Easy and Fun Microscope Experiments

Although a microscope is well associated with serious and important activities performed by medical professionals and scientists, this device can actually be used for fun and educational experiments that can be carried out by your kids.

These activities can be a good bonding session between parents and their children over the weekend. Aside from the enjoyment it brings, performing these experiments can be an informative experience that children and teens can benefit from for their educational development or homeschooling lessons.

For your reference, below are some of the fun and safe experiments that you and your kids can do with a microscope. Prior to doing any of these investigations, it is presumed that you have your own or at least access to a microscope and some other cheap microscope tools.

1. The Onion Experiment

onionepidermis100x2You will find this experiment fascinating as the thin, transparent epidermis layer you can extract from onions will be a good specimen to look at down a microscope.

It will enable you to observe nuclear division and determine the different stages of the cell cycle i.e. whether the cell is in mitosis or interphase.

This is easy and does not require a lot of materials to perform. Adult supervision is recommended though as onions can be toxic to kids if ingested.

Materials Required: Glass microscope slide and coverslip, fresh onion, tweezers, knife, water and dropper, methylene blue (optional)

Instructions: 

  1. The first step is, of course, to cut and peel a fresh onion, ensuring you cut as small as possible. Once cut into small portions, peel some onion skin away for analysis.
  2. The next step here is to put a few drops of clean water onto the slide using a dropper; this is required to prevent the onion specimen from getting dry
  3. Next, use your tweezers to collect a piece of the thin membrane from the onion; it is the transparent layer or part of the onion under the skin; while this is generally safe, do not let young kids do this unsupervised as onion samples could hurt their eyes and result in a dramatic onion crying scenario; gloves may be worn as needed
  4. Now, use your tweezers to put the samples or thin layers of onion onto the slide with a few drops of water; it is optional to put a few drops of methylene blue onto the sample to stain the specimen and make its internal structures more visible, however, you should still be able to make out cell structures such as the nucleus
  5. Put the coverslip on the specimen or the slide; press it gently to make sure that there are no bubbles or air on the sample that you will use
  6. Place the slide with the specimen on the microscope’s platform. Be ready to get amazed by what you will see

2. Experiment using Pond Water

algaeconjugateAnother easy and fun experiment to do with a microscope, which your kids can also perform, is to use nearby pond water or any other body of water in the local vicinity. You can examine the hidden world of your pond, allowing you to observe what your naked eyes can’t see.

Although this is generally safe to be performed – even by kids, make sure that proper guidance is given as pond water is not always the safest substance and is obviously not acceptable for drinking.

You can also use different samples of water such as clean, dirty and pond water for analysis and comparison of the local ecosystem and water quality.

Materials Required: Collection container, pond water, dropper, concave microscope slide with a coverslip, stick or spoon

Instructions: 

  1. Collect a sample of water from a nearby water source, as well as drinking water for a clear comparison, you can easily use a bucket or cup for collection
  2. To prepare your pond water sample, use a stick to stir the water gently in order to mix all the components; this will ensure all the contents are seen – it will be more interesting to see a good mix of particles and creatures under the microscope
  3. Once the water has turned murky, scoop a small sample using your container
  4. Using your dropper/pipette, collect a small sample from the jar and put a few drops on the concave slide; be sure to use one slide per sample of water collected for an accurate analysis
  5. Place the coverslip carefully on the sample or the slide
  6. Put the slides with the sample water under the microscope. Is there anything interesting to see? Let us know!

If you live near the coast, you could also try investigating your local marine life, as there are some truly fascinating creatures. Take the Gnathophausia zoea (see
image) this is an infant crab and fascinating to observe under the microscope. The sinister-looking spines are there to make it look less appealing to hungry predators such as larval fish.

3. Fiber Experiment

620px-esem_color_woolAnother interesting item to use for a safe and intriguing microscope experiment are fibers.

The benefit of this one is that it is perhaps the safest and can be done by kids for a fun and engaging experience. You can use many different fabrics for this activity.

You can even try extracting fibers from your old jeans or your mom’s unwanted sweater for analysis.

