Main Page/BPHS 4090/In-Vivo Spectrocopy - Revision history

Gloria at 18:56, 18 December 2020

2020-12-18T18:56:54Z

Gloria at 17:03, 18 December 2020

2020-12-18T17:03:39Z

Gloria at 16:57, 18 December 2020

2020-12-18T16:57:38Z

Gloria at 16:56, 18 December 2020

2020-12-18T16:56:52Z

Gloria at 16:50, 18 December 2020

2020-12-18T16:50:35Z

Gloria at 16:46, 18 December 2020

2020-12-18T16:46:57Z

Gloria at 16:33, 18 December 2020

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Gloria at 16:22, 18 December 2020

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Gloria at 16:10, 18 December 2020

2020-12-18T16:10:00Z

Gloria at 16:04, 18 December 2020

2020-12-18T16:04:35Z

@@ Line 76: / Line 76: @@
 <p>Most of the diffuse reflection you will measure originates from the epidermal layer, which contains no blood vessels or capillaries.  The blood diffuses through the dermal layer and into the epidermis, essentially meaning that there is a homogeneous distribution of Hb in the epidermal layer.  For the purposes of this lab, you can consider the epidermal layer to be a perfect diffuse reflector, with a uniform distribution of melanin and Hb ‘absorbers’ present in a given volume.  Since the diffusely reflected light is interacting with melanin and Hb as it is scattered within the epidermal layer, there is information within this light regarding the absorption properties of Hb and melanin.</p>
-<h1>Part A: In-Vivo Spectroscopy</h1>
+<h1>Experiment: In-Vivo Spectroscopy</h1>
 <h2>Methods </h2>

@@ Line 6: / Line 6: @@
 <li>PC with spectrometer software</li>
 <li>[[Media:Spec_White_Reference.JPG|White reflectance reference target]]</li>
-<li>[[Media:Spec_Light_Source.JPG|White LED illumination source]] and housing unit</li>
+<li>[[Media:SpecLightSource.JPG|White LED illumination source]] and housing unit</li>
 <li>[[Media:Spec_Pressure_Cuff.JPG|Blood pressure compression cuff]]</li>
 <li>Stopwatch timer (can use windows clock for this)</li>

@@ Line 155: / Line 155: @@
 <h2>Data Analysis</h2>
-Using your data sets and a program of your choice you can analyze your data.  The following section, "Calculating Theory" describes he analysis.
+Using your data sets and a program of your choice you can analyze your data.  The following section, "Calculating Theory" describes the analysis.
 <h2> Calculation Theory </h2>

@@ Line 119: / Line 119: @@
 <h2>Blood Oxygenation Time Series Data Collection </h2>
-To obtain the time-series blood oxygenation of the forearm you will need the blood pressure compression cuff.  Pressure to the cuff is increased by pumping on the bladder, and a silver release valve in front of the bladder allows the pressure to be released.  Perform a trial run of the experiment without saving the data and observe the live spectrum during the procedure.  Once you are comfortable with the timing and have set up the parameters as described below collect data for 3 trials using the following procedure:
+To obtain the time-series blood oxygenation of the forearm you will need the blood pressure compression cuff.  Pressure to the cuff is increased by pumping on the bladder, and a silver release valve in front of the bladder allows the pressure to be released.  Perform a trial run of the experiment without saving the data and observe the live spectrum during the procedure described below.  Once you are comfortable with the timing and have set up the parameters as described below collect data for 3 trials using the following procedure:
 To start the measurements follow the steps below:</p>
@@ Line 150: / Line 150: @@
 </ol>
-<p>After completing the above steps, repeat them while taking measurements of your lab partner’s inside forearm.  After those measurements are completed, record spectra from the inside of your palm, then your partner’s palm.  In total, you should have 4 complete data sets, but it may be a good idea to take 2 measurements from each site (8 in total between you and your partner).  This way if you find some error during data processing, you have another set to analyze and won’t have to go back to the lab and run the experiment again.</p>
+<p>After completing the above steps, repeat them two more times on the forearm. In total, you should have 3 complete data sets, </p>
-<p>Before leaving the lab please ensure that you have copied all of your data to a usb stick, or emailed it to yourself.  You will also need to take with you a copy of the excel macro spreadsheet (.xlm file), which will be used to import all the spectra and calculate everything you need to quantify the change between oxy- and deoxy-haemoglobin.  Finally, please ensure that the light source and spectrometer are properly shut down and stored away before you leave the lab.</p>
+<p>Before leaving the lab please ensure that you have copied all of your data to a usb stick. Finally, please ensure that the light source and spectrometer are properly shut down and stored away before you leave the lab.</p>
 <h2>Data Analysis</h2>
-<h2> Using the Excel Analysis Spreadsheet</h2>
+Using your data sets and a program of your choice you can analyze your data.  The following section, "Calculating Theory" describes he analysis.
@@ Line 256: / Line 239: @@
 </ol>
-<h1>Part B: Cytochrome C</h1>
+<h1>Cytochrome C - Additonal Information</h1>
 <p>Two molecules: hemoglobin, cytochrome c, studied in the lab using the spectrophotometry technique, are shown below. They are members of a large family of metalloporphyrin molecules. The active component of these molecules is the heme group, consisting of the transition metal ion, surrounded by four nitrogen atoms. The heme is part of many proteins, whose functions are diverse and include oxygen transfer and storage (hemoglobin), electron transfer, for example, in cellular respiration (cytochrome) and energy conversion in the photosynthesis process (chlorophyll). </p>

