An Electron Microscopic Study
By: Kevin Mille
I. Brief Specimen Description
Drosophilae are very common flies with red eyes and whose bodies are banded with yellow and dark gray stripes. These flies appear near open wine and around fruit, hence their common name, the fruit fly. Typically, Drosophilae are approximately 3 – 5mm in length. The compound eye structure of the drosophila is quite unique and complex; this was the main interest behind this experiment. The compound eye structure is composed of approximately 700 - 800 eye units, otherwise known as ommatidia. Each eye unit is covered with a cornea. Around the eye cells there are large hairs that are fixed in between facets. These hairs utilized by the fly for mechanic-sensory. The ommatidia contain a cluster of 7 or 8 photoreceptors cells, known as rhabdomere, that are used to total internally reflect light to a sensor. A cross section of this can be seen in Image 1a below. Each receptor has the ability to absorb a different wavelength of light ranging from UV (345nm and 375nm), blue(437nm and 480nm) and green (508nm).
Image 1a. (Source 3)
Overall the compound eye structure of the common fruit fly is quite unique and complex. This project is aimed at taking a closer look at the eye structure and making it more appealing and apparent to the common viewer.
There are two main objectives that were accomplished during this project. The first was to create a motion video by streaming multiple images together and the second to colorize images of flies’ eyes to make a piece of artwork.
The original video that was planned to be made was going to take the viewer on a journey throughout the layers of a flies’ eye. This was to be achieved by using a microtome to cut into the eye a few nanometers at a time. Unfortunately, due to difficulties with using the microtome, properly cut samples were never achieved. Thus, the final video compiled only takes the viewer to a journey to the surface of the eye.
Sample Preparation Techniques
1. Critically Point Drying (CPD)
The first process in preparing the samples for imaging is to fix the soft tissue of the flies with Glutaraldehyde. Once this process is complete the flies are ready to be critically point dried. Since biological samples are composed with a lot of water and must be dried for imaging. Simply air drying a sample will not allow for the sample to remain properly intact and can result in the destruction of important structural detail. Dehydration is very important since the specimen will go under vacuum and the water will be forced out completely destroying the sample. A critical point dryer will replace water with miscible fluid. This miscible fluid is then replaced by a transitional fluid. The transitional fluid will be driven past the critical point through an increase in pressure and temperature. As the pressure is released the gas will escape leaving the sample perfect intact and dry.
Since my goal was to image the All of the samples were coated originally with 100 angstroms of gold. This amount of gold was inadequate due to the fact the specimens were ‘charging’ when being viewed. Therefore I recoated all samples until there was at least 180 angstrom surface of gold on the specimen. This yielded far superior results.
Different accelerating voltages can have different affects on the sample and coating as well. By using an electron flight simulator program one is able to predict how deep the electrons will penetrate through the coating and into the substrate, as shown below in images 2a - 2c. This penetration depth is important since the electron beam can damage a sample. Some fly eyes were damaged as a result of either a too high magnification or viewing a fragile section of the image at a high magnification. Some of the corneas on the eye would become deformed and were depressed by the beam. Most of the higher quality pictures were obtained using a 10kV accelerating voltage in order to get a higher resolution image. Unfortunately there was damage caused to the areas being viewed. A 5kV or 4kV accelerating voltage helps to reduce this damage with a more shallow penetration depth as you can see below.
Image 2a. Electron Flight Simulator
(10kV Accelerating voltage, 180angstrom Gold coating, Organic Biological substrate)
Image 2b. Electron Flight Simulator
(5kV Accelerating voltage, 180angstrom Gold coating, Organic Biological substrate)
Image 2c. Electron Flight Simulator
(4kV Accelerating voltage, 180angstrom Gold coating, Organic Biological substrate)
3. Secondary Electron Images (in-chamber and in-lens detectors)
All of the images used a mix of an in-chamber and in-lens secondary electron detector in order to achieve the best results. By using both detectors and mixing their images, higher quality images with a more appealing contrast and brightness can be captured.
4. Light Microscope
A light microscope is another tool that is used to obtain color images. Due to the short depth of focus the microscope has, quality images were difficult to obtain as you can see below. These images did show the true color of the eye and lenses that could be used to make the colorized eyes more realistic.
Post Imaging Techniques
Since SEM images are obtained using electrons, color images are impossible to obtain. This does not mean that color images can not be created to not only make the image more eye appealing but even bring the image to a more artistic level. Through the use of Adobe Photoshop, the following images were colorized and created. Roll over the images to see them prior to the colorization.
Using multiple images and framing them together a video can be developed. The video shown below has 109 images framed together to bring the viewer from a 56x magnification to a 30,000x magnification. One problem with the creation of this video is that it
Drosophilia Video (56x - 30,000x)
Microangela Electron Microscope Image Gallery<http://www.pbrc.hawaii.edu/bemf/microangela/>.
Spatial and temporal aspects in biological signal transduction<http://www.ub.rug.nl/eldoc/dis/science/m.postma/>.