Materials Required: Pieces or thread from samples of clothing such as jeans and sweaters, 2 to 3 plain microscope slides with a coverslip (one slide per type of fabric), water and dropper (optional)

Instructions: 

  1. Using the dropper, put a few drops of water on the slide; this is optional as you can still see the fiber even if it’s dry, but putting a few drops of water on the sample will make it more visible and easier to stabilize on the slide
  2. Using your tweezers, get a large sample or single thread from the fabric and put them on the slide; you may want to use different fabrics for comparison, but make sure to use a different slide for each type of fabric
  3. Put on the coverslip and gently press it down on the sample, removing any of the air bubbles by applying a little pressure using your fingers
  4. Now, put the slides on the microscope’s platform one at a time and compare how each fiber looks when magnified under a microscope

4. Human or Animal Hair Experiment

This one is as interesting as the fiber experiment. It is also pretty straightforward as you do not need any 600px-pixie-hair_lichen_981340459samples other than hair strands that you can easily collect from around the home.

To make it more fun and exciting, get hair samples from each member of your family and be amazed how each hair strand can look so different under a microscope.

You can also get a sample from your pet for a neat comparison between human and animal hair, observing the differences between texture, color and size.

Materials Required:  Hair strands, plain microscope slide with a coverslip, tweezers, water and dropper (optional)

Instructions:

  1. As you have learned from the previous sample, putting a drop of water on a dry specimen is optional, but it can make the sample more visible; do this if you wish by putting a few drops of water on the slide using your dropper. However, don’t worry – you can also use a dry sample for this too, although, be aware that it may be unstable on the slide and as a result more difficult to view down the scope
  2. Use your tweezers to get a few hair samples and put them on the glass slide; use a different slide for each hair sample, this will allow you to make accurate comparisons
  3. Next, cover the specimen using the coverslip
  4. Finally, put the slides under your microscope and be thrilled to see how hair strands are unique from one another depending on the age of the person you got it from, the hairstyle,  the color and the species! Make a note of any other differences and carry out research like a real scientist!

Microscopy and Related Techniques

800px-kidney_cd10_ihcSince microscopes offer the ability to see tiny things in an incredible amount of detail, it’s not so surprising that they are used by medical professionals to diagnose and treat diseases on a daily basis.

The two main categories of microscope used in clinical settings are the basic light microscope and the more advanced electron microscope.

The smallest visible object that can be seen by the human eye is less than 100 micrometres, and since animal cells range from 10 to 100 micrometres, the microscope is an essential tool in cellular pathology. The most commonly used being the compound microscope.

Biopsy Analysis

When a disease is suspected a tissue specimen is regularly required in the process of deriving a diagnosis and confirming the presence of a disease state. This process begins with a biopsy, which can include a wide range of different techniques, common biopsies include core biopsies from breast, prostate and renal tissue, as well as excision biopsies from the skin.

labpict11Once the biopsy has been removed, it is immediately placed into a specimen pot with a fixative, such as 100% formalin. The purpose of this measure is to preserve the cells in a life-like state, it achieves this in a variety of ways including prevention of autolysis, putrification as well as maintaining antigenicity. It is then transported to the pathology laboratory for processing.

Prior to the specimen being placed into the cassette(s), the specimen dissection takes place. Typically, the areas of interest are sampled, and if applicable, the lymph nodes and tumor margins are also sampled as these can help guide treatment options and prognosis.

For example, if cancer cells are still remaining outside of the margin, further surgery may be required; additionally, if cancer cells are discovered in the lymph nodes, this can indicate metastasis.

cassetteAlthough many samples warrant just a single cassette, there are often many cassettes per patient sample. Once labeled, cut and placed into their cassettes, they can be processed, which today is typically a heavily automated process.

In short, this involves a series of steps including tissue sample dehydration, clearing, embedding in paraffin wax and staining with a histological stain. Dehydration is an important step since the beginning stage i.e. formalin is not miscible with the end stage i.e. paraffin wax.

This involves the tissue being submerged in a series of graded alcohol’s to remove water e.g. 70%, 90%, and 100% ethanol. Once the dehydration process is complete, clearing is the next step, which removes the dehydrating agent e.g. ethanol and replaces it with a solvent miscible with wax e.g. xylene.

The paraffin wax is crucial to the embedding process, which involves placing the tissue into a mold, and hot paraffin wax is then added and left until cool and solid. The specimen now embedded in wax can be efficiently cut into sections using an apparatus referred to as a microtome, and 4µm slices are the typical thickness for histological samples. The resultant and extremely delicate sections are immediately placed onto a water bath and then onto glass slides ready to be stained.

