Assignment 2 Part 4: Fluorescent imaging of actin

From Course Wiki
Jump to: navigation, search
20.309: Biological Instrumentation and Measurement

ImageBar 774.jpg

3T3 Actin.png

3T3 Actin plus CytoD.png

3T3 cells. Actin stained with Alexa Fluor 568 Phalloidin.

Same cell type incubated with cytochalasin D, an inhibitor of actin polymerization, prior to fixing and stained.


In this part of the lab, you will use fluorescence microscopy to investigate how an inhibitor of actin polymerization called cytochalasin D (CytoD) affects the structural properties of mammalian cells. To make the cells' actin cytoskeleton visible, you will exploit a mushroom toxin called phalloidin that has been conjugated with the fluorescent dye Alexa Fluor 568. Phalloidin binds to polymerized filamentous actin (F-actin) much more tightly than to actin monomers (G-actin), and stabilizes actin filaments by preventing their depolymerization. By comparing Alexa Fluor phalloidin-stained untreated cells and CytoD-treated cells, you will observe and quantify the effect CytoD has on the actin stress fiber network. You will compare the structure of cells that have been treated with CytoD with that of untreated cells.

20.309 130904 PhalloidinActin.png Alexa568.jpg
Example of a NIH 3T3 fibroblast cell labeled with rhodamine phalloidin Excitation and emission spectra for Alexa Fluor 568

Warning.jpg Wear gloves when you are handling biological samples.

Procedures for fixing and labeling cells

3T3 Swiss Albino

You will be given two plates of cells:

  • one dish will be left untreated, and then fixed and labeled with phalloidin;
  • the second dish will be first treated with CytoD, and then fixed and labeled with phalloidin.

A key technique to keep in mind when working with live cells - to avoid shocking them with "cold" at 20°C - is to be sure that any solutions you add are pre-warmed to 37°C. We will keep a warm-water bath running for this purpose, in which we will keep the various media.

You are provided with 3T3 cells, which were prepared as follows: Cells were cultured at 37°C in 5% CO$ _2 $ in standard T75 flasks in a medium referred to as DMEM++. This consists of Dulbecco's Modified Eagle Medium (DMEM - Invitrogen) supplemented with 10% fetal bovine serum (FBS - Invitrogen), 1% of the antibiotic penicillin-streptomycin (Invitrogen), 1% non-essential amino acids, and 1% glutamine. The day prior to the fluorescence imaging, cells were plated on 35 mm glass-bottom cell culture dishes (MatTek, equipped with coverslip suited for optical microscopy studies).

Please read through the protocol for the two dishes below. Think about what you need to do so you can handle the dishes in parallel and save yourself some time.

Below is the protocol to stain 3T3 cells with Alexa Fluor 568 phalloidin:

  • It is optimal for your cells to be ~60% confluent. If cells are too crowded, they will not stretch properly and show their beautiful actin filaments. Thus, you'll want to image cells that are stretched out and not overlapping much. Note also that these cells remain alive until the addition of formaldehyde, therefore requiring that any buffer/media added be pre-warmed.
  1. Pre-warm 3.7% formaldehyde solution and phosphate buffered saline at pH 7.4 (PBS) in a 37°C water bath if available. Keep the formaldehyde wrapped in foil to protect from light.
  2. Retrieve an aliquot of cytochalasin D from the freezer and warm to 37°C.

Dish 1--CytoD Treated Cells

  • Prepare only ONE dish to be treated with cytochalasin D (CytoD). The other dish will be an untreated control.
  1. Remove the medium with a pipette and wash ONE dish 2X with 2 mL of pre-warmed PBS. Pipet into the dish gently to avoid washing away cells.
  2. Add 1 mL of the pre-warmed 10 μM CytoD solution to the same cell culture dish and incubate at 37°C for 20 minutes (and not a minute longer!). Afterwards, wash 2X with PBS.

