Difference between revisions of "20.109(S07): Measuring calcium in vivo"

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	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 you'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 you'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].
		+	Consider the image that appears on the front cover of this experimental module.
		+	[[Image:Macintosh_HD-Users-nkuldell-Desktop-SgnlMeasrmnt_coverart_S07.jpg|thumb|300 pixels|center| MES cells expressing YFP, 400X magnification]] Once 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 gleened 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.
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	==Protocols==		==Protocols==
	===Microscopy===		===Microscopy===

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Revision as of 12:05, 28 December 2006

Introduction

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 you'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.

Once 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 gleened 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.

Protocols

Microscopy

1. visualize cells vs controls
2. modulate Ca++ with ionophores/chelators

DONE!

For next time

Reagents list