Haematoxylin and eosin (H&E) is the principal and routine stain used on all received samples, and the diagnosis of malignancies is based largely upon this procedure.

Additionally, there are many other stains, termed “special stains” that are utilized to identify other structures not possible with H&E, for example, silver stains, which are commonly employed as a connective tissue stain to identify and observe disturbed patterns of reticulin fibers in cirrhosis and some tumors.

Looking through the Microscope

compoundpartsOnce the patient sample has reached the final stage of tissue preparation i.e. been microscopically prepared and stained, it is ready to be observed by a pathologist and the results reported. There are 3 important questions asked of every specimen, including what’s the diagnosis? As well as the prognosis? And the resulting treatment?

In short, the diagnosis involves the clinician looking for any structural and morphological changes in the tissue, and checking if they fit with the data set. Furthermore, the pathologist will need to request further special staining if they can’t be sure with an H&E stain alone.

In addition to a diagnosis, the prognosis and treatment are also points of interest, for example, if neoplasia was observed, the pathologist would need to answer whether it was malignant or benign, as well as the grade, whether it was metastatic and provide potential treatment options.

Immunohistochemistry

300px-immunohistochemicalstaining2At approximately day 4 immunohistochemistry may also be employed for Identification of a certain antigen in tissue by an antibody specific to that antigen. The site of antibody/antigen binding must then be labeled for microscopic visualization, and this technique is particularly useful for tumor typing, prognosis, and therapy.

Immunohistochemistry (IHC) is a technique which detects antigens in cells or tissue by utilizing the ability of antibodies to bind to specific antigens in biological tissues.

IHC staining is commonly utilized in the diagnosis of neoplasia, as for example, specific molecular markers can be detected, which are characteristic of a particular cellular event e.g. cellular proliferation or apoptosis or a particular tissue, helping to differentiate between a primary of a secondary tumor. Thus, common immunohistochemistry investigations include primary and secondary tumor typing and the confirmation of metastasis.

The visualization of the antibody-antigen interaction can be achieved in numerous ways, for example, the antibody can be conjugated to an enzyme such as peroxidase, which catalyzes a color-producing reaction. Additionally, there are other labels that can be conjugated to the antibody including enzyme-Horse radish peroxidase + Chromogen-Diaminobenzidine tetrahydrochloride (DAB), as well as fluorescent labels.

A good example of IHC utility in the diagnosis of neoplasia is the identification of specific markers for diagnosis, tumor typing, and confirmation of metastasis, which often involves the use of particular antibody panels. For example, a CK7, CK20, and TTF-1 antibody panel, which is useful in the diagnosis of lung tumors and for the differential diagnosis of primary pulmonary adenocarcinomas from extrapulmonary adenocarcinomas metastatic to the lung.

The History of Optical Microscopes

swiftrightsiderMicroscopes are amazing tools that have enabled man to make new scientific discoveries, diagnose and treat human disease, as well as make intricate things that require powerful magnification, resolution, and illumination.

The uses of optical microscopes are almost endless, but they weren’t invented overnight, in fact, they have a long and vibrant history involving numerous significant milestones and innovations. So where did it all start?

Since time began, man has imagined what it would be like to see things beyond the naked eye. The exact time at which man started to use lenses is unknown, but there is evidence of that for over 2000 years glass making light bend has been known.

In the 2nd Century, BC Claudius Ptolemy documented a stick seeming to bend when submerged in water. In 50-80 AD Emperor Nero used emeralds to watch Gladiators and lenses were first used for spectacles by D’Armato late 1200’s.

The First Microscope

jan_baptist_van_helmont_portraitIn the 1590’s, Zaccharias Janssen and his father Hans began to use lenses for the first time. They made a very infantile microscope consisting of lenses within a tube and observed the object at the end of the tube appeared enlarged. Although this wasn’t a working microscope, it certainly paved the way for new innovations.

Between 1632-1723 Antony Van Leeuwenhoek was the first man to create a working microscope. It was very simple, yet achieved the highest magnification so far at a power of x270. This was the first microscope that was really useful.

In the 17th century, the first compound microscope was developed, this is a microscope which utilizes two lenses, an objective lens, and an eyepiece or ocular lens. This advanced magnification significantly as in effect one lens is able to be magnified by the other, creating a superior microscope.

