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How to make macro images

Oliver Kim — Sat, 29 May 2010 10:00:47 +0000

Aperture: f/19.9, Exposure time: 10sec.

Aperture:f/22.6, Exposure time: 20sec.

This time I’d like to talk about a topic which is only indirectly related to microscopy: macro imaging. Taking high-quality macro images can be quite a challenge and can involve quite a bit of trial and error until one has found the ideal conditions. The pictures of the rose have been taken with a Sigma 70-300mm DG APO objective and a Canon EOS 450D camera. No artificial light was used, the exposure times were therefore quite long.

In order to obtain good looking macro images one has to take several measures:

Technical set-up

A solid, stable tripod: A heavy and stable tripod is absolutely necessary. Even the slightest vibrations (people walking, wind, shutter vibrations, etc.) will translate into a blurry image. I used a tripod designed for heavier video cameras.
High f-stop values: Macro images generally have a low depth of field. In order to increase the depth of field it is necessary to increase the f-stop values. This again results in longer exposure times.
Reducing shutter vibrations: Shutter vibrations can be minimized by using the mirror-lock up function of the camera. When the live-view feature of the camera is enabled, the mirror is in the “up” position. During release, the mirror does not have to swing up (because it is already up) and this significantly reduces vibrations. Not every SLR camera has a mirror lock-up feature, however. In this case, one can use a trick to completely eliminate shutter vibrations. Adjust the camera to have a long exposure time (let’s say 20 seconds). Cover the objective with a black cardboard, without touching the objective. Then release the shutter and wait 1 second for the system to stop vibrating. Then remove the black cardboard, this starts the exposure. One second before closing of the shutter cover the objective again. The opening and closing of the shutter (and mirror swing) will then take place while you have the black cardboard in front of the objective. Amateur astronomers use the same technique for taking vibration-free images of the night sky. They cover the telescope aperture with a dark cloth (without touching the telescope, of course).
Long exposure times: This may come as a surprise for some. Long exposure times can result in more steady images because the duration of the vibrating camera/tripod system (after release) are much shorter than the total exposure time during which a steady image reaches the sensor of the camera.

Composition, artistic aspects etc.

Sufficient ambient light: To prevent hard shadows, I decided to use the natural, indirect light of the room to take the picture of the rose. This results in longer exposure times, which makes a very stable tripod a necessity.
Contrast: Make sure that the main object is set off from its background. This can be achieved either by blurring the background, or by making sure that the background has a distinctly different color.
Exposure time: If you use a black background, then the camera may expose longer than necessary. The black background “fools” the camera into thinking that the image is too dark. This may result in the object being overexposed. Underexpose by 1-2 stops if the main object is too bright.
Post processing: Crop the image and adjust the colors so that the background is completely black (if you use a black background). This will increase the impact of the picture.

Volvox

Oliver Kim — Sat, 22 May 2010 10:00:18 +0000

Volvox is a fresh water green algae and a member of the Chlorophyta. The picture shows a spherical volvox colony, each ball can contain hundreds, if not thousands of individual cells. The picture shows six daughter colonies inside the main colony. The main colony disintegrates and the daughter colonies are then released. Volvox is a nice example of an organism which shows first signs of multicellularity. Larger colonies can be up to 1mm in diameter and can be seen with the unaided eye.

Reproduction

Volvox reproduces both sexually and asexually. During asexual reproduction cells from the equator of the colony move to the inside and divide to form daughter colonies. The daughter colonies will grow and multiply. The mother colony will then rupture and release the offspring.

During sexual reproduction, Volvox colonies form sperm and egg cells (ova). The sperm cells will swarm out to find ova in other colonies. The fertilized egg cells will then form new colonies.

Growing and observing Volvox

Microscopists who are interested in observing Volvox should try to investigate water samples from ponds and puddles. It is also possible to grow Volvox at home. Volvox likes to grow in nutrient-rich water. Dilute some plant fertilizer in water and add some pond water containing Volvox (or other green algae that you want to grow). Place the container on the window sill for several days but prevent direct sunlight as this may cause overheating, and drives out the CO2 for photosynthesis from the water. Alternatively, you can also use a plankton net to catch the colonies.

