Protocols

Protocols
These are the protocols and methods for data collection and analysis for members of the Beetles Project.
Last updated 28/8/17. Any newer protocols supersede all previous ones.
Collecting Spectral Data
To collect spectral of the elytra, the following equipment is required:

360° goniometer with 0.25° divisions for the angle of incidence
Clamp, for holding elytra pieces, composed of aluminium
Microscope slides to hold the elytra pieces in the clamp
Ocean Optics USB-650 Red Tide Spectrometer
SpectraSuite Software
Laptop with Spectrasuite Software installed
Ocean Optics HL-2000 Tungsten Halogen Light Source
Scissors
Plain paper
Ruler (15cm length)
Screwdriver
Metal wire (1mm diameter)
Voltcraft infrared thermometer (measures from -30°C to 260°C with accuracy ±2°C)
Binder clip

Calibration
Usually the goniometer-spectrometer is calibrated, meaning that the light source hits the sample and light is reflected to the receiver of the spectrometer. However, if the setup is not calibrated, most or almost no light will reach the receiver and data cannot be collected. This is the method for calibration:

Plug the Ocean Optics HL-2000 Tungsten Halogen Light Source into a power socket and then turn the device on by flicking the switch on its side. The light source should be in a fixed position on one of the arms of the goniometer.
Turn the arm of the goniometer on which the receiver, which is connected to the spectrometer, so that the light source is shining directly into the receiver.
Hold a piece of paper directly in front of the receiver, as this will allow you to see where the beam of light is shining. If the light is horizontally not in line with the centre of the receiver, turn the arm of the goniometer so that the beam of light is directly in line with the centre of the receiver. If the light is vertically not in line with the receiver, raise or lower the light source and the receiver using the screws on the two arms. After this, the light source should be in line with the receiver, as shown in Figure 1 .
Once the receiver and light source are exactly in line, rotate the dial on the goniometer so that the angle between the two arms is shown to be 180°.

Preparing the sample:

Take the C. wallacei elytron that you want to collect the spectral data of and cut it in half, as shown in Figure 2, using scissors if it is not done so already.
Take two microscope slides and place the edge of the elytron that was not cut between the slides halfway along the length of the slides, so that the side of the elytron that was cut is exposed. Use a binder clip to keep the elytron piece in its position between the slides, as shown in Figure 3.
Unscrew the screws on the aluminium clamp and then place the two microscope slides, with the elytron between them, in the clamp, and then tighten the screws on the clamp in order to fix the slides in position. Be careful not to tighten the screws too much as this may damage the elytron or the microscope slides. The microscope slides can be placed in the clamp with the elytron piece either facing backwards or facing forwards, and the correct direction is with the elytron piece facing the direction from which the heads of the screws are visible. The reason for this is that the elytron piece is further away from the light source and receiver when facing the other way, and the receiver has been calibrated for only one distance from the sample. The elytron piece should be closer to the light source than it would be if the clamp were facing the opposite direction.

Calibrating the sample with the goniometer-spectrometer setup:

Plug the Ocean Optics HL-2000 Tungsten Halogen Light Source into a power socket and then turn the device on by flicking the switch on its side. The light source should be in a fixed position on one of the arms of the goniometer.
Place the clamp, which is holding the elytron piece between the microscope slides, into the centre of the goniometer so that the light beam is shining directly to the exposed section of the elytron piece, and tighten the screw on the goniometer to keep the clamp in position.
Ensure that the elytron piece is perpendicular to the light beam by lining up a ruler with the clamp and the light source and seeing if they line up. Rotate the clamp if the elytron piece and the light source do not line up.
The light source should be shining at the centre of the exposed section of the elytron piece. If the light beam is shining above or below this point, raise or lower the clamp and then tighten the screw at the centre of the goniometer. If the light beam is to the left or to the right of the centre of the exposed section of the elytron piece, unscrew the screws on the clamp that are holding the microscope slides, and then move the slides left or right in order to line up the light beam.

Checking the temperature:

Using an infrared thermometer gun, measure the temperature of the elytron piece by pointing the gun at the centre of the exposed section of the elytron wing. Repeat this at least three times and if the temperature displayed on the device is consistently within room temperature range (15°C-25°C), continue with the collection of spectral data. If the temperature is not consistently within this range, turn on a heater or a fan and continue with the experiment once the temperature of the elytron piece is within the room temperature boundaries.

Removing the dark spectrum:

Turn on the computer which has the SpectraSuite software and plug in the USB cable of the spectrometer before opening the software. Data cannot be connected with SpectraSuite if the USB is not plugged in before initiating the software.
On SpectraSuite, set the integration time to 100 milliseconds, scans to average to 5, and boxcar width to 10.
Turn all the lights in the room off, pull down any blinds on any windows, reduce the screen brightness of the laptop screen and have the screen facing away from the spectrometer, and pull down the blind in front of the goniometer-spectrometer setup.
Turn off the light source.
Click on the “Store Dark Spectrum” icon (a black bulb) and then click on the “Remove Dark Spectrum” icon.

Preparation for collection of spectral data:

Pull up the blind in front of the equipment, turn on the light source, and turn on a light in the room.
Rotate the arm of the goniometer on which there is the receiver so that the angle between the two arms of the goniometer is 20°.
On the computer screen there should be a spectrum of light in the SpectraSuite Program. The spectrum may have a peak that is greater than the limit of the spectrometer (4000), or a very small peak. To adjust the intensity peaks, the top of the clamp with the elytron piece can have its angle adjusted in order to increase or decrease the amount of light reflected, hereby resulting in a different intensity peak. In addition to this, a wire can be used to prop up the elytron piece against the clamps. The arrangement of the equipment should now be as shown in Figure 4:
Tighten all screws of the set up in order to ensure no screws can become loose during the collection of spectral data and hereby skew results.
Open a text file with a suitable name which contains the following information:

Description of the elytron you are about to collect spectral data of.
The date.
The temperature of the elytron piece in Celsius measured using the infrared thermometer.
The SpectraSuite settings (integration time 100 milliseconds, scans to average 5, boxcar width 10).
The angles of reflection you are collecting data for.
The interval between the angles of reflection for which you are collecting spectra.
Whether or not the active spectrum has been removed.

Once the text file is made, store it in a suitable location.

Collecting spectral data:

Pull down the blind in front of the window and the blind in front of the setup, turn off all lights in the room, and reduce screen brightness of the computer to a minimum whilst having the screen face away from the spectrometer.
Click on the “Convert Active Spectrum to Overlay” icon. This will add the active spectrum as an overlay.
Turn on the light in the room, pull up the blind in front of the goniometer-spectrometer setup, and then increase the angle shown on the dial by 1°. Make sure you do not accidentally move or change the position of anything else of the setup as this could render the spectral data invalid.
Repeat this process of adding overlays at increasing angles until you reach the angle of reflection of 39°. There should be a clear trend in the spectra, but if you see that one of the spectra is completely different to the others, delete the spectrum and collect another spectrum for the angle. If this does not remove the anomaly, delete the spectral data you have so far and start again at 20°, as something must have changed at some point during the collection of spectral data.
Set the angle of reflection to 40°, but do not click on the “Convert Active Spectrum to Overlay” icon. Instead, click on the “Copy Spectral Data to Clipboard” icon. What this does is copy all the spectra on the screen, including the active spectrum. This is why an overlay for the active spectrum should not be added for the angle of reflection of 40°, as therefore two copies of this spectrum would be copied to the clipboard. This also why it should be specified in the text file whether the active spectrum has been removed.
Paste this data into the text file which was created earlier, and also paste this data into a suitably named new Excel file.

Creating overlays using Excel:

The data in Excel will have 42 columns: 21 columns will be the wavelengths of light in nanometres and will be identical (350-1000). The other 21 are the relative light intensities of the corresponding wavelengths of light at different angles of reflection. As the 21 wavelength columns are identical, only one copy is needed to produce a graph, so remove the 20 other columns.

Insert a new row above the first row, and then above each column, except the wavelength column, write down the angle of incidence. The first column (from left to right) will be the relative light intensities at 20° angle of incidence.

Select all the data in the sheet and then select to make a line graph with the data. This will produce an untitled graph with an overlay of the spectra at different angles of incidence.

With this graph you should:
1. Add a title
2. Label the axes “Relative light intensity” and “Wavelength of light (nm), Angle of incidence in degrees)
3. Set the minimum value of the y-axis to 0
4. Set the interval of the values of the x-axis to a suitable value depending on the size of the graph (usually 25 or 50)

Creating angle of incidence vs relative light intensity peak graphs in Excel:

Click on a cell underneath a column of relative light intensities, and then enter the code “ =MAX(first cell, last cell) ”. This code will return the maximum value between “first cell” and “last cell”.
Drag this cell by the bottom right corner so that the code is replicated underneath every column.
Paste the angle of incidences above each maximum value, and then a graph can be produced by selecting the row with the angles of incidence and the rows with the maximum relative light intensities. A title and axis labels should be added to the graph.

Creating angle of incidence vs wavelengthmax in Excel:

Click on a cell underneath a column of relative light intensities, and then enter the code “ =MATCH(MAX(first cell, last cell), first cell, last cell,0)+349 ”. This code will look up an item, which in this case is the maximum value in the column, in the column itself and return the relative position of the item. 349 is added to the number that is returned because the wavelength of light is shifted up by 349 relative to the numbering of the boxes. The 0 in the code specifies that the number being looked up in the list should be exactly equal to the number that it is looking for.
Drag this cell by the bottom right corner so that the code is replicated underneath every column.
Paste the angle of incidences above each maximum value, and then a graph can be produced by selecting the row with the angles of incidence and the row with the wavelengths of the maximum relative light intensities. A title and axis labels should be added to the graph.

Other statistical analyses

You can work out the standard deviation of a spectrum with the following code, where A2-A652 refers to the wavelengths and B2-B652 refers to the relative light intensities. Note: this may not work for spectra with many negative light intensity values, in which case more coding is required:=(SUMPRODUCT(($A$2:$A$652)^2, B2:B652)/SUM(B2:B652)-(SUMPRODUCT($A$2:$A$652,B2:B652)/SUM(B2:B652))^2)^(0.5)
You can work out spectral richness for with the following code, where A1-U1 arae the angles of incidence (21 angles) and A2-U2 are the wavelengthmax values: =SLOPE(A2:U2,$A$1:$U$1)
You can find the trough within the infrared range, where B2-B652 refer to the light intensities you are looking up and where B576-B620 is the range where you are looking for a minimum value: =MATCH(MIN(B$576:B$620),B$2:B$652,0)+349

Heat treatment with glass kiln

Please speak to Mr McMachan of the Langton Design department for help with heat treatment. For previous investigations, elytron pieces were heated in the glass kiln on top of ceramic paper with a plate overhead. The rate of heating was 1000°C/hour, with 30 minute soak time at the desired temperature, with the samples being removed once the kiln was at room temperature.