The limit of every microscope is its resolving power or resolution, in simple terms, this is the smallest distance that can be distinguished between two points. This is different from magnification, which refers to the size of the image only.

In the middle of the 17th century, Robert Hooke discovered the cell, one of the most significant biological discoveries. Hooke is also attributed to using the first microscope with three lenses, which is still used today. He observed “cells” in cork and named them after monastic rooms he said reminded him of what he saw.

Modern Microscopes

zeiss-libra-232x300In the late 1800’s microscope design improved greatly due to the work of the German company Ziess, Ernst Abbe, Otto Schott, and August Koehler.

These individuals solved many of the barriers with older microscopes giving rise to new, super powerful microscopes that had better optics and lighting than ever before, being able to achieve powerful magnification and resolution.

In 1902-Ives developed binocular eyepieces, and in 1935-Zeiss phase contrast microscopy, leading to the best optical microscopes to date.

All microscopes are limited by resolution, and due to the nature of light itself, resolution and magnification are limited. To overcome this barrier, the electron microscope was developed that replaces light photons with an electron beam. This has led to magnifications of x1,000,000 and resolution of less than 2nm, which is an incredible feat.

How to Clean Your Microscope

leica-microscope-repair-3Microscopes are highly sophisticated and often expensive pieces of equipment that have the potential to last many years. However, even though the structure of microscopes is typical very robust and constructed from metal, they can become dirty.

Additionally, the optics are the most delicate parts of the microscope and by far the most difficult to clean and maintain, that’s why it’s so important to know exactly how to clean and protect microscope lenses, as without a functional lens it is practically useless.

microscopedustcoversFirst things first, as we’ve all heard prevention is better than cure, and in the case of cleaning microscopes, this is very true.

Dust is the number one enemy of microscopes, particularly of the lenses and other glass components, that’s why it’s always advised that you should avoid contamination, to begin with. This is best achieved by using a dust cover, a lot of microscopes come with these upon purchase, however, if not they are generally inexpensive to buy.

Locating the Impurities

Next, if you notice any impurities within your field of view when looking down your microscope, you want to determine the cause. Before considering cleaning your microscope optics, it’s a good idea to double check if it’s not caused by something less obvious, such as an incorrect diaphragm setting. If after checking the components, you are sure there are impurities, you will want to proceed to clean your microscope.

It’s not always simple to pinpoint the exact location of the dirt or impurity, to determine if the dirt is on your camera lenses you can simply begin turning your lenses. If the position of the dirt changes when you rotate the camera, then it is located elsewhere.

To examine other components of your microscope, it’s a wise idea to isolate each of them so you can investigate carefully and rule them out one by one without confusion.

Removing the Dirt

air-bulbDirt is a very generic term, yet when cleaning your microscope you need to be aware of the different types of dirt or impurities. There are non-permanent impurities such as dust, dead skin cells and other tiny fragments.

More persistent impurities include water-soluble and solvent-soluble types, you can also discover combinations of the two.

So, how do you remove different types of dirt from your microscope? Well after determining the type of dirt you’re dealing with you should opt for compressed air to remove non-permanent dirt while cleaning liquids should be reserved for more stubborn impurities.

Compressed air is ideal because it removes dirt easily, without the need for harsh rubbing which has the potential to scratch or spread dirt on your optics.

If the dirt is ore stubborn, you will need to use a cleaning liquid and an appropriate cloth, always use a gentle cloth that won’t cause damage. Next, it’s vital that you don’t use the cloth alone, as rubbing away at stubborn, caked on dirt can cause scratches and damage your lens.

Additionally, you should only use water and 100% solvents, no mixtures, and avoid solvents containing ammonia and acetone. A good technique is to apply water using your breath, then gently rubbing using soft cotton wool, then if appropriate apply a small amount of solvent, this should do the job well.

Different Types of Laboratory Microscopes and Their Functions

img-microscopeThere are many different types of microscopes used in modern pathology laboratories and research departments around the world, these typically include stereo, compound, digital, and pocket microscopes as well as an electron, and fluorescence microscopes.

Light Microscopy is the cornerstone in all laboratories as it provides substantial magnification, enabling the professional to observe the specimen as required and with ease.

How does light microscopy work?

light-microscopy-3769-300x200As the name suggests light is the principal behind these types of microscopes, and the size of the image seen is determined by the angle of light entering the eye.

Therefore a glass lens is used as it slows the light causing the wavelength of the light to become shorter and as a result light bends (refraction), the amount bent is called the refractive index.

The lens within the light microscope serves to focus light rays at a specific place called the focal point, this is the distance between the center of the lens and focal point is the focal length. The strength of the lens is related to focal length as the shorter the focal length, the greater the magnification.

Normal light microscopy is called bright field, however, there are also specialist types of light microscopy methods called dark-field microscopy, phase-contrast microscopy, polarised light microscopy as well as fluorescence and stereo microscopy.

Types of Light Microscopes

The Compound Microscope

The compound microscope is a light microscope that utilizes photons (light) and lenses to magnify the object under observation. It differs from other types of microscopes as it utilizes multiple lenses and can achieve a high magnification typically reaching 1000x magnification. The lenses are called the eyepiece lens and the objective lens.

Compound microscopes have many uses in modern laboratories, for example in pathology departments they are commonly used by physicians to observe patient specimens e.g. tissues and cells and to look for cellular changes that could help to diagnose or screen for diseases such as cancer.

The Stereo Microscope

A stereo microscope is another common microscope found in laboratories, yet due to its low magnification has limited use. Stereo microscopes are typically below x100 magnification, yet they allow specimens to be viewed in three dimensions.

They are commonly used in tissue inspection and microsurgery.

The Electron Microscope

zeiss-libra-232x300The electron microscope is an extremely powerful microscope capable of magnifications of x1,000,000 and resolutions of about 2 nm.

They utilize the same principles as the light microscope, yet instead of a light source, a beam of electrons is used.

In laboratories, they are used by highly trained professionals to investigate and observe various markers of cell differentiation to identify tumors types, and in renal disease, to monitor disease progress.

They also have a critical role in the diagnosis of renal disease and a range of other conditions. Additionally, they are often used for microorganism identification.

Fluorescent Microscopy

The fluorescence microscope utilizes fluorescence and phosphorescence to observe and study various molecules of interest.

The specimen absorbs the light then re-emits it with a longer wavelength, this is usually achieved by attaching fluorochromes to the tissue or biomolecule of interest. A fluorochrome or fluorophore is a fluorescent chemical that can re-emit light when the light is “excited”.

This results in biomolecules that can easily be observed and tracked under the microscope, this is commonly used to confirm the presence of certain proteins e.g. growth factors important in the treatment of particular types of cancers.

Disadvantages of Electron Microscopes

electron-microscopeElectron microscopes (EM’s) are very sophisticated and powerful pieces of equipment that have revolutionized the world of science and medicine.

Thanks to the EM for the first time scientists have been able to observe and produce genuine images of viruses, bacteria and other cells in mind-blowing detail.

Electron microscopes utilize a beam of electrons in order to create an image of much higher magnification and resolution when compared to standard light microscopes.

There are two types of electron microscope: scanning electron microscope (SEM) and transmission electron microscopes (TEM) which both use electrons to produce an image but use them in different ways.

Scanning Electron Microscope

SEM’s uses a primary electron beam which scans across the surface of the specimen and interacts and excites secondary electrons on the surface of the sample. This emits a signal that allows the SEM to build an image.

Transmission Electron Microscope

TEM’s emit a high voltage electron beam through a thin slice of the specimen and the structure of the specimen is constructed and magnified by a photographic plate, fluorescent screen or a sensor which records the spatial variation and density of the resulting electron beam.

Disadvantages of Electron Microscopes 

Electron microscopes are a fantastic way to study samples in high detail and are used to create images on a wide range of samples including; cells, molecules, microorganisms, metals, crystals and more.

However, electron microscopes do have a few disadvantages which would prevent them from being used outside of the clinical or research lab environment.

Cost – The first of these disadvantages is the expense. Not only are the cheapest of SEM’s still quite an expensive piece of equipment (lowest price: $2,500) but replacement parts for them can set you back too. For a laboratory grade professional SEM you are looking at a price around the $10,000 mark whereas TEM’s require a quote request form the manufacturers.

Training – Another disadvantage is the amount of training and knowledge required to operate an electron microscope. For instance, samples must be prepared and observed in such a way to minimize artifacts and when the resulting image is analyzed one must be able to recognize these artifacts to obtain proper results. This requires a good amount of professional training and experience.

Space – The electron microscopes are large and are usually kept in a designated room which is fitted appropriately for the microscope itself. A room designated for the microscope would ensure there is no interference with the electrons that the machine utilizes and produce much better images.

Living cells – In comparison to the light microscope, the electron microscope can only observe an image of a fixated sample, unlike the light microscope which can view specimens when they are alive and motile.

Therefore it is not as useful as the light microscope for studying the behaviors, motility or moving structures of cells and microorganisms. This is perhaps the biggest disadvantage, as medical research often requires living cells in order to track various biomolecules and monitor important changes.

What is an Electron Microscope?

zeiss-libraLight microscopes have many important uses, especially for medical professionals and researchers who use them to help make a diagnosis and make new discoveries.

However light microscopes have limited resolving power, this is the ability to distinguish small or closely adjacent images. The reason for this is due to the nature of light itself.

Therefore the obvious solution was to use a beam of electrons instead of light, giving rise to electron microscopy, an extremely powerful microscope that has revolutionized science.

The electron microscope can achieve a resolution of about 2 nanometers (incredibly small) and a mind-blowing magnification of x 1,000,000. There are two distinct types of electron microscopy, called transmission electron microscope (TEM) and scanning electron microscope (SEM).

History of EM

dos1924, Louis De Broglie introduced the theory of electron waves, the foundation of electron microscopy.

In 1931, German engineers Ernst Ruska and Max Knoll developed the transmission electron microscope, capable of four-hundred-power magnification.

Ruska realized that electron wavelengths are far shorter than light wavelengths and understood that, if he was able to discover a technique to apply this knowledge, he could grow a far more superior microscope.

Both Knoll’s and Ruska worked together to develop the first-ever electromagnetic lens, which worked by focusing a beam of electrons instead of a traditional illuminator to generate a magnified image.

In 1939, the first EM was produced commercially by Siemens under the guidance of Ruska who worked as an electrical engineer and in 1986 Ruska was awarded the Nobel Prize for Physics.

How does it work?

picture2-300x212An EM works by generating an electron beam which enables the user to examine objects at the nano-scale. They work on the same principle as a light microscope except instead of light or photons they utilize electrons.

The basic operation of an electron microscope involves an electron gun producing a beam of electrons from an electric current, the electrons are accelerated using an anode plate to focus on the object.

All EM’s are composed of an electromagnetic and/or electrostatic lenses, which are made up of a solenoid, which is a coil of wire wrapped around the exterior of a tube. The specimen is then placed into a vacuum chamber as electrons do not travel well in the air.

In a light microscope, the glass lenses refract the light passing through them causing magnification, however, in an electron microscope the coils bend the electron beams in a similar way.

An image is then produced, called an electron micrograph, this is usually seen on a computer screen as electron microscopes typically come accompanied with software for image analysis and observation.

Specimens used in an EM need specialized preparation before being placed in the air free chamber, the exact method depends on the kind of specimen and analysis, which include:

  • Cryofixation
  • Fixation
  • Dehydration
  • Embedding
  • Sectioning
  • Staining

The use of an electron microscope and the preparation of specimen requires specialist training and knowledge as inexperience can lead to contamination with other artifacts.  Specimens are typically embedded in plastic epoxy resin and sectioned using glass knives on an ultramicrotome.

Transmission Electron Microscope

The transmission electron microscope (TEM), was the very first type of EM developed and the primary EM used in diagnosis. It is an incredibly powerful microscope that works on the same principle as the light microscope, except photons are replaced by an electron beam. It can produce images at the nanoscale.

The electron beam is passed through the specimen at high speeds (transmission) which produce a high-resolution image. This produces a black and white 2D image that can be viewed via the screen.

Scanning Electron Microscope

The scanning electron microscope (SEM) has no relation to light microscopy and has a lower resolution than TEM. It can only produce an image of the specimen surface (topography), the main advantage of TEM is producing 3D images.

The SEM microscope utilizes multiple solenoids which scan the surface of the specimen and the specimen reflects electrons back when irradiated with the electron beam.

The electrons are detected by converting energy into light-measured by a photomultiplier and the magnification can be increased by increasing the strength of the electron beam. The backscattered electrons give information of the subsurface and an image is produced.

Specimen processing for the SEM is highly specialized, firstly it is dried with alcohol, then soaked with liquid CO2, and the CO2 is converted into gas and slowly released. It is then sputter coated with a film of metal causing the specimen to become electrically conductive.