The video shows the crystallization of Vitamin C (dissolved in water) in time lapse, under crossed polarizing filters.

Some technical information:

• The frames in the video were taken with a time interval of 2 sec.
• The colors were not adjusted at all. No contrast enhancement, no levels were corrected, no white balance.
• The pictures were taken with a digital SLR camera at low magnification (4x objective and 2.5x photo projection ocular).
• The original 3:2 aspect ratio image was cropped to 16:9. Software used: PHATCH
• Exposure time: 0.5 sec or 1 sec. The relatively long exposure time was used to compensate for vibrations (this allowed the vibrations to swing out).

Bright-field microscopy is useful for specimens, which possess a sufficiently high natural color contrast with the background, or for specimens that can easily be stained by dyes. Now, it is possible to increase the contrast by closing the condenser aperture diaphragm. This, however, results in a reduction of the resolution and introduces diffraction artifacts. The natural colors also become less visible, as the whole image darkens. To overcome these limitations of bright-field microscopy, different optical contrasting techniques were invented.

• Dark Field Microscopy: This is one of the easiest and cheapest contrast-enhancing techniques. The main light beam is not able to reach the objective (and therefore the eye), resulting in a black background image. Light is capable of striking the specimen, however. This light is then scattered into various directions, and is also picked up by the objective. The specimen will appear bright on a dark background. Dark-field illumination can be achieved in two ways. Either a specialized dark-field condenser is used, or a so-called patch-stop filter is inserted into the filter holder of the condenser. The patch-stop possesses a central black area which blocks the main light of the illumination system. The patch-stop may not result in a satisfactory image quality for all magnifications, it is advised to experiment with the size of the central black area. For more information: Darkfield Microscopy.
• Rheinberg Illumination: This contrast enhancing technique is closely related to the dark-field method. In this case the patch-stop filter is modified in such a way that the central black area is replaced with a strongly colored, transparent film. The color of the central area of the filter represents the background color of the microscopic image. The peripheral area of the filter possesses a different color. Specimens will then possess the color of the peripheral area. These filters can be easily made by printing the filter using a color printer on an overhead transparency.
• Phase contrast microscopy: This system was invented by Frits Zernike (who received the Nobel Prize for this invention in 1953). Transparent, colorless objects can differ from their surrounding medium (for example water, or the mounting medium) in that they possess a different refractive index. Using bright field microscopy alone, these objects would nearly be invisible. The phase contrast optics of a microscope is able to convert the differences in the refractive index into a difference in brightness. Depending on the system used, the specimens will either appear bright on a dark background, or dark on a bright background. Phase contrast microscopes need special phase contrast objectives and a dedicated phase contrast condenser. In many cases, the phase contrast objectives can also be used for regular bright-field work, with a slight decrease in image quality. Phase contrast microscopy is commonly used for the observation of bacteria, which are otherwise difficult to see.
• Nomarski Differential Interference Contrast (DIC): The theoretical background of this method is complex. The light of the microscope is split up into two beams by a specialized prism which is located beneath the condenser. One beam passes through the specimen, the other beam does not. The two beams therefore have to pass through different refractive indexes and are then allowed to interfere with each other. The result is an image which gives the impression of being three-dimensional. A cell, for example, will appear to be illuminated from the side, with one corner darker than the other. The individual cell organelles will appear to stand out (or be depressed). The 3-dimensional appearance is an illusion, formed by the shadows and highlights. The formed image is similar to oblique illumination.
• Polarization: This contrast enhancing method is commonly used when viewing bifringent speciems, such as starch grains, crystals and cellulose. The light from the illumination system passes through a polarizing filter and then through the bifringent specimen. These specimens are able to interact with the light in such a way, that the light is split into two components. This light continues, and passes through a second polarizing filter, where it is allowed to interfere. The specimens will appear as bright, colorful objects on a dark background. The colors can change when the filters are rotated. Dedicated polarizing microscopes possess a rotating stage and tension-free objective lenses. Possible tension in glass modifies the plane of the polarized light.
• Fluorescence: Certain specimens, such as chloroplasts or cell walls of plant cells, have the tendency to glow in a visible color when flooded with ultraviolet (UV) light. It is also possible to selectively stain the different parts of a cell with flurochomes (fluorescing stains) to visualize them. The UV light can either be passed through the specimen either from the bottom or from the top (“epi-illumination”). It is recommended to use fluorite objectives, otherwise the glass elements, the lenses, will start to glow as well.
• Oblique Illumination: In this method, the illumination system of the microscope is placed-off center. The light strikes the specimen from the side. The specimens appear darker on one side compared to the other side. It is also possible to use a patch stop filter which allows light to pass through only one side. The effect is, that the specimen seems to create a shadow and appears three-dimensional. See Oblique Illumination for sample images.
• Using Color Filters: Color filters absorb the complimentary color. A red filter will result in green chloroplasts to appear dark. A blue “daylight” filter is commonly used as well. It will absorb the red parts of the spectrum and will enhance the contrast of objects that possess a red color. The blue filter will also increase the resolution, as it allows only the passage of the shorter wavelengths.

When the polarizing filters are turned into a crossed position, then they will not allow light to go through. This is the position used for microscopy.

When the polarizing filters are turned into a parallel position, then they will allow light to go through.

Place one polarizing filter on top of the light source, and the other one on top of the specimen.

Polarization microscopy of crystals is an aesthetically rewarding experience. Obtain two linear polarizing filters. Make sure that the two filters will not let light go through if crossed. Many polarizing filters sold in photography stores are circular polarizing and they will not work. It is best to test the filters first, or to buy polarizing filters from a school supplies company.

Place one filter on top of the light source and the other filter on top of the specimen, beneath the objective. Then rotate the filter of the light source into a crossed position. Be careful – The filter changes the focal distance and focus. Be careful of not smashing the objective into the filter when refocusing. For safety, only use this system with the low power objectives.

There are a wide range of different samples that can be viewed under polarized light:

• Various crystals
• Potato starch grains
• House dust: many components of dust are de-polarizing the light and these components will appear bright on dark background, similar to dark-field illumination.
• Transparent materials (plastics) that contain tensions. The tensions turn the plane of polarization of light and will result in colorful images.

It is possible to purchase dedicated polarization optics. These optics are tension free and will deliver a completely dark image when used with crossed polarization filters. Regular achromatic bright field objectives (as commonly used in schools) are not tension free and there may be a slight background illumination even when the filters are completely crossed. For practical purposes, this is of no relevance.

Vitamin C (ascorbic acid) in polarized light.

Vitamin C (ascorbic acid) in polarized light.

Citric acid (citrate) in polarized light.

Materials: Any one of these substances: Vitamin C, Salycilic acid (the active component in Aspririn), citric acid, tartaric acid or table salt, destilled water, pure alcohol, a set of liniar polarizing filters

Method: for Vitamin C, tartarc acid, table salt

1. Dissolve A knife-tip of the substance in a few milliliters of destilled water. Shake until all of the substance is dissolved
2. Evenly spread the solution on a clean slide
3. Place the slide on a warm, but not hot plate or rest the slide for several hours (or over night) for the water to evaporate. It is possible to follow the formation of the crystals.

Troubleshooting Vitamin C

Problem: As the water evaporates, it starts to retract and does not form a nice thin layer of crystals on the slide. The crystals are thick and dense.
Solution: This is due to the surface tension of the water. Carefully spread the water all the way to the corners of the slide. The water should actually contact all the corners. The water is then not capable of retracting as it evaporates. The addition of surface tension reducing substnces (such as soap) may impair the crystalization process.

Method for Aspirin

1. Dissolve A knife-tip of the substance in a few milliliters of pure alcohol. Shake until all of the substance is dissolved.
2. Evenly spread the solution on a clean slide
3. Place the slide horizontally on a table and wait until the alcohol has evaporated. This should only take a few minutes. Crystal formation can be observed.
4. Careful: do not ingest! Only use very small amounts.

Method for Citric acid

1. Place a few grains of crystal on a microscopic slide and put a cover slip on top.
2. Carefully place the slide on a hot plate. The crystal will melt and will spread between the slide and the cover-slip. Do not overheat. The substance should not start to smoke or change its color.
3. Remove the slide and rest over night. Crystal formation can take several hours.
4. Alternatively, the melt can be spread out over the slide without a cover slip. Crystal formation can then be easily initiated by carefully touching the (cold) melt. Dust, crystal parts still sticking on the the fingers will initiate the crystalization.

Troubleshooting Citric Acid
Problem: Crystals do not form
Solution: This is quite possible, but not the rule. If both slide and cover slip are too clean, then there is not place for crystal formation to start. Use more citric acid the next time, so that some of the substance flows out beneath the cover slip. This can then be a place to initiate crystal formation by carefully scratching the substance with a sharp object. It could also be that the citric acid was overheated.

Problem: There are many bubbles between cover slip and slide
Solution: Bubbles are difficult to avoid completely, and in many cases they make the specimen more interesting (and beautiful) to observe. It is possible to melt the crystals first, and then press the cover slip on top of the melt. This also reduces the bubbles.