8.jpg
diff --git a/book/src/chapter_live_cell_mito/startup/step_9.jpg b/book/src/chapter_live_cell_mito/startup/step_9.jpg
Original file line number	Diff line number	Diff line change
		@@ -1 +1,3 @@
		# Live Cell Fluorescence Imaging of Mitochondria

		In this section we will use a Zeiss Axio Observer 7 widefield fluorescence microscope to perform live cell imaging of mitochondria in U2OS cells. The cells have been modified to express [StayGold](https://www.fpbase.org/protein/staygold/) fluorescent protein that is targeting the mitochondrial matrix.