Types of Objective Lens

Types of Objective Lens & Their Functions

Light microscopes are relatively complex pieces of equipment in nature with multiple different parts, some which are more complex than others.

The lenses of the microscope are fundamental to its function as they provide the magnification power that allows the microscopic specimen to be seen or observed in greater detail. The two main types of lenses found in light microscopes today are called the objective lens and the ocular (or eyepiece lens).

The ocular lens, which is also called the eyepiece lens, is positioned at the top of the optical tube, while the objective lens is positioned at the bottom. Both of these lenses have important roles in magnification, but the objective lens also has other defined roles, such as resolving power.

Objective Lens Microscope Function

The majority of light microscopes have an objective lens of some kind, which includes both compound microscopes and stereo microscopes. These types of microscopes are also the same in that each type has an eyepiece or ocular lens.

Magnification Power: 

The objective lens and the ocular or eyepiece lens are in combination responsible for magnification of the specimen being observed.

For example:

Total magnification = Objective magnification X ocular magnification

Therefore, for 10X objective and 10X ocular the total magnification = 10 X 10 = 100X

This means that the specimen being observed is now 100X it’s actual size.

Resolution:

Resolving power is also a very important metric since magnification power is of little importance if the resolution is not high. Resolution is defined as the ability to distinguish 2 points as two points.

For example, if you are looking down a microscope, the resolution power relates to the space you can see between two points. A very low resolution would result in a blurred image and would prevent proper observation of the specimen.

While the total magnification is determined by both the objective and ocular lens, the resolution is determined by the objective lens alone.

Types of Objective Lenses

The majority of compound microscopes come with interchangeable objective lenses, which have different magnification powers. This commonly includes 4x, 10x, 40x, and 100x objective lenses.

Scanning Objective Lens (4x) 

Combined with the eyepiece lens, this lens will provide the lowest magnification power. For example, 10x eyepiece lens, multiplied by the 4x objective lens gives a total magnification of 40x.

This objective is often referred to as the scanning objective lens since the low power provides enough magnification to give the observer a good overview of the entire slide and sample.

Low Power Objective (10x)

This objective lens is the next lowest powered and is often the most helpful when it comes to analyzing glass slide samples. The total magnification for this lens is equal to 100x magnification (10x eyepiece lens x the 10x objective equals 100).

Since it still provides a good amount of magnification at a good distance from the slide, there is a limited risk of it breaking the glass and potentially ruining the sample. Hence, why it is often preferred before going for a high powered lens.

High Power Objective Lens (40x)

This is referred to as the high powered objective lens since it is ideal for observing the small details within a specimen sample. The total magnification for this lens is equal to 400x magnification (10x eyepiece lens x the 40x objective equals 400).

Oil Immersion Objective (100x)

This objective lens will achieve the greatest magnification and has a total magnification of 1000x (10x eyepiece lens x the 100x objective equals 1000). However, since the refractive index of air and the glass slide are slightly different, a special oil must be used to help fill the gap between the two. Without a drop of oil, the objective lens will not work properly and you will not achieve the desired magnification and resolution.

What is the Difference between Ocular and Objective lenses?

As previously mentioned, the ocular or eyepiece lens is located at the top of the eyepiece tube and is where you position your eye to observe the specimen. The ocular lens typically has a low magnification (10x) and works in combination with the objective lens to achieve a greater magnification power.

The objective lens is at the bottom of the eyepiece tube and is responsible for both total magnification of the specimen, as well as the resolving power of the microscope.

Bacteria Under the Microscope

Bacteria Under the Microscope

Bacteria are single-celled organisms that are defined as prokaryotes, these are organisms that have cells with no defined nucleus or other specialized organelles.

In total, there are estimated to be millions of species of bacteria, which are diverse in shape, size and many other defining features. By visually inspecting bacteria for these physical characteristics bacteria can be identified with the help of a light microscope.

Microscope Bacteria Preparation

Culturing Bacteria Species  

Before certain bacteria can be seen under a light microscope, they first must be grown in a nutrient-rich culture media. Since bacteria have different nutrient requirements, the exact type of media used depends on the bacteria that is being selected for.

These are some common types of culture media used in modern microbiology laboratories:

  • Nutrient Agar media – This is the most common type of nutrient media used and is a non-selective media that allows the growth of a wide variety of different species. This includes aerobic, anaerobic and microaerophilic microorganisms.
  • Selective Agar -This a type of agar that inhibits the growth of certain bacteria while allowing the growth of another e.g. Mannitol salt agar is selective for Gram-positive bacteria.
  • Differential Culture Media – This is a kind of media that is used to differentiate between bacteria. For example, blood agar is commonly used to identify bacteria that are responsible for hemolysis of the blood e.g. Streptococci.

Slide Preparation

Once the bacteria has been left to incubate and grow on the agar plate, it then is viewed under the microscope. In order to do this, a bacterial smear must be performed. This involves placing a thin layer of bacteria onto a glass slide, typically before staining it.