@@ Line 96: / Line 96: @@
 </p>
 </td></table>
-<h2>Using the reference sample to calibrate the system. </h2>
+<h2>Using the reference sample to calibrate the system</h2>
 <p> Before we are ready to collect data we must first acquire a set of calibration data sets using the spectrometer to account for the natural spectral shape of the light source and optical system being used. This will be accomplished by measuring the diffuse reflectance of a ‘white’ reference target, which should have been supplied to you at the start of the lab.  This target will allow you to measure the natural spectrum of the LED being used, and this data will later be used to normalize the diffuse reflectance skin spectra and remove any artifacts from the light source and fiber collection system.</p>
 <table width=380 align=left><td>
@@ Line 116: / Line 116: @@
 </ol>
 <br style="clear: both" />
-<p>You should now have a single text file in your save to directory with the reference spectrum data.  Now, remove the white reference target and place it back in its container.  We are ready to begin spectral measurements.  For this you will need the blood pressure compression cuff.  Pressure to the cuff is increased by pumping on the bladder, and a silver release valve in front of the bladder allows the pressure to be released.  Perform a trial run of the experiment without saving the data and observe the live spectrum during the procedure.  Once you are comfortable with the timing and have set up the parameters as described below collect data for 3 trials using the following procedure:
+<p>You should now have a single text file in your save to directory with the reference spectrum data.  Now, remove the white reference target and place it back in its container.  We are ready to begin spectral measurements.
 To start the measurements follow the steps below:</p>
@@ Line 133: / Line 136: @@
 <table width=380 align=left><td>
 <p align=justify>[[File:Acquisition_parameters.png|1040px|border|center]]
-<b>Figure 10</b> - Save reference sample spectrum window settings.
+<b>Figure 11</b> - Save acquisition parameters for time-series data collection.
 <br clear=right>
 </p>

@@ Line 138: / Line 138: @@
 </td></table>
 <li>Place the compression cuff just above your elbow, covering you bicep.  Ensure that the pressure is fully released by opening the release valve for a few seconds then close it shut.</li>
 <li>Place the reflectance scan head on the inside of your forearm (you should hold it in place while your lab partner operates the spectrometer software)</li>
-<table width=380 align=left><td>
+<li>Click on the save icon in the toolbar (see figure 9) to start data collection.  (This icon will remain red during data collection)</li>
 <li>After 20-30 seconds, begin inflating the pressure cuff to capacity.  This will restrict blood flow.  The pressure should be maintained to at least 200 mmHg on the pressure dial.</li>
 <li>After maintaining at least 200 mmHg for 90 seconds of constriction, release the pressure valve to allow blood to re-circulate.</li>
-<li>Continue monitoring the directory you are writing spectral files into.  When you reach file 49 you are done.</li>
+<li>Continue monitoring the directory folder you are writing spectral files into.  When you reach file 49 you data collection is complete and the save icon on the toolbar will no longer be red.</li>
-<li>Press “Terminate All” to remove the completed data capture.  If you forget to do this, you will not be able to set up another time series for the next set of measurements.  If you find that this occurs, re-open the “Manage Spectrum Exports” dialogue will allow you to terminate the process and use the spectrometer again.</li>
 </ol>
 <p>After completing the above steps, repeat them while taking measurements of your lab partner’s inside forearm.  After those measurements are completed, record spectra from the inside of your palm, then your partner’s palm.  In total, you should have 4 complete data sets, but it may be a good idea to take 2 measurements from each site (8 in total between you and your partner).  This way if you find some error during data processing, you have another set to analyze and won’t have to go back to the lab and run the experiment again.</p>

@@ Line 104: / Line 104: @@
 </p>
 </td></table>
-<p>  Once you have the proper gain settings for the reference spectrum, save the spectrum window, click File → Save Project (or hold down “ctrl + alt + s”) Provide a name and save it in a new folder in the Documents folder.  Click on the "Configure Graph Saving" icon located in the toolbar above the graph (see Figure 9).  Set the parameters as shown in Figure 10.
+<p>  Place the 'white' reference target on the table and place the housing unit on top.  You should see a spectrum on the graph.  Once you have the proper gain settings for the reference spectrum as described above, save the spectrum window, click File → Save Project (or hold down “ctrl + alt + s”) Provide a name and save it in a new folder in the Documents folder.  Click on the "Configure Graph Saving" icon located in the toolbar above the graph (see Figure 9).  Set the parameters as shown in Figure 10.
 </p>
 <ol>
 <li>Under “Save Options” select to “Save every scan”</li>
 <li>Check the “Stop after this many scans” box, and enter “1” in the box beside it.</li>
-<li>Under “File Options” select “ASCII (with header data)”</li>
+<li>Under “File Options” select “ASCII (with header data)” for the file format.</li>
 <li>Select your “Target Directory” and select the folder your created in the Documents.</li>
 <li>Enter “Reference” for the “Base Filename”</li>
@@ Line 116: / Line 116: @@
 </ol>
 <br style="clear: both" />
-<p>You should now have a single text file in your save to directory with the reference spectrum data.  Now, remove the white reference target and place it back in its container.  We are ready to begin spectral measurements.  For this you will need the blood pressure compression cuff.  Pressure to the cuff is increased by pumping on the bladder, and a silver release valve in front of the bladder allows the pressure to be released.  To start the measurements follow the steps below:</p>
+<p>You should now have a single text file in your save to directory with the reference spectrum data.  Now, remove the white reference target and place it back in its container.  We are ready to begin spectral measurements.  For this you will need the blood pressure compression cuff.  Pressure to the cuff is increased by pumping on the bladder, and a silver release valve in front of the bladder allows the pressure to be released.  Perform a trial run of the experiment without saving the data and observe the live spectrum during the procedure.  Once you are comfortable with the timing and have set up the parameters as described below collect data for 3 trials using the following procedure:
 <ol>
-<li>Open the “Manage Spectrum Exports” dialogue by either going to File → Save → Pause/Resume Export or pressing “ctrl + s”.</li>
+<li>Click on "Configure Graph Saving" icon on the toolbar.</li>
-<li>Highlight any processes and select “Terminate All”</li>
+<li>Under “Save Options” select to “Between saved scans sait at least 6 seconds”</li>
 <li>Check the “Stop after this many scans” box, and enter “50” in the box beside it.</li>
-<li>Under “File Type” select “Tab Delimited”</li>
+<li>Under “File Options” select “ASCII (with header data)” for the file format.</li>
-<li>Select your “Save To Directory”</li>
+<li>Select your “Target Directory” and select the folder your created in the Documents.</li>
-<li>Enter “Back of Hand” for the “Base Filename”</li>
+<li>Enter “Forearm#” for the “Base Filename” (Insert 1, 2 or 3 for each trial #.</li>
 <li>Ensure “Padding Digits” is set to “5”</li>

