Main Page/BPHS 4090/microscopy II - Revision history

Mgeorge: moved Main Page/microscopy II to Main Page/BPHS 4090/microscopy II

2011-07-19T12:39:06Z

moved Main Page/microscopy II to Main Page/BPHS 4090/microscopy II

Mgeorge: Protected "Main Page/microscopy II" ([edit=sysop] (indefinite) [move=sysop] (indefinite))

2011-07-15T19:05:48Z

Protected "Main Page/microscopy II" ([edit=sysop] (indefinite) [move=sysop] (indefinite))

Sbilling: 28 revisions

2011-01-17T21:02:11Z

28 revisions

Junk at 20:08, 26 November 2010

2010-11-26T20:08:59Z

Junk: Protected "Main Page/microscopy II" ([edit=sysop] (indefinite) [move=sysop] (indefinite))

2010-09-17T17:29:12Z

Protected "Main Page/microscopy II" ([edit=sysop] (indefinite) [move=sysop] (indefinite))

Junk at 17:50, 10 September 2010

2010-09-10T17:50:22Z

Junk at 17:49, 10 September 2010

2010-09-10T17:49:23Z

Junk at 17:39, 10 September 2010

2010-09-10T17:39:31Z

Junk at 17:32, 10 September 2010

2010-09-10T17:32:49Z

Junk at 17:27, 10 September 2010

2010-09-10T17:27:12Z

@@ Line 1: / Line 1: @@
 <h1>Required Components</h1>
 <ol>
-<li>Stage micrometer slide</li>
+<li>[[Media:Micrometer_Slide.JPG|Stage micrometer slide]]</li>
-<li>Chroma fluorescent slide kit</li>
+<li>[[Media:M2_Chroma_Slide_Kit.JPG|Chroma fluorescent slide kit]]</li>
-<li>Fluorescent bead slide</li>
+<li>[[Media:M2_Bead_Slide.JPG|Fluorescent bead slide]]</li>
-<li>Fluorescent microchannel slide</li>
+<li>[[Media:M2_Microchannel.JPG|Fluorescent microchannel slide]]</li>
-<li>Fresh onion</li>
+<li>[[Media:Slide_Tools.JPG|Fresh onion]]</li>
-<li>Knife and tweezers</li>
+<li>[[Media:Slide_Tools.JPG|Knife and tweezers]]</li>
-<li>Toothpick or popsicle sticks</li>
+<li>[[Media:Slide_Tools.JPG|Toothpick or popsicle sticks]]</li>
-<li>Blank slides and coverslips</li>
+<li>[[Media:Slide_Tools.JPG|Blank slides and coverslips]]</li>
-<li>Syringe of vaseline with pipette tip end</li>
+<li>[[Media:Slide_Tools.JPG|Syringe of vaseline with pipette tip end]]</li>
-<li>Distilled water</li>
+<li>[[Media:Slide_Tools.JPG|Distilled water]]</li>
-<li>Immersion oil</li>
+<li>[[Media:Immersion_Oil.JPG|Immersion oil]]</li>
-<li>Nikon upright microscope and CCD camera</li>
+<li>[[Media:CCD.JPG|Nikon upright microscope and CCD camera]]</li>
 <li>Usb stick for transferring data and images for processing and report generation</li>
 </ol>

@@ Line 554: / Line 554: @@
 intensity by the exposure setting used for each of the images. In other words, if
 you used a 100 ms exposure for the 40x oil and 400 ms exposure for the 40x air:
-<table width=250 align=center><td>
+<table width=280 align=center><td>
-<p align=justify>[[File:Fl_eqn5.png|250px|center]]
+<p align=justify>[[File:Fl_eqn5.png|280px|center]]
 <br clear=right>
 </p>
@@ Line 575: / Line 575: @@
         <li>Save all images and clean the slide and put it away.</li>
         </ol>
-</ol>
 <h1>Questions</h1>
@@ Line 594: / Line 594: @@
 <ol>
 <li>1.	The optical path of a DIC microscope and its main components are shown in the figure below.  Alongside this figure are actual images of what the components look like when removed from the microscope.
-<table width=350 align=center><td>
+<table width=450 align=center><td>
-<p align=justify>[[File:Fl_a1_1.png|350px|center]]
+<p align=justify>[[File:Fl_a1_1.png|450px|center]]
 <br clear=right>
 </p>