Dish 1 (CytoD) & Dish 2 (Untreated) Stained in Parallel

  1. Remove the medium from the untreated dish (Dish 2) with a pipette and wash 2X with 2 mL of pre-warmed PBS. Pipet gently to avoid washing away cells. This assumes you've already washed your Cyto D plate (Dish 1).
  2. Carefully pipet 400 μL of 3.7% formaldehyde solution onto the cells in the central glass region of each dish and incubate for 10 minutes at room temperature. This "fixes" the cells, i.e. cross-links the intracellular proteins and freezes the cell structure.
  3. Wash each dish 3X with 1.5 mL PBS (note that this PBS solution no longer needs to be pre-warmed as the cells are dead).
  4. Extract each dish with 1.5 mL 0.1% Triton X-100 (a detergent) for 3-5 minutes. (Extraction refers to the partially dissolution of the plasma membrane of the cell.)
  5. Wash the cells 2X with 1.5 mL PBS.
  6. Incubate the fixed cells with 1.5 mL 1% BSA in PBS for 20 minutes. (BSA blocks the nonspecific binding sites.)
  7. Wash each dish 2X with PBS.
  8. Add 200 μL of Alexa Fluor 568 phalloidin solution (pre-mixed in methanol and PBS) to each dish. Carefully pipet this just onto the center of the dish, cover with aluminum foil, and incubate for 45 minutes at room temperature.
  9. Wash 3X with PBS.
  10. You can now store the sample at +4°C (regular refrigerator) in PBS for a few days, wrapped in parafilm and foil.

Flat field correction

  1. Perform flat-field correction on the images.
    • Divide the image by a normalized version of your reference image minus the dark image (see this page for more detail).
  2. Include the original, reference, and flat-field corrected images in your lab report.

Report requirements

General guidelines

  • Each part of the microscopy lab requires its own submission. Only the final report will include all 3 sections. You may revise any part of your report until the final deadline. Update the apparatus section to reflect any changes you make during the course of the lab.
  • On each due date, one member of your group should submit a single PDF file to Stellar in advance of the deadline. The filename should consist of the last names of all group members, CamelCased, in alphabetical order, followed by a hyphen, the name of the assignment, with a .pdf extension. Example: CrickFranklinWatson-MicroscopyPart1.pdf.
  • Use the outline below to break your report into sections.
  • Keep your lab report short. Use bullet points, tables, and other organizational tools when appropriate. Brevity is not an excuse for imprecise, incomplete, or unclear communication.
  • Be selective in what images you include in your report. Where images are required, include one or a few that convey the character of the dataset. Size images appropriately (does it really need to take up half a page?). At the same time, ensure plots images are readable. A good trick for reducing plot size while maintaining clarity is to adjust the font size and line values so the plot remains clear even when it is small. It helps to save images and plots in an uncompressed format; import and resize them in a way that retains the full quality; and create your final PDF file with settings that do not compress plots and pictures.
  • An outstanding error discussion is an essential element of a top-notch report.
  • Include any computer code you developed in an appendix at the end of your report. Indicate the source of any code used that you obtained from outside sources.
  • Refer to this page for more detail on how to write an excellent report, and to this page for an outline of the final microscopy report.

Part 2 report outline

  1. Microscope documentation
    1. Include an updated block diagram of your microscope.
  2. Images
    1. Include a figure with an images of the 3.26 μm fluorescent microsphere samples, and the stained cell samples with and without Cyto-D.
      • For each sample, create 1 figure with 5 panels.
      • The panels of the figure should be: A) unprocessed image; B) reference image; C) dark image; D) flat-field corrected image; and E) histogram.
      • In the caption, specify the exposure and gain settings. Each image should have a scale bar. State the dimension of the scale bar in the caption.
      • For panel E, plot histograms of the unprocessed, dark, reference, and corrected image on the same set of axes. Plot log10( count ) on the vertical axis and intensity on the horizontal axis. Use a line plot instead of a bar chart for the histogram.
    2. Image profile
      • For one reference, dark and cell image set, plot an intensity profile across the same diagonal. You may also use a bead image, along with it's unique reference and dark images. The intensity of your three images should be on the same scale, i.e., 0 to 65,535 or 0 to 1. Place all three profiles on a single set of axes for comparison. (Use the improfile command in MATLAB.)
  3. Discussion
    1. How did your beam expander design affect your images?
    2. What differences did you observe between the cells with and without CytoD?

Optical microscopy lab

Code examples and simulations

Background reading