For making permanent mounts, it’s probably best to use a water-based mounting medium such as glycerin gelatin. Solvent based media may dissolve the chlorophyll of the chloroplasts and may cause the cells to lose water and shrink.

Dandelion parachute ball up close

Oliver Kim — Fri, 14 May 2010 22:00:18 +0000

Dandelion macro image.

This is one of the first tries taking pictures with my new Sigma objective, and I have to admit that I’m very satisfied with the lens. The lens does not include image stabilization, a steady tripod is therefore a must. Contrast was slightly enhanced to make the background (my computer screen!) completely black. Mirror lock-up was used to further minimize vibrations. At a focal length of 300mm even the smallest vibrations cause a blurry image.

Lens: Sigma 20-300mm APO DG MACRO (macro up to 1:2)
Camera: Canon EOS 450D
Exposure Time: 2 sec. (long time to minimize vibrations)
Aperture Value: 6.62 EV (f/9.9)
ISO Speed Rating: 100
Focal Length: 300.0 mm

Ranunculus (Buttercup) pollen

Oliver Kim — Sat, 01 May 2010 19:28:12 +0000

Ranunculus repens pollen in dark field

Ranunculus repens pollen in bright field

Ranunculus repens

Spring time is pollen time! Here are two images of Ranunculus repens (the Creeping Buttercup or Creeping Crowfoot) pollen, the top one in dark field, the bottom one in bright field. This plant is poisonous and can cause skin irritation. The name “Crowfoot” comes from the shape of the leaves, which resemble the claws of a crow.

Now a few words concerning sample preparation. The pollen was collected by dusting the flower over a microscopic glass slide. The pollen was briefly dried in open air (about 1 hour) and then permanently mounted in Euparal mounting medium. The standard mounting medium for pollen is Glycerin gelatin, which is water based. I assume that the drying and the Euparal caused the pollen to shrink somewhat, but I yet have to make a comparison with fresh pollen.

Human hair under the microscope

Oliver Kim — Tue, 23 Feb 2010 11:00:38 +0000

Two intertwined human hair under the microscope. The original image. The field iris diaphragm is still visible at the corners.

The power of image clean-up: dust and dirt removed.

Today I’d like to show you a nice microscopic picture, which I took several years ago of two human hair. The pictures on the right show you the original, unprocessed image at the top, and a second cleaned-up image with a nice background on the bottom. The top image shows darkened corers from the field diaphragm (from Köhler illumination). I closed the diaphragm quite a bit in order to increase image contrast (I intended to crop the image anyway). Then I did some processing in PhotoShop (or was it GIMP?). I selected the hair to remove dust and dirt and placed it on a nice blue gradient background. The most difficult part of the project was the making the hair knot and finding of an appropriate focus of the relatively thick sample. I did not do any image stacking (combining several images into one to get one final image which is sharp throughout).

Bacteria in phase contrast

Oliver Kim — Sat, 06 Feb 2010 09:00:44 +0000

Cocci in packets

Cocci in pairs and packets of four.

Short rods

Rods-slightly curved cells

The four pictures on the right show different bacterial species in phase contrast.

About phase contrast

Bacteria are transparent and therefore difficult to see using regular bright-field microscopy. The bacterial cells will appear just as bright as the surounding medium and there is no color contrast. Phase contrast optics provides a solution. Phase contrast optics convert the differences in optical density (i.e. the refractive index) of the bacterial cells into different shades of brightness. The optics achieves this by interference of the light which passes through the specimen (the bacteria) with the light that goes around the medium. Phase contrast optics therefore work only if the cells have a different refractive index compared to the medium.

How the bacteria were prepared

The bacteria were grown in pure culture in an appropriate microbiology laboratory. A colony was then suspended in saline (salt water) of right concentration and then microscoped with a 1000x magnification in oil immersion (using a 100x oil objective).

