20.109(S07): Measuring calcium in vivo

Introduction

excitation and emission spectra for a fluor, from Invitrogen

principle of fluorescence

Fluorescent microscopes can reveal aspects of cellular structures that other techniques cannot address. Information about a cell's shape, its cellular compartments and the components of those compartments are all routinely collected thanks to laboratory microscopes no different than the one we'll use today. A number of useful fluorescent molecules are available for these purposes, each with distinct excitation and emission properties. In every case, the fluor is excited by light at one wavelength which boosts an electron to a higher orbital and it is the energy emitted by that electron as it relaxes that is detected. Ongoing research is directed at improving both the spectral properties of the fluorescent molecules themselves and at improving microscopy techniques, for example through better digital image analysis and through novel setups that circumvent detection limits imposed by light's diffraction, for example the recent description of PALM microscopy.


Consider the image that appears on the front cover of this experimental module.

MES cells expressing YFP, 400X magnification

One could qualitatively describe the appearance of the cells under the fluorescent microscope ("a mixture of non-fluorescent and fluorescent cells, some more brightly fluorescent than others"). A more quantitative measurement of the fluorescence associated with each cell might be gleaned from sorting the cells using flow cytometry. With that technique, the spectral signature of individual cells can be assessed, at an astonishing rate (hundreds of thousands of cells/minute), and the population can be "binned" according to the brightness of their signal. But lost is information regarding localization of the signal inside each cell, and real time changes in fluorescence of single cells cannot be assessed with this technique.

Digital processing of the image above might allow for a more quantitative analysis of its appearance. Fast digital cameras with impressive resolution and color fidelity are widely available, as are software packages that analyze pixel distributions in images. Some software will even "guess" at the value between pixels to improve resolution. Imaging artifacts arise, however, due to the light emitted from other objects in the focal plane as well as out-of-plane light that adds to the object being analyzed see, for example, Barlow and Guerin, 2007. In addition, since fluorescence microscopy relies on an excitation wavelength to stimulate the fluor, there can sometimes be "bleeding" of from the shoulder of the excitation energy into the detection window.

Protocols

Microscopy

The cells you transfected last time were fed yesterday and are ready for you to wash and visualize today. The washing will be done in the cell culture facility using sterile technique but the cells will be treated with Ca²⁺-mobilization agents and then visualized in the main teaching lab.

In the cell culture facility

1. Prepare the sterile hood in the cell culture facility as you are now accustomed to doing.
2. Inspect your cells at the inverted microscopes, looking at the color of the media, the cell's density and distribution, etc.
3. Aspirate the media from the wells, taking care not to touch the cells themselves with the tip of the aspirator.
4. Wash the cells by adding ~0.5 ml of PBS to each well and then aspirating the liquid.
5. Fill each well with exactly 500 ul of OptiMEM, measured with your P1000, to each well.
6. Clean and close down the sterile hood and move, with your cells, into the main teaching lab.

In the main lab

1. At your bench add the calcium-mobilizing agents you have chosen to the wells. Recall that the well closest to the slide's label was not transfected and so should remain untreated. You and your partner should also choose one transfected well to leave untreated.
2. Once the cells have been treated, remove the liquid with your P1000, taking care not to touch the cells themselves with your pipet tip. Discard the liquid into a conical tube for disposal.
3. Use the "key" that is available to slide the wells off the slide itself, carefully peeling the plastic wells off and covering the cells with a long coverslip to keep them from drying.
4. Examine the cells under the fluorescent microscope, first finding the cells in the white light and then examining them under the fluorescent ("FITC") light. You will have to turn off the white light or cover the source with a box-top to see the fluorescent signal. You may also want to turn off the room lights and work with only the task light on.
5. Some comparisons you will want to make (and record your observations in words in your notebook and/or with digital photos) include:
   • how do the mock transfected cells compare with the transfected but untreated cells
   • how do the treatments affect fluorescence
   • how do clumps of cells vs spread cells compare
   • how homogeneous is each sample
   • what magnification gives you the most and most reliable information

Before you leave lab today, please upload at least one image to the discussion page associated with today's lab. Include a description of the image, written as though you are submitting a figure for a journal article. Specifically, you should include a title for the image (often the conclusion you would like the reader to draw) and some information about the image for readers who know nothing about the experiment you've performed (cell type, magnification, intent of the experiment, conclusion you can draw). Be sure to include your names or team color as well.

DONE!

For next time

1. Print out the image and description that you uploaded to the wiki.
2. Look through the list of journal articles that are collected for the final day of this module. You will be presenting one of these articles to the class, so begin to review both the guidelines for oral presentations and the list of possible articles for presentation. If there is one you like, please sign-up for the article by putting your (initials/lab section/team color) next to the article on the list. You may present an article that is not on the list, but please email NK with your idea first.
3. Revise your first draft of your module 1 essay. It is due in one week.

Reagents list

• PBS
• OptiMEM