Materials Required: 

  • Bacteria sample
  • Distilled water
  • Light microscope
  • Bunsen burner
  • Inoculating loop
  • Glass Slide
  • Felt marker

The Procedure: 

  • Using your felt marker, place a small mark where you will be placing the bacteria and making the smear.
  • Using an inoculating loop, place a small drop of water whee you are going to place the bacteria and perform the smear.
  • The smear is now ready to be prepared. Firstly, place the inoculation loop into the blue portion of the flame of the bunsen burner. Allow the loop to glow red and then cool.
  • Next, immediately remove a bacteria colony from the agar plate or broth using the loop. Remember to maintain good aseptic technique to prevent contamination of the sample.
  • Mix the bacteria on the loop with the water you placed on the glass slide earlier and then flame your loop once again.
  • Finally, allow the slide to air dry.

Staining Procedure 

When looking at bacteria under the microscope much of the bacteria can appear transparent without staining. Staining allows different structural components of the cells to be visualized including the cytoplasm, cell wall, and membranes. Common stains used on bacteria include crystal violet, methylene blue, and safranin.

The Procedure: 

  • Place your prepared slides next to each other on a paper towel or staining rack.
  • Cover the slides in the chosen stain and leave for 1-2 minutes.
  • Remove excess stain by running a gentle stream of water over the surface of the glass slide.
  • Place the glass slide on the microscope stage and begin viewing the specimen. Begin with a low magnification and gradually increase to observe greater detail.

Microscope Bacteria Identification

Upon viewing the bacteria under the microscope, you will be able to identify the bacteria based on a wide variety of physical characteristics. This mainly involves looking at their shape and size.

There are a wide variety of different shapes, yet the three main types are cocci, bacilli, and spiral.

Cocci – These are the most common type of bacteria. They are named cocci due to their spherical shape, although they commonly appear in groups. For instance, diplococci refer to pairs of cocci, streptococci refer to chains and staphylococci refers to clusters of cocci.

Bacilli – These are rod-shaped bacteria and just like cocci they can be found on their own or grouped. For example, diplobacilli refer to two bacilli next to each other, while streptobacilli refers to chains of bacilli.

Spiral – These are simply spiral-shaped bacteria. Examples include spirillium, which are thick, durable spirals and spirochetes, which are slender and flexible.

Gram Postive or Gram Negative?

Another useful way of identifying a bacteria is by determining whether it is a gram negative or gram positive. This is achieved through the staining process and stains such as crystal violet dye, iodine, and the counterstain safranin.

Gram Postive Bacteria – These are bacteria that have a thick peptidoglycan layer which the stain (crystal violet-iodine complex) is attracted to. As a result, the stain is retained resulting in them having a purple/bluish appearance when viewed down the microscope. Examples of gram-positive bacteria include Listeria, Streptococcus, and Bacillus. 

Gram-Negative Bacteria – These are bacteria that do not have a thick layer of peptidoglycan and so the crystal violet-iodine complex is unable to be trapped within the structure. Instead, the bacteria can hold the safranin, which results in them having a red appearance under the microscope. Examples of gram-negative bacteria include proteobacteria and cyanobacteria.

The gram staining technique is an extremely useful technique, that will help identify a bacteria at the basic level, but not a species level.

Examples of Bacteria Under the Microscope

Escherichia coli: 

Escherichia coli (E.coli) is a common gram-negative bacterial species that is often one of the first ones to be observed by students. Most strains of E.coli are harmless to humans, but some are pathogens and are responsible for gastrointestinal infections. They are a bacillus shaped bacteria that has a very fast growth (they can double every 20 minutes), which is one of the main reasons they are used in research.

E.coli-colony-growth
Escherichia coli (E.coli)

Staphylococcus Aureus: 

Staphylococcus Aureus (S. Aureus) is another round-shaped bacteria that is extremely common. It is abundant on the skin, in the nose and the respiratory tract. Just like E.coli, there are pathogenic strains of S. Aureus that are responsible for skin infections, abscesses, and respiratory infections.

This bacteria is ideal for the gram staining technique since it is a gram-positive bacteria, this means it has a thick peptidoglycan layer that will trap crystal violet and so will appear bluish/purple under the microscope.

Staphylococcus Aureus
Staphylococcus Aureus (S. Aureus)

What Type of Microscope is Best for Bacteria Observation?

Since bacteria are incredibly small and transparent, they can be extremely difficult to observe under a standard bright-field compound microscope. Instead, it’s common for researchers and scientists to use a phase contrast microscope instead.

A phase contrast microscope uses an optical technique that works by the use of a device to read the difference in thickness of the subject affecting the phasing of light resulting in a high contrast image. Staining bacteria also help microscope users to see bacterial structures that would have otherwise been invisible to the naked eye.

Compound Microscope vs. Stereo Microscope

Compound Microscope vs. Stereo Microscope

 

Stereo microscopes and compound microscopes are two distinct types of light microscopes that have many similarities, but some fundamental differences.