If one takes too much liquid, then the cells start to float in and out of focus and it is not easily possible to capture the shape of the individual cells. A similar problem can occur if the cells are much smaller than the film of liquid between the slide and cover slip. The evaporation of the liquid from the edges of the cover slip will cause a constant movement of the cells and make it difficult to take a steady picture. In this case it is necessary to heat-fix the bacteria. A colony was then suspended in saline and dried at room temperature. The slide was briefly pulled through the flame of a bunsen burner, with the bacteria on the opposite side of the the flame. This heating process fixed the bacteria to the glass slide. Immersion oil was then directly applied to the slide and the bacteria were observed without cover glass. One disadvantage of heat fixing is, that during the drying process the bacteria may aggregate (as the volume of liquid decreases) and it may become more difficult to see individual cells.

About the photographs

The pictures were taken on analog B/W film and then digitized with a camera and an adapter (see the following post for more info on the set-up: Digitizing photographic slides with a digital camera ). The negative was then inverted and the contrast levels adjusted. The soft, slightly blurry appearance of the pictures shows that we are already at the limits of the resolution. The images were not sharpened. Notice the bright halo around the bacterial cells. This is typical for phase contrast microscopy.

Trichinella spiralis – the “pork worm”

Oliver Kim — Thu, 04 Feb 2010 11:00:33 +0000

Encapsuled Trichinella spiralis larva in muscle. The larva is cut diagonally.

Longitudinal cross-section.

The larva (circular patterns) in cross-section.

Trichinella spiralis is the smallest nematode parasite in humans. It causes the disease trichinosis. It is also one of the most wide spread parasites of the world. It can be contracted by eating raw or half-cooked pork or wild game animals.

Life Cycle of Trichinella spiralis

T. spiralis larva are encapsuled in the muscle of the host animal. The pictures on the right show different cross-sections of this stage.
A person who eats raw or undercooked meat will take these larva up into the body. The larvae are released by the stomach acid and pass into the intestine, where they mature and start to reproduce.
The offspring larvae travel in the circulatory system throughout the body and settle in the muscles, where they encyst again.

Mitosis stages of the Lily

Oliver Kim — Sun, 06 Dec 2009 15:33:52 +0000

Interphase. The nucleus is visible.

Prophase. Chromosomes are starting to form.

Metaphase. The chromosomes align at the equator of the cell.

Anaphase. The two sister chromatids are separated.

Possibly metaphase or anaphase seen head-on. The chromosomes are possibly pointing towards the viewer.

Telophase. The spindle fibers are still visible between the two nuclei. The cytoplasm has not yet divided.

Background Information: Mitosis is cell division in eukaryotes. During mitosis the chromosomes are visible. Interphase is not considered part of cell division. The following stages are Prophase, Metaphase, Anaphase and Telophase.

Image Information: Higher resolutions are, unfortunately not available. A magnification of 400x was used to obtain these images. The cells were treated with a dye that has a high affinity for DNA.

Buttercup (Ranunculus repens) Root

Oliver Kim — Sat, 28 Nov 2009 16:56:14 +0000

Vascular tissue of a Buttercup, Ranunculus, root.

Image Information: The root was microtomed and stained. The triangular structure on the left is the vascular tissue, used for transporting substances up and down the plant.

Background Information: Ranunculus is a large genus encompassing about 400 different species. They possess bright yellow or white flowers and some have orange or red flowers. All members of the genus are poisonous. The toxin is inactivated when dried, hay used for livestock is therefore safe.

The Tick (Ixodidae)

Oliver Kim — Sat, 28 Nov 2009 14:42:25 +0000

The tick (Ixodidae) in dark field, edited and placed on white background.

Image Information: The tick was treated with alcohol and preserved in permanent mounting medium (Eukitt). Darkfield images were then taken under 40x magnification and stitched together with panorama software. The tick was cut out and placed on white background.

Background Information: Ticks are parasites that feed on blood. They are known to transmit a variety of diseases, such as Lyme disease (borreliosis) and tick-borne encephalitis.

Zoom into the image here: Virtual microscope: The Tick.

The Dog Flea (Ctenocephalides canis)

Oliver Kim — Sun, 01 Feb 2009 13:04:30 +0000

The Dog Flea (Ctenocephalides canis) is an external parasite which can be found in the fur of both dogs and cats.

A portait image of the flea.

Image Information: The presented image is composed of several different overlapping images stitched together. Stacking to increase the depth of field was not performed.

Background Information: The adult female flea reaches a size between 2 and 4mm. Males are a little smaller, 2-3mm. They are wingless insects with a flattened body. Their chitin exoskeleton is resistant to pressure. Both sexes feed on the host’s blood. The fleas produce eggs out of which the larvae hatch. The larvae are between 1-6mm long (depending on the stage of the life cycle), and do not possess legs.

Papaya (Carica papaya) in Polarized Light

Oliver Kim — Sat, 31 Jan 2009 22:35:19 +0000

Microscopic image of a Papaya fruit (Carica papaya) in Polarized Light. The fruit was squeezed between the microscope slide and cover slip.

Image Information: A piece of papaya was squeezed between the microscope slide and cover glass. The sample was observed in polarized light (see Simple Polarization Microscopy). The cell walls (made of cellulose) are clearly visible using this method.

Background Information: Carica papaya, also referred to as the “big melon”, is a native of tropical Americas (southern Mexico, Central America and northern South America). Papayas are not only used as food: They contain the enzyme Papain, which is a protease and able to break down proteins. It is used in biotechnology. Papain ointment can also be used to treat cuts, rashes, stings and burns.

Wood of the Spruce Tree (Picea)

Oliver Kim — Sun, 25 Jan 2009 21:02:56 +0000

Wood of the Spruce Tree (Picea) - a conifer.

The annual rings can be seen well.

Image Information: The two pictures show a microtome cut of a piece of spruce wood. The image was taken under bright-field.

Background Information: Spruce trees belong to the genus Picea. This genus contains about 35 separate species. Spruce trees are evergreen coniferious trees and can be found in the colder, northern regions of the earth (the taiga). Most spruce trees are between 20 and 60 m tall, but they can reach a height of up to 95 meters. The wood of spruce trees (known as whitewood) has a range of different applications. It is used in paper manufacture and also in musical instruments. The needles of the tree can be boiled to make tea, rich in Vitamin C. The needles are also a source for essential oils and can be used to produce spruce tip syrup.

Spirogyra Algae

Oliver Kim — Sun, 25 Jan 2009 12:51:45 +0000

Microscopic picture of the algae Spirogyra. The algae possesses a spiral shaped chloroplast, which is clearly visible in the cell.

Image Information: The image of Spirogyra is taken from a permanent slide. Several pictures were stacked and the contrast was enhanced.

Background Information: Spirogyra is the genus name of a fresh water algae, of which there are over 400 individual species. The spiral chloroplast is characteristic for this genus. The organism can be found in clean ponds of high nutrient content (such as due to fertilization of nearby fields). It then grows to form slimy filamentous masses of algae. Spirogyra is capable of reproducing both sexually and asexually. A filament may fragment into smaller pieces, each one capable of forming new cells. During sexual reproduction two cells align with each other and form congugation tubes which connect the two cells and allow for the exchange of genetic material, forming zygospores.

Vitamin C (Ascorbic Acid) Crystals

Oliver Kim — Sat, 24 Jan 2009 19:03:51 +0000

Image Information: Both images depict Vitamin C (Ascorbic Acid) crystals under low magnification using crossed polarizing filters. A dilute solution of pure vitamin C was evaporated on the microscope slide. Crystals formed randomly, delivering a nearly unlimited source of beauty.

Background Information: Vitamin C is an birefringent, optically active molecule. The crystals have a refractive index which is different into different directions. Due to the interference of light, some wavelengths (colors) are absorbed resulting in a colorful image. For the method, view Simple Polarization Microscopy and Growing Crystals.

Sand from the Kalahari Desert

Oliver Kim — Sat, 24 Jan 2009 18:25:35 +0000

Sand from the Kalahari desert under the microscope. Dark field illumination.

Image Information: This image is a stack of six separate pictures. This way the depth of field could be increased. The bright spots on the dark background is dust, which becomes especially visible using dark field illumination. The image was slightly sharpened. Under the microscope it is evident that the individual sand grains are transparent, something which is not evident when looking at the sand with the unaided eye.

Background Information: Sand is made mostly of silicium dioxide (SiO2). Glass is made of the same material. The red patches on the individual sand grains are made of iron oxide. The Kalahari Desert covers large areas of Botswana, Namibia and parts of South Africa.

Elderberry (Sambucus)

Oliver Kim — Thu, 22 Jan 2009 18:14:17 +0000

Microscopic image of the cross section through the stem of an elder plant (Sambucus)

Image Information: This is a bright field image of the cross section of an elder plant. The inner part of the stem has a styrofoam-like consistency and is commonly used for making microtome cuts when preparing microscopic specimens.

Background Information: The elder (or elderberry) belongs to the genus of Sambucus. There are between 5 and 30 species in this genus.

Shaving Foam

Oliver Kim — Thu, 22 Jan 2009 16:14:08 +0000

Shaving foam under the microscope. Dark field illumination.

Image Information: Here I compressed some (very stiff) shaving foam between glass slide and cover glass. Quite a bit of pressure was necessary to form a single layer of bubbles. The image was made using dark field illumination.

Background Information: I used shaving foam from the can… Very interesting chemical compounds can be found in the foam, at least they sound interesting: Isobutane, Sorbitol, Glycerin, Tocopheryl acetate, Allantoin, Propylene Glycol, TEA-Palmitate, Polyquaternium-7, Stearate, Ceteth-20 ….. and of course Aqua (water) and perfume. The propane is probably the propellant (is this can of shaving foam explosive as well??). A little bit of research reveals some interesting facts:

Tocopheryl acetate: this is a Vitamin E derivative which protects the skin from ultraviolet (UV) light. It is commonly found in ceams and other products that are applied to the skin.
Allantoin: this substance has a moisturizing effect on the skin and increases its smoothness. It binds substances that irritate the skin and therefore protect the skin.
Propylene Glycol: this one seems to be a pretty versatile compound. It is used as a moisturizing agent as well as for de-icing aircraft.
Polyquaternium-7: An anti-static agent. It also forms a film around hair to protect it (it is also found in many shampoos).

Potato Stach Grains

Oliver Kim — Sun, 18 Jan 2009 17:30:52 +0000

Potato starch grains in dark field.

Potato starch grains in bright field.

Image Information: Here I would like to show you two images of potato starch grains taken with different optical contrasting methods. The top image was taken in dark field, the bottom one in bright field. The purple or red structures are the starch grains of the potato (Solanum tuberosum). This is a nice example on how the addition of a simple field-stop filter can result in drastically different images. The contrast of the images was adjusted and both images were sharpened slightly. Image stacking was not necessary.

Background Information: The starch grains of potatoes are also called amyloplasts, they are found inside the cells of the potato tuber. Starch is a polysaccaride, made of long chains of glucose molecules. The glucose was originally produced by the leaves of the potato plant. Starch can be present in the form of either amylose or amylopectin. It is not water soluble and therefore suitable for storage.

Female Pine Cone (Pinus)

Oliver Kim — Sun, 18 Jan 2009 11:57:59 +0000

Image Information: The image on the right shows the longitudinal cross-section of the female cone of a pine tree (Pinius). The specimen size is approximately 20mm from left to right, so I had to assemble several individual pictures together to produce the final image. The resulting original image is quite large (10000×7000 pixels). The bottom two pictures are enlargements of the original. Of course there was also a bit of clean-up work involved removing disturbing dust and to brighten the background.

Background Information: Most pine trees carry male and female cones (monoecious), only few carry cones of only one type (sub-dioecious). Each pine cone has scales, each scale (if fertilized) is able to carry two seeds. Some scales (those at the tip of the cone) are sterile and smaller and do not produce seeds.

I also recommend the following post: Virtual microscope: female pine cone (Pinus)

Some histology: Human Scalp (Head skin)

Oliver Kim — Sat, 17 Jan 2009 17:12:26 +0000

Microscopic image of human head skin (scalp) with hair roots. The roots are colored red.

Image Information: This time I photographed the cross-section of human head skin, a commercial permanent slide. The final image was assembled from 6 separate pictures.

Background Information: The individual hair roots / hair follicles can be seen nicely as red, elongated structures. The surface of the skin is towards the bottom. Not all of the hair seem to reach the outside. This is because the microtome cut was not absolutely parallel to the hair (the hair was cut diagonally). We only see a two-dimensional cross-section of a three dimensional structure. I do not know what the red, round, curly structure is towards the middle-left, I assume some kind of gland (either sweat or oil).

Kiwifruit Mystery

Oliver Kim — Sat, 17 Jan 2009 15:52:48 +0000

Funny round bubbles inside a kiwifruit. What are they?

Image Information: Who would have guessed, that the image shows a kiwi fruit Actinidia deliciosa? Honestly, I had problems identifying the individual cells under the microscope. I guess that this is due to the fact that I had to squeeze the fruit between the slide and the cover slip, destroying many of the cells. I could clearly see interesting bubble shaped structures, but the function is not clear (chlorophasts? They are green, after all.) I stacked several pictures, to increase the depth of filed, and I also improved the contrast. The result is an image that looks at least as refreshing as the whole fruit…..

Click on Observing a kiwi fruit to read the procedure.

Root of a Monocot

Oliver Kim — Sat, 17 Jan 2009 15:34:28 +0000

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Root cross section of a monocot plant, Zea mays, maize, corn.

Image Information: This is another panorama photomicrograph, assembled from four individual images. The image shows a cross-section of Zea mays, maize, a monocotyledonous plant (“monocot”).

Background Information: The picture shows epidermis (the outside layer of cells), endodermis (inside ring-shaped layer of smaller cells) and vascular tissue, the larger cells inside the endodermis, which carries water and nutrients. The water of the soil has to reach the vascular tissue towards the center of the root and is forced to go through the endodermis, which acts as a “filter”.

Hydra, a fresh-water polyp

Oliver Kim — Sat, 17 Jan 2009 15:21:04 +0000

Microscopic image of a fresh-water polyp, Hydra sp. 27 individual images were stacked together to produce one final sharp image.

Image Information: This image nearly crashed my computer – it is a stack of 27 separate images to increase the depth of field. The computer worked nearly an hour on this picture. Without stacking, some of the tentacles would not be in focus. I think that less pictures would have given a similar result. The picture on the right shows the fresh-water polyp Hydra sp. The specimen is about 5mm in length, the picture shows about half of the organism.

Background Information: The Hydra belongs to the taxon Cnidaria and is a relative of the sea anemones, corals and jelly fish. Its tentacles are used to catch food. It is sessile, this means that it is attached to a solid surface and does not move. The mouth of the hydra is located towards the left of the image, where the tentacles attach to the body.

Head of a Fly

Oliver Kim — Sat, 17 Jan 2009 13:06:20 +0000

Microscoic image of the head of a fly, several pictures combined to one.

Image Information: Today I started to go through several permanent slides which I borrowed – and the result can be seen on the right. This is the head of Musca domestica, the common house-fly. I took seven overlapping pictures and assembled them with a stitching software (which are commonly used for making panoramas). The pictures were stitched in two dimensions, not every panorama software is able to do this. Many are only able to stitch the images horizontally only. Naturally I also had to do some cleanup work in Photoshop, removing the dirt and dust of the background. The specimen was sufficiently thin, so I did not have to combine the different focus levels into one final sharp image.

Background Information: The house fly is one of the most widely distributed insects and can be a vector for the transmission of a range of different illnesses. An adult female lays about 500 eggs during its lifetime. Maggots hatch within one day. They will feed on decaying organic matter and grow into a fully sized larva. Pupa will form and after metamorphosis the adult flies will emerge. Adult flies are 6-9mm long, and will mate already 36 hours afterwards to complete the life-cycle.

Mystery Object in Dust (Anthrenus sp.)

Oliver Kim — Sun, 04 Jan 2009 09:11:07 +0000

Image Information: The image shows a strange looking object which in the dust of my apartment. What could it be? One thing should be clear: it’s biological origin, otherwise I can not explain its regular structure. The bottom image is a stack of several individual images to increase the depth of field. I also performed a color adjustment to increase the contrast. The round spherical structure above the date is an air bubble.

Background Information: In order to find out more about the structure, I posted a comment on a German microscopy forum, and I obtained some interesting responses. Apparently it is a bristle of an insect larva of Anthrenus sp., according to one member of the (forum). And indeed, another check of the dust turned up an empty exoskeleton of an insect, with a large number of bristles. The same structure was also seen by another microscopist here. Anthrenus sp. is also known as a “carpet beetle”, and is known to eat textile material. Not a good thing to have it around in a household.