Electron microscopes use electrons to illuminate a sample and form highly magnified images. There are several types of electron microscopes, and each produces different types of images. For instance, a transmission electron microscope (TEM) transmits electrons through a very thin sample, providing a two dimensional image of structures inside cells. In contrast, a SEM scans the surface of a sample with a beam of electrons, producing images of cell surfaces that appear three-dimensional. The SEM at NOAA is used to examine the surface of cells and tissues of aquatic organisms.
The magnification of light microscopes is limited by the wavelength of light, which is about 400–700 nanometers. A nanometer is one billionth of a meter. Light microscopes with good quality lenses can magnify up to 1000 times. The wavelength of electrons is thousands of times shorter than light, so electron microscopes can magnify images thousands of times greater than light microscopes. Many SEMs can magnify up to 300,000 times or greater. However, magnifications used to examine biological samples are generally range from about 30–80,000 times.
The SEM produces a beam of electrons that scans across the surface of a sample. The electrons generated by the SEM interact with the sample, causing additional or secondary electrons to be emitted from the sample surface. A detector collects the secondary electrons, and using technology similar to a television or computer, the electrons are converted into an image that is viewed on a cathode ray tube or a computer screen.
Other types of information can also be obtained from the SEM with appropriate detectors. For instance, additional electrons are reflected back from the sample surface, these are called backscattered electrons and they provide information about the composition of the sample. X-rays are also emitted and specialized detectors can measure the energy of the X-rays to provide information about the elemental composition of the sample. The images on this web page were produced with secondary electrons, which is the most common method used to examine surface features of biological samples.
Conventional SEMs examine samples in a vacuum because air molecules interfere with the path of the electrons. Samples examined under vacuum cannot contain water, so biological samples must be carefully preserved and dried before they can be examined with a conventional SEM. Samples must also be conductive to properly interact with the electron beam. Low vacuum or environmental SEMs have also been developed to examine uncoated wet samples. However, they operate at relatively low magnifications and usually use backscattered electrons to form images. The images were photographed using conventional SEMs that produce high resolution images.
Most anything that is dry, conductive and fits on the sample platform can be examined. SEMs are frequently used to look to look at a variety of materials, including fractured metal surfaces, quality control for microchip production, analysis of rocks and minerals, forensic investigations and much more. SEMs are also frequently used to study plants and animals. Samples that are already dry and conductive need little preparation. Wet, non-conductive samples must be carefully dried and coated with a thin layer of metal to make them conductive.
Soft tissues such as fish eggs, embryos, zooplankton and fish skin were placed in a chemical fixative. The water in the samples was then removed by placing the sample in a bath of 35% or 50% ethanol (ETOH) for 5-30 minutes, depending on sample size. The bath was repeated several times with increasing concentrations of ETOH (75%, 85%, 95%) ending with several washes of 100% ETOH. This dehydration process gradually replaces the water in the sample with ETOH. The ETOH was then removed through a process called critical point drying. This process consisted of placing the dehydrated sample into critical point drying equipment, where it was rinsed and immersed in liquid CO2. The temperature and pressure of the liquid CO2 was then gradually raised to its “critical point” where the CO2 achieves a phase change from liquid to dry gas without the effects of surface tension. This prevents wrinkling or shriveling that typically happens if the sample was simply air-dried. The CO2 gas was then slowly vented, leaving dry tissues free of distortion.
The dried samples were glued to a specimen holder, and coated with a thin, conductive layer of gold and palladium. The coating process was conducted using vacuum evaporating equipment, where the sample was placed under a vacuum and small amount of the metal was heated until it melted. The metal molecules float in the vacuum and coat the surface of the samples. The coating thickness was controlled by adjusting the length of time the metal vapor was in contact with the sample.
Diatoms may be prepared for SEM in a number of ways. Diatoms have a hard covering or frustule made of silica, so they do not need to be dehydrated. However our studies focused on species identification, so we prepared the samples by first removing the thin layer of organic material from the diatom surface so that the details of the frustule could be easily observed. Most of the diatom images were prepared by passing water samples containing the diatoms through small filters, and the diatoms were collected on the surface of the filters. The filters were rinsed with hydrochloric acid and potassium permangenate to remove the organic material from the diatoms. The filters containing the diatoms were then rinsed with water and air-dried. The holes of the filter membrane can be seen underneath some of the diatom images. The filters were glued to the SEM stub with colloidal graphite paint, and coated with a thin layer of gold and palladium to make the surface conductive.
The organisms seen in these images came from Puget Sound, Pacific Ocean waters, or from laboratory studies. They came from research projects conducted at NOAA Fisheries Service, NWFSC from 1975 to 2010.
Human eyes see color only within visible light wavelengths of 400–700 nanometers (a nanometer is one billionth of a meter). The wavelength of electrons is thousands of times shorter, well outside the range of visible light. Therefore images from electron microscopes do not have any color. Artificial color can be added through filters or digital enhancement. However for this book, we feel that the original black and white images are the best way to show the incredible structures present on these marine organisms.
An AMRAY 1000 SEM was used from 1975 to 2003. It used analog technology and film to record the images. We used Polaroid type PN55 film, it produced both an instant positive photograph as well as a 4 × 5 inch negative. A Polaroid camera back positioned over a CRT tube on the AMRAY SEM was used to expose the film. For many years, the 4 × 5 inch negatives were used to print images in a chemical darkroom. With the advent of digital technology, the 4 × 5 negatives were digitally scanned for use in this book.
The SEM was replaced in 2003 with a JEOL 6360LV SEM. This SEM is digital, so images photographed after 2003 were digitally recorded.