Spider Silk

An Electron Microscopic Study








Background Information

Spider Silk is a natural fiber secreted by spiders for the prodcution of webs and egg sacs as well as transportation.  The silk is secreted from glands inside the spiders spinnerets, located on the back of a spiders abdomen.  Spider silk is renouned for being stronger than steal by mass and is surprisingly elastic and has generated interrest for an array of applications.  Allegedly, these properties are a result of both its structure and chemical make up.  One source claims that the structure is a combination of crystaline sections linked by irregular elastic amino acids. (Wikipedia) 


Figure 1:Spider Silk Structure Figure 2:Structure Schematic
(http://www.xs4all.nl/~ednieuw/Spiders/InfoNed/webthread.html)               http://en.wikipedia.org/wiki/Spider_silk#Properties

The most common amino acids are thought to be glycine (C2H5NO2 ) and alanine (C3H7NO2).

The size of spider silk, around 1 um in diameter, makes visual investigation impossible.  This project is an exploration of spider silk with an electron microscope.

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Methods

I. Sample Collection
            Silk was collectd from common spiders from around the University of Rochester campus as well as near by parks.  A sample stub was prepared with double sided tape and then pushed through the webs.  For comparrison, spider silk was also wrapped around a human hair (special thanks to my roomate  for his generous contributions to science) before being placed on the stub.  One of the spiders who produced the web for this project was also collected.

II. Sample Preperation
Silk samples were first prepared by painting the edges of the stub with a conductive carbon-based adhesive.  Then, specimens were coated with a gold and palladium alloy in a sputter coater for various lengths of time from thirty seconds to two minutes.
         The spider was first submerged in gluderaldehide to fix the sample.  Then the sample was submerged in Hexamethyldisilazane (HMDS), a chemcial used to dry the sample without destroying soft tissues.  After the HMDS evaporated off, the sample was mounted on a sample stup and sputter coated as described for the silk samples.

III.  Data Collection   [Click on a Section Title to see those results]

          
The University of Rochester's LEO 982 FE-SEM was the instrument used to collect the raw data for this project. 

             A.  Secondary Electron Images were taken with various microscope parameters.  These parameters can be viewed in the display bar of the micrograph images.  In addition, the signal mixer was used to mix the in-chamber and in-lens SE detectors which provides for more control in signal processing.  Some of the microscopes software was utilzed in making point-to-point measurements of sample features.

             B.  Anaglyphs   were created of the spiders spinneretts.  In order to create the anaglyphs, two images need to be taken.  The first image is taken and a distinct feature at the center of the screen is marked with a dry erase marker.  Then, the sample stage is tilted about 4 degrees.  Then, the sample is repositioned so that the distinct feature lines back up with the mark made on the screen and a second image is captured.  The images were then processed  in Photoshop in the following way.  Both images are converted into RGB color format and the zero tilt image is given a red hue and the tilted image is given a blue hue.  Then, the images were layered one on top of the other and the opacity of the top image is adjusted to 50% so that the images are superimposed.

    C.  Colorization  of micrographs was done using Adobe Photoshop.  This is more artistic than anything else.

           D.   X-ray Analysis    was conducted on the silk as well as a control group of just the sample stub and tape.  The SEM's EDAX detector was utilized along with the help of the software included.
  
            E. The Electron Flight Simulator program was also used to provide some insight on the beam interaction with the sample.  For the purposes of the simulation, the spider silk was modelled as a carbon based organic compound about a micron thick, as per the direction of Brian McIntyre.  The model also includes a 10 nanometer layer of Gold and Palladium alloy on top of the spider silk model to simulate the sputter coating.  Part of the reason for this anaylsis was to invesitgate the problem with the webs being destroyed by the electron beam.




                                           Figure3: Micrograph of a spider silk fiber being destroyed by the electron beam.

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Results and Discussion   [Click on a Section Titles to go back to the corresponding Methods]

A.  Secondary Electron Images   

Taking images of just the spider silk gives a good idea of the structure and size of the silk.  During the course of the project I saw single-stranded silk, double-stranded (above) silk and multi-stranded silk.


                                 Figure 4:  Multi-Standed and Single-Stranded Spider Silk (From the Same Spider)

To be a little more quantitative...



                                                    Figure 5:  Multi-Standed Fiber, Measured with SEM software

And my favorite:


                          Figure 6:  Multi-Stranded Silk.  I count 17 strands that can be seen, but there must be more.

 

To give you a better sense of the size, here are micrographs of spider silk on human hair (thanks again to Jim Morphis):








Figure 7:  Spider Silk laid over human hair.


    

Now for where the silk comes from.




    Figure 9:  Spinnerrette Arrrays on the back of the spider's abdomen.

A little closer...



      Figure 10:  Several spinnerettes in an array.

And a little closer....



Figure 11:  A single spinnerette



B.  Anaglyphs 



Figure 12:  Spinnerette Anaglyph

After putting on 3-D glasses, try focusing on smaller objects in the background until your brain superimposes the images, creating a3-D effect.


Figure 13 : Spinnerette Anaglyph 2

Cool.  They work okay.  This is trickey for high mag images.


C.  Colorization 


                    


   Figure 14:  Spider Web Art 

      

 

                                                                                                               

Figure 15:  Spinnerette Colorized




D. X-ray Analysis 



  Figure 16: X-ray spectrum from Spider Silk                 Figure 17: X-ray Spectrum from Control Group

By comparing the x-ray spectrum of the spider silk to that of the control group we can see that the only significant difference is that the spider silk spectrum has a small nitrogen signal at base of the carbon signal.  The carbon, oxygen, gold, and palladium signals can be considered to come mostly from the stub, tape, and coatings.  The presense of Nitrogen is in agreement with the background information that the amino acids glycine (C2H5NO2 ) and alanine (C3H7NO2) are present in spider silk. 


 

E.   The Electron Flight Simulator



Figure 18:  EFS, 5kV Accellerating Voltage



Figure 19:  EFS, 10kV Accellerating Voltage




Figure 20:  EFS, 20kV Accellerating Voltage

The analysis shows that a 5 kV accellerating voltage causes the beam to penetrate into the sample only about 450 nm.  The 10 kV beam penetrates all the way through the sample, but a lot of the energy is dissipated into the silk.  The 20 kV beam penetrates through the sample again, but this time with much less difflection into the sample and so much less energy dissipated into the spider silk.

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Conclusion 
        Spider Silk is some pretty cool stuff.  Its diameter varies but it generally around a micron in diameter, about 80 times smaller than the human hair donated by my roomate, Jim.  It can be made up of a single strand or several strands, each as small as 100 nanometers.  X-ray analysis confimed the presense of nitrogen, which is in agreement with the theory that the silk is composed of the amino acids glycine and alanine.  It's fragility may require selecting low energy electrons to prevent damaging the sample or high energy electrons which pass through the sample readily.  Sputter coating, of course, is also  useful.  I had also never seen the spinnerets of a spider before and did not realize how many there were.  There was a lot of interresting structure to see on the spide itself.

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