20.109(S07): Measuring calcium in vivo: Difference between revisions
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==Introduction== | ==Introduction== | ||
Fluorescent microscopy relies on excitation of the fluor at one wavelength which boosts an electron to a higher orbital. The energy emitted by the electron as it relaxes is detected. <br> | Fluorescent microscopy relies on excitation of the fluor at one wavelength which boosts an electron to a higher orbital. The energy emitted by the electron as it relaxes is detected. <br> | ||
[[Image:Macintosh HD-Users-nkuldell-Desktop- | |||
[[Image:Macintosh HD-Users-nkuldell-Desktop-fluorescenceprinciple.png | [[Image:Macintosh HD-Users-nkuldell-Desktop-excitation emission.jpg|left|thumb| excitation and emission spectra for a fluor, from Invitrogen]] | ||
[[Image:Macintosh HD-Users-nkuldell-Desktop-fluorescenceprinciple.png|right|thumb| principle of fluorescence]] | |||
Microscopy 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. Furthermore, as research to improve microscopy techniques continues, breakthroughs are reported that improve [http://www.microscopyu.com/articles/digitalimaging/drentdigital.html digital image analysis] and that increase detection limits imposed by light's diffraction [http://www.hhmi.org/news/palm20060810.html , for example the recent description of PALM microscopy]. | Microscopy 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. Furthermore, as research to improve microscopy techniques continues, breakthroughs are reported that improve [http://www.microscopyu.com/articles/digitalimaging/drentdigital.html digital image analysis] and that increase detection limits imposed by light's diffraction [http://www.hhmi.org/news/palm20060810.html , for example the recent description of PALM microscopy]. | ||
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[[Image:Macintosh_HD-Users-nkuldell-Desktop-SgnlMeasrmnt_coverart_S07.jpg|thumb|500 pixels|left| MES cells expressing YFP, 400X magnification]] One could <b>qualitatively </b> 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.<br> | [[Image:Macintosh_HD-Users-nkuldell-Desktop-SgnlMeasrmnt_coverart_S07.jpg|thumb|500 pixels|left| MES cells expressing YFP, 400X magnification]] One could <b>qualitatively </b> 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.<br> | ||
Digital processing of the image above might allow for a more <b> quantitative </b> 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. 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. | Digital processing of the image above might allow for a more <b> quantitative </b> 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 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17131356&query_hl=1&itool=pubmed_docsum 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. | ||
Revision as of 06:45, 28 December 2006
Introduction
Fluorescent microscopy relies on excitation of the fluor at one wavelength which boosts an electron to a higher orbital. The energy emitted by the electron as it relaxes is detected.
Microscopy 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. Furthermore, as research to improve microscopy techniques continues, breakthroughs are reported that improve digital image analysis and that increase 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.
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
- visualize cells vs controls
- modulate Ca++ with ionophores/chelators
Before you leave lab today, please upload at least on 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. 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!