It’s certainly worth being aware of these key differences as they impact factors such as the magnification power, working distance and the depth of field, as well as imparting some other very useful qualities. As a result, each is intended for viewing different types of objects and therefore for different applications.

In brief, stereo microscopes offer a lower magnification, but useful qualities such as 3D visualization and depth perception. Therefore, making them ideal for visualization of larger objects.

Whereas, compound microscopes provide a much greater magnification power and so are better suited to inspect the microscopic structures in much smaller specimens.

Stereo Microscopes Defining Features 

Stereo microscopes also referred to as dissecting microscopes provide a relatively low magnification, typically 50x and below.

Stereo microscopes have two optical paths, one which is offset from the other. This is because stereo microscopes use two separate objective lenses and eyepieces. This means that different axis of an object or sample can be analyzed at the same time. This key difference provides it with two very useful features – depth perception and 3D visualization.

Stereo microscopes use a type of illumination referred to incident light illumination (light is reflected off the surface of the object). This allows the visualization of the surface of the object.

These components provide stereo microscopes with superior depth perception and a great working distance. The depth perception relates to the distance between two points in a specimen that both remain clear and focused when viewed.

For these reasons, stereo microscopes are mainly used for viewing opaque objects, including, insects, minerals, jewelry, coins, watches, hardware, and many other large specimens.

Microscope Anatomy

Compound Microscopes Defining Features

Compound microscopes are able to provide a much larger magnification, which can range from 40x to 1000x or more. They achieve this by magnifying in two stages, the first that uses the ocular lens and the second which uses interchangeable objective lenses, which are often 4x, 10x, 40x and 60x magnification.

Unlike stereo microscopes, compound microscopes only have a single optical path. This means that the specimen can be viewed through the eyepiece at a greater magnification as the objective lenses can be interchanged.

However, since it does not allow provide two different axes of the same specimen to be viewed at once it lacks the depth perception and 3D visualization of a dissecting microscope.

Compound microscopes also use transmitted light illumination (light is passed through the sample).  This often means that samples have to be dissected into smaller slices to enable the light path to travel through the sample and illuminate more of the microscopic structures.

These attributes make compound microscopes well suited for the observation of smaller samples, often sliced into a section to allow the light to pass through them.  This commonly includes biological samples such as bacteria, plant cells, and tissues.

Do you Need a Compound or Stereo Microscope? 

Although the general rule is that compound microscopes are used for smaller objects and stereo microscopes are used for viewing larger objects, it really depends on the type of objects you are going to be analyzing on a regular basis. Essentially, you can use either type of microscope to view many types of a specimen.

However, sometimes one microscope is more appropriate. The two most important factors are the size of the object and how much light can pass through the object.

Stereo microscopes illuminate objects from above, which allows the surfaces of many large objects to be viewed in a good amount of detail. They even allow you to view the object stereoscopically (3-dimensional), hence their name.

These dissecting microscopes also offer enough magnification power to make viewing the surface of large objects possible.

However, for those interested in viewing much smaller structures and internal structures of a specimen will require a compound microscope. These microscopes offer a much greater magnification power and illuminate objects from below. These are great for those individuals wanting to view cells, bacteria, fungi, tissues, organs and other small biological specimens.

STEREO VS COMPOUND

Binocular vs. Monocular Heads

Many people assume that a compound microscope with a binocular head (with two eyepieces) is a stereo microscope. The reality is that both stereo and compound microscopes can have a binocular head, but only a stereo microscope is able to offer 3D-visualization. The reason for this is that they provide two different optical paths which are offset from each other.

How are Compound and Stereomicroscopes Different? 

By now, you are probably well aware of the major differences between compound and stereo microscopes (also called dissecting microscopes). However, in summary, here is a brief overview of the differences:

Compound Microscope Key Features: 

  • Use transmitted light illumination (light is passed through the sample), typically from below the object.
  • Have a greater magnification power, which can exceed 1000x
  • Have a single optical path
  • Use a single ocular lens and interchangeable objective lenses

Stereo Microscope Key Features: 

  • Use incident light illumination (light is reflected off the surface of the object), typically from above the object.
  • Have two different optical paths, allowing stereoscopic (3D visualization) of the specimen and depth perception
  • Have a lower magnification, typically 50x and below
  • Use two objective lenses and two ocular lenses

Final Thoughts

Both stereo microscopes and compound microscopes can both be used for a wide variety of biological and hobby based activities. Although many models look similar in design, it’s important to remember that they have some very important differences. As you may have expected, the key differences lye within their optics.

Compound microscopes named so due to their two sets of lenses (objective and ocular) offer superior magnification power and so are ideal for looking at small specimens.

On the other hand, stereo microscopes, named so due to their stereoscopic ability are more suitable for analyzing larger objects, where 3D visualization and depth perception would be useful.

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.

Microscope Spot

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.