Darkfield Microscopy

Brightfield microscopy uses light from the lamp source under the specimen gathered in the condenser, and then shaped into a cone where the apex is focused on the plane of the specimen. In order to view a specimen in a brightfield microscope, the light rays that pass through it must be changed enough to be able to interfere with each other (contrast) and therefore, build an image. At times, a specimen will have a refractive index very similar to the surrounding medium between the microscope stage and the objective lens. When this happens, the image can not be seen. To visualize these biological materials well, they must have a contrast caused by the proper refractive indices, or be artificially stained. Since staining can occasionally kill specimens, at times darkfield microscopy is used instead.

While brightfield microscopy illuminates the sample with a filled cone of light, in darkfield microscope the condenser is designed to form a hollow cone of light. The objective lens sits in the dark hollow of this cone and light travels around the objective lens, but does not enter it. The entire field of view appears dark when there is no sample on the microscope stage. However, when a sample is on the stage the sample appears bright against a dark background. It is similar to back-lighting an object in order to make it stand out.

 Illustration courtesy of Washington State University.

The illustration above shows how the light is directed differently when using a darkfield microscope or a brightfield microscope.

There are several different types of darkfield microscopes, including stereo darkfield microscopes, darkfield biological microscopes and darkfield metallurgical microscopes. Darkfield is commonly used to better view bacteria, blood cells, diamonds and precious stones, and various types of algae.

You can learn more about darkfield microscopy and view different darkfield microscopes here.

Keep Your Microscope Clean

Whether your microscope cost a few hundred dollars or several thousand, it is a precision instrument and a few standard practices are required to care for it.  There is no reason for a microscope to perform less over time and your images should continue to be as sharp as when you first acquired it with only a small amount of maintenance.

The most important thing is to keep the microscope clean, so cover it when not in use.  Most microscopes have dust covers which come with the microscope. These protect the eyepieces and objectives from dust and debris that degrades the image and over time will affect the working mechanisms.  If you don’t have a cover you can order a replacement or use an over-sized clean plastic bag that will cover the instrument. Even though the microscope can be and should be cleaned when needed, it is preferable to maintain a protected environment for the optical elements.

Store the microscope in the most protected place possible.  A closed cabinet or microscope case is ideal.  A garage or hot attic could be disastrous.  A microscope uses rubber-like seals on the objectives which will degrade in a hot and dry area. Also, the internal grease used to coat the gears in the focusing and stage control will also dry out.

If you use the oil immersion lens (@100x), make sure you wipe the immersion oil off before putting the microscope away.  Wipe away any spills on the stage. If not cleaned, the oil will harden and attract dust and other debris.

This microscope image captured at 40x shows the distracting debris in the field of view and would be considered a poor photo-micrograph.

This is a much better microscope image where the debris has been cleaned off the microscope objective.

The dust on the eyepieces can be cleaned with an air blower (included in this microscope cleaning kit), or canned air canister. Be very careful to hold the can upright as you use it. Keeping your microscope clean from the start will pay off many times over.

Penicillium Mold under a Fluorescence Microscope

Penicillium mold is a common contaminant that causes food spoilage and is an indicator organism for indoor dampness. Penicillium molds with rapidly growing colonies have structures that resemble brushes and commonly have a strong musty odor.

This image of penicillium mold was captured with a fluorescence microscope using the Lumenera Infinity 2-3 CCD microscope camera.

Penicillium is one of the first fungi that grows on water-damaged materials, and can often be found in air conditioning ducts or places where insulation is moist for extended periods of time. Allergic reactions to Penicillium mold include hypersensitivity pneumonitis, and a variety of severe lung complications. It may cause sarcoidosis and fibrosis.

Some species of Penicillium are beneficial to humans. Cheese such as Roquefort, Brie, Camembert, and Stilton are ripened with species of Penicillium and are safe to eat. The drug penicillin is produced by Penicillium Chrysogenum, a commonly occurring mold in most homes.

Cerebellum under the Microscope

The cerebellum (Latin for little brain) is the region of the brain that plays an important role in motor control. It is also involved in some cognitive functions such as attention and language. The cerebellum does not initiate movement, but it contributes to coordination, precision and accurate timing.

The cerebellum appears to be a separate structure attached to the bottom of the brain. The surface of the cerebellum is covered with finely spaced parallel grooves, which covers up the fact that the cerebellum is a continuous thin layer of neural tissue, tightly folded like an accordion. Within this layer are several types of neurons - and it is this complex network of neurons that allows them their massive signaling capability.

The cerebellum receives input from sensory systems, other parts of the brain, and the spinal cord and then uses this input to fine tune motor activity. Because of this fine-tuning function, if there is damage to the cerebellum, it does not cause paralysis, but instead produces disorders in fine movement, equilibrium, posture and motor learning.

Cerebellum captured under a laboratory microscope using a CCD microscope camera.

On the microscopic level, each part of the cortex of the cerebellum contains the same set of neuronal elements, laid out with highly stereotyped geometry. There are two types of neurons that play primary roles in the cerebellar circuit: Purkinje cells and granule cells.

E. Coli

Escherichia Coli (known as E. Coli) is a rod-shaped bacterium commonly found in the lower intestine of warm-blooded organisms. E. Coli brings to mind images of serious food poisoning in humans and massive produce recalls. However, most E. Coli strains are harmless. The harmless strains are part of the normal flora of the gut and can benefit the host by producing Vitamin K and by preventing the establishment of pathogenic bacteria within the intestine.

In 1885, Theodor Escherich, a German pediatrician, first discovered Escherichia and Salmonella in the feces of healthy individuals and called it Bacterium coli commune.

 E. Coli captured with the MW5-CCD camera on a BA310 biological microscope.

E. Coli cells are typically rod-shaped and are about 2 micrometers (um) long and 0.5um in diameter. Optimal growth of E. Coli occurs at 98.6°F (37°C), but some laboratory strains can multiply at temperatures up to 120°F (49°C).

E. Coli normally colonizes an infant's gastrointestinal tract within 40 hours of birth, arriving with food or water or with the individuals handling the child. In the bowel, it adheres to the mucus of the large intestine. As long as these bacteria do not acquire genetic elements encoding for virulence factors, they remain benign organisms that neither benefit, nor harm the other.

Dicots under the Microscope

Dicotyledons (dicots) are a group of flowering plants whose seed typically has two embryonic leaves. Flowering plants that are not dicots are monocots, having one embryonic leaf.

FEATURE	MONOCOT	DICOT
# of parts of each flower	In 3s	In 4s or 5s
# of furrows or pores in pollen	1	3
# of cotyledons (leaves in the seed)	1	2
Arrangement of vascular bundles in stem	scattered	concentric circles
Roots	adventitious	develop from radicle
Arrangement of major leaf veins	parallel	reticulate

A magnolia is an example of a dicot.

 Dicot captured using a student biology microscope using the MW5-CCD microscope camera.

Monocot under the Microscope

Monocots are one of two major groups of flowering plants (angiosperms) that are typically recognized. The other being dicots. Monocot seedlings typically have one seed-leaf, in contrast to the two seed-leaves in dicots.

This monocot was viewed under a biological microscope and captured with the MW5CCD microscope camera.

There are over 59,000 species of monocots. The largest family in this group is the orchid family.  A distinctive property of a monocot's flower is that it is trimerous - meaning the flower parts in threes or multiples of three. For example, a monocot flower typically has three, six, or nine petals. Many monocots also have leaves that have parallel veins in them. For example, look at the slice of an onion - it has parallel veins in the cross section.

The tallest monocot in the world is the wax palm, shown below.

Image courtesy of Diego Torquemada.

If you are interested in viewing your own monocot prepared microscope slide, there is one included free with all student high power microscopes in Microscope World's prepared slide kit.

Microscope Field of View

A microscope's field of view is the diameter, in mm, of what you see when looking through the microscope.

The field of view can be found if you know the eyepiece magnification field number (FN) and the objective magnification. You can view complete formulas to calculate microscope field of view here. If you are using a stereo microscope with an auxiliary lens you will also need to know the magnification of this lens.

Your field of view will always increase (ie you can view more of your specimen) as you decrease your magnification (or "zoom out" when using a stereo microscope). As you increase magnification, the field of view gets much smaller.

Sugar Crystals under the Microscope

These images of sugar crystals were captured for a customer using the DC5-420T digital stereo microscope.

10x magnification.

20x magnification.

30x magnification.

40x magnification.

Cardiac Muscle

Cardiac muscle is a type of striated muscle found in the walls and foundation of the heart. It is one of three types of muscles - the other two being skeletal and smooth muscle.

Contractions of the cardiac muscle, are what pump blood out of the atria and ventricles throughout the body. The cardiac muscle is highly resistant to fatigue and has a high number of mitochondria, which enable continuous aerobic respiration and a good blood supply to deliver nutrients and oxygen.

The above image of cardiac muscle is included in the Musculoskeletal Histology Slide Kit from Microscope World. More of the prepared slides in this kit can be viewed here.

Viewing Micron Sized Medical Parts

With the advances in medicine, small parts are often manufactured for less-invasive procedures. These small parts require quality assessment prior to use. A customer recently approached us with the need to view a needle tip that was 5 microns in size. Microscope World configured a system that involved using a microscope camera on a video microscope with a 20x metallurgical objective lens.

The needle tip under the microscope system.

Sow Bug

The sow bug, also known to many children as the "rolly polly" bug, and to others as a woodlouse, is a crustacean with a rigid, segmented, long exoskeleton with fourteen jointed limbs. This exoskeleton is something that the sow bug must actually shed as it grows.

This sow bug was captured with the National Optical 420AH trinocular stereo zoom microscope, using a consumer digital camera adapter.

The sow bug is not typically considered a household pest, as they do not spread diseases and do not harm wood structures like termites do. However, sow bug presence can often indicate dampness issues in a household.

Fabric Under the Microscope

Textile manufacturers often use stereo microscopes to view their fabric weaves. The stereo microscope can help find flaws or areas where the stitching is uneven. When a variety of colors are woven into the fabric, the microscope can help ensure the pattern is aligned.

Fabric captured at 10x magnification using the SMZ-168 stereo zoom microscope.

Same piece of fabric at 60x magnification using the same microscope and a 1.5x auxiliary lens.

Hydra

Hydra are predatory animals that belong to the phylum Cnidaria (class: Hydrozoa). They can be found in most unpolluted fresh-water ponds, lakes and streams. The easiest way to collect hydra is by using a net to gather a water sample near weeds growing on the edge of the pond.

Typically a few millimeters in length, hydra are often studied extensively by biologists because of their ability to regenerate. Hydras have a tubular body, as shown above.

Hydra tentacles (also called cnidae) are covered in stinging cells called cnidocytes. These are used when contacting prey to discharge a dart-like thread containing neurotoxins into the prey. (They are too small to injure humans). Hydra do not have a brain or true muscles, and they mainly feed on small aquatic invertebrates such as Daphnia and Cyclops.

If you have access to a microscope this summer, here is a fun project.

1. Gather some fresh pond water (if you have access to salt water, gather some of that as well!)
2. Make sure you label your jars of water if you collect pond water from multiple locations.
3. Place a small drop of pond water on a depression slide and place a cover slip on top.
4. Using a student microscope, first focus at 40x, then move up to 100x and finally 400x. You will be able to view most hydra at lower magnifications. At the higher magnifications, you may see some bacteria. 
5. If you are unable to view any samples with your first slide, simply clean the slide and place a new drop of water on the slide and try again. Sometimes it takes a few tries before you get a good sample.
6. Take note of the images you see. Can you identify the organisms? If you are unsure of what you are viewing draw a picture of the sample and research it, or ask your science teacher to help you identify it.