@@ Line 524: / Line 524: @@
         </li>
         </ol>
-<li>b>(OPTIONAL)</b> Fluorescence of microbeads
+<li><b>(OPTIONAL)</b> Fluorescence of microbeads
       <ol style="list-style-type:lower-latin">
       <li>Obtain the fluorescent microbead slide. There are six regions marked on the slide, the
@@ Line 554: / Line 554: @@
 intensity by the exposure setting used for each of the images. In other words, if
 you used a 100 ms exposure for the 40x oil and 400 ms exposure for the 40x air:
-<table width=150 align=center><td>
+<table width=250 align=center><td>
-<p align=justify>[[File:Fl_eqn5.png|150px|center]]
+<p align=justify>[[File:Fl_eqn5.png|250px|center]]
 <br clear=right>
 </p>
@@ Line 566: / Line 566: @@
         </ol>
 <li><b>(OPTIONAL)</b> Skin slide

@@ Line 524: / Line 524: @@
         </li>
         </ol>
-<li><b>Optional</b> Fluorescence of microbeads
+<li>b>(OPTIONAL)</b> Fluorescence of microbeads

← Older revision		Revision as of 17:32, 10 September 2010
Line 481:		Line 481:
	</ol>		</ol>
	<li>Liver slide		<li>Liver slide
		+	<ol style="list-style-type:lower-latin">
		+	<li>Obtain the fluorescent liver slide.</li>
		+	<li>Using the 20x objective, find some areas with bright staining and move to the 40x (oil)
		+	and 100x objectives.</li>
		+	<li>Try and observe the peroxisomes using the lower resolution objectives (10x/20x). Do
		+	you see them as donuts or spots?</li>
		+	<li>Using the 40x (oil) and 100x objectives acquire fluorescence images using all three filter
		+	cubes, particularly focusing on getting good clean images of the peroxisomes, which
		+	should look like small ring shaped objects with these objectives.</li>
		+	<li>Save all images and clean the slide and put it away.</li>
		+	<li>Peroxisome measurement and RGB image generation.
		+	<ol style="list-style-type:lower-roman">
		+	<li>Input the spatial calibrations you calculated previously for these objectives and
		+	generate and RGB merged image using the method described above.</li>
		+	<li>Measure the size of the peroxisome, which should be around 200-400 nm. Do
		+	your measurements agree?</li>
		+	</ol>
		+	</li>
		+	</ol>
		+	<li>Gut slide
		+	<ol style="list-style-type:lower-latin">
		+	<li>Obtain the fluorescent gut slide.</li>
		+	<li>Browse around the slide and locate some villi.</li>
		+	<li>Acquire images using 10x and 20x objectives with all filter cubes as well as by sliding the
		+	DIC polarizers and prisms in place and acquiring a DIC image (note: DIC is only possible
		+	using the 20x and 100x objectives on this system).</li>
		+	<li>Repeat with the 100x objectives.</li>
		+	<li>It may be useful to take the DIC images first to make sure that you see some goblet cells
		+	in the field of view on the camera.</li>
		+	<li>Save all images and clean the slide and put it away.</li>
		+	<li>Image manipulations in ImageJ.
		+	<ol style="list-style-type:lower-roman">
		+	<li>Produce a fluorescence/DIC overlay in ImageJ of the frames you acquired using
		+	the method described above.</li>
		+	<li>Using spatially calibrated images, measure the size of the microvilli on the
		+	columnar epithelial cells. They should be approximately 400 μm in length, and
		+	barely visible on the 100x objective.</li>
		+	<li>Also measure the diameter of the mucus secretions in the goblet cells.</li>
		+	<li>Both of the above measurements may be easier to perform on the original
		+	greyscale data rather than the colour composite.</li>
		+	</ol>
		+	</li>
		+	</ol>
		+	<li><b>Optional</b> Fluorescence of microbeads

← Older revision	Revision as of 12:39, 19 July 2011
(No difference)

← Older revision	Revision as of 19:05, 15 July 2011
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← Older revision	Revision as of 21:02, 17 January 2011
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← Older revision	Revision as of 17:29, 17 September 2010
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@@ Line 425: / Line 425: @@
 <ol style="list-style-type:lower-latin">
     <li>Prepare another cheek sample and onion sample as you did in the previous lab and observe them in DIC using the 20x and 100x DIC objectives.  Be careful not to transfer any oil onto the 20x objective.</li>
-</li>
+</li></ol>
 <li>Fluorescence excitation and photobleaching of sub-resolution bead mixtures.
 <ol style="list-style-type:lower-latin">