diff --git a/book/src/SUMMARY.md b/book/src/SUMMARY.md index e753d4f..7a3de80 100644 --- 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 index a2811cc..a428fb6 100644 --- 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 index 731f17e..bff48fc 100644 --- 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/book/src/chapter_image_acquisition/epifluorescence-microscope.svg b/book/src/chapter_live_cell_mito/epifluorescence-microscope.svg similarity index 100% rename from book/src/chapter_image_acquisition/epifluorescence-microscope.svg rename to book/src/chapter_live_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 deleted file mode 100644 index e0dba54..0000000 --- a/book/src/chapter_live_cell_mito/materials.md +++ /dev/null @@ -1,3 +0,0 @@ -# Materials - -Add 2 \\( \mu \\)L of 1 mM Mitotracker Green to 2 mL of DMEM. diff --git a/book/src/chapter_live_cell_mito/startup/README.md b/book/src/chapter_live_cell_mito/startup/README.md new file mode 100644 index 0000000..e3452a8 --- /dev/null +++ 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. 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