@@ Line 89: / Line 89: @@
   A spectrometer essentially consists of some light collection optics coupled to an optical fiber (not shown), the light collected by the fiber is passed through a lens and slit assembly mounted inside the spectrometer (2 & 3) before striking a collimating mirror (4).  This mirror produces a collimated beam of light, which is then reflected off of a diffraction grating (5) and is focused by a second mirror (6) and onto a linear CCD array (7).  A CCD (Charge-Coupled Device) is a light-sensitive detector which produces a voltage proportional to the light striking the active area, or pixels.  The diffraction grating will reflect light of different colour at slightly different angles, and thus red light is focused towards the side of the CCD indicated by (8) and blue light is focused towards (9).  Reading out the voltage levels on the pixels across the CCD therefore allows us to measure the spectrum of light collected by the fiber.  When light is spread across the CCD in this fashion, the wavelength range striking each pixel on the CCD is very small, typically less than 0.25 nm per pixel, which allows for the visualization of very fine spectral features.</p>
-<p>Software operation of the spectrometer will be quite basic for our purposes, the software is pre-installed on the PC you will be using for data collection, and a shortcut named "OceanView" can be found on the desktop. Click [[Media:OceanViewManualNew.pdf| Ocean View User Manual]] and review the document prior to the lab. Double click on the icon to start the program and select "Quick View". Ensure that the cable is connected to the PC and the fiber optic cable is connected between the spectrometer and light delivery/collection housing.  The main parameters we will have to change are the exposure, averaging and boxcar settings.  Typically it is good to use 4x averaging and 2-4x boxcar averaging.  The exposure setting will vary based on the individual, but should be in the range of 50 - 100 ms.  The ideal gain setting will have the most intense pixel values at ~50,000 levels of grey (see vertical axis in Figure 9) when the "white" reference target is used as the diffuse reflectance sample with the light housing.</p>
+<p>Software operation of the spectrometer will be quite basic for our purposes, the software is pre-installed on the PC you will be using for data collection, and a shortcut named "OceanView" can be found on the desktop. Click [[Media:OceanViewManualNew.pdf| Ocean View User Manual]] and review the document prior to the lab. Double click on the desktop icon to start the program and select "Quick View". Ensure that the cable is connected to the PC and the fiber optic cable is connected between the spectrometer and light delivery/collection housing.  The main parameters we will have to change are the exposure, averaging and boxcar settings.  Typically it is good to use 4x averaging and 2-4x boxcar averaging.  The exposure setting will vary based on the individual, but should be in the range of 50 - 100 ms.  The ideal gain setting will have the most intense pixel values at ~50,000 levels of grey (see vertical axis in Figure 9) when the "white" reference target is used as the diffuse reflectance sample with the light housing.</p>
 <table width=560 align=center><td>
 <p align=justify>[[File:Software_Interface.png|1040px|border|center]]
@@ Line 96: / Line 96: @@
 </p>
 </td></table>
-<p> Before we are ready to collect data we must first acquire several calibration data sets using the spectrometer to account for the natural spectral shape of the light source and optical system being used (this is the spectrum shown in Figure 10).  This will be accomplished by measuring the diffuse reflectance of a ‘white’ reference target, which should have been supplied to you at the start of the lab.  This target will allow you to measure the natural spectrum of the LED being used, and this data will later be used to normalize the diffuse reflectance skin spectra and remove any artifacts from the light source and fiber collection system.</p>
+<h2>Using the reference sample to calibrate the system. </h2>
 <table width=380 align=left><td>
-<p align=justify>[[File:Save_spectrum.jpg|320px|border|center]]
+<p align=justify>[[File:Save_spectrum.png|320px|border|center]]
 <b>Figure 10</b> - Save Spectrum setup.
 <br clear=right>