Education in Microscopy and Digital Imaging

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• Introduction
• Dirt and Debris
• Optical Surfaces
• Cleaning Agents
• Immersion Oil
• Avoid Problems
• Print Version

Introduction

Microscopes often represent a significant investment of funds and are sophisticated optical instruments that require periodic maintenance and cleaning to guarantee successful microscopy and perfect images. When neglected, exposure to dust, lint, pollen, dirt, and failure to remove immersion oil in a timely manner can seriously damage optical performance. Over the years, a variety of cleaning procedures have been recommended, but many users remain unsure as to which of these will yield the best results. The choice of the best cleaning method depends on the nature of the optical surface and the type of substance to be removed.

A microscope that remains unused for a long period of time can accumulate dust and debris from the air, which can lead to deterioration of image quality even though the instrument may be practically new. Even when carefully covered for protection during periods of inactivity, however, microscopes that are used regularly are subject to build up of contaminants. Some of these are unavoidably introduced from the environment and others by the microscopists themselves, especially in areas where the hands, eyelashes, and even moisture from breathing contact the instrument over time. The critical areas include:

Some optical surfaces are more sensitive to dirt than others. The front lens of the objective is particularly significant, as when the optical elements at or near the conjugate field (image) planes are dirty, damaged, or defective, artifacts are likely to appear in sharp focus superimposed on the specimen image (see Figure 2). Ironically, the higher the quality of optical components, such as the condenser and collecting and relay lenses, the more these blemishes interfere and contribute to optical noise. Before every critical use, check the front lens of the objective for dirt.

For any dry objective, the smaller the free working distance and the smaller the surface area of the concave front lens, the greater the danger of soiling the front lens with embedding media, immersion liquids, or dust. The front lens of an immersion objective should be cleaned to remove residue both after use and before applying fresh immersion liquid. The mixing of different immersion media, as well as different lots of the same medium can result in blurred images. Though the utilization of immersion oil is essential in maximizing microscope optical performance, its improper use or the failure to immediately remove the oil after each use constitutes the most serious contaminant that must be dealt with in instrument maintenance, and thus will be discussed in further detail in a later section.

Microscopes that are equipped with digital cameras may develop a degradation in captured image quality or exhibit image artifacts caused by accumulation of contamination either on filter elements that are sometimes utilized in the camera adapter or on the optical glass window that may be incorporated to seal the camera housing and protect the charge coupled device (CCD) or complementary metal oxide semiconductor (CMOS) image sensor. In practice, if dark specks or similar in-focus artifacts are observed in digital images, and they are not in the specimen plane, their most likely cause is particulate contamination on the image sensor or an associated filter surface. The cameras always need to be handled with the utmost care and protected from dirt according to the manufacturer's recommendations.

back to top ^Dirt and Debris

Familiarity with the best possible results obtainable with a specific technique and procedure permits a user to recognize the consequences of dirty optics. The ability to compare the expected image and the image actually obtained with respect to optimal sharpness, contrast, and the absence of contamination-dependent visual artifacts allows the user to immediately recognize when the microscope is dirty or not. If the image sharpness or contrast is not optimal, then there is a high probability that the microscope optics are not clean. In order to determine the location of the dirt, proceed as follows:

The affected optical surface is identified when a suspected optical component is moved and the dirt follows this movement. The single exception to this rule is the camera. Dirt located within the camera (usually on the optical window protecting the image sensor) will not move when the camera is moved. A macroscopic check for larger dust particles and scratches on optical surfaces can be carried out using either a magnifying glass (with a magnification of 3x to 6x) or an eyepiece viewed through the reverse aperture (in effect, peering through the opposite end of an eyepiece). A soiled objective front lens is easily identified by examining an evenly lit surface through the rear aperture of the objective. The internal lens organization produces an enlarged image of the smallest contaminants present on the external surface of the front lens. The final check should always involve an assessment of the improvement in image quality.

It is crucial to differentiate between dust (for example, glass dust from slides, flakes from skin of the microscopist, fibers from clothing, pollen from spring and summer flowering) and other dirt (liquid or dried-out embedding or immersion media (see Figure 3), culture solutions, residue from improper cleaning attempts, fingerprints and grease). Dust particles can either rest loosely or can be more or less stuck to optical surfaces. Other dirt is either water-soluble or may only be completely removed using organic solvents. However, a blurred image may not always be due to dirt. Using an objective with a large numerical aperture in conjunction with a cover slip of the wrong thickness can result in blurred images (spherical aberration). Dry objectives of high numerical aperture normally have a correction collar, which permits compensation for the spherical aberration. Many highly corrected immersion objectives also require specially selected cover slips with a thickness of 170 micrometers, if the best image is required.

Immersion objectives should only be used in conjunction with a suitable, bubble-free immersion oil. Although the viscosity of immersion oil minimizes any immediate migration into unintended locations, if it is not removed promptly and is allowed to accumulate, the effects of gravity and capillary forces will ultimately result in the oil moving into parts of the substage mechanism and microscope stand, and perhaps even into the objective. This accumulation may not be readily visible, and can go unnoticed until mechanical or optical problems become severe enough to require service by a microscope repair facility. The occasional recommended use of the aromatic amine, anisole, as an immersion medium results in loss of sharpness and contrast. Anisole can attack the cement of front lenses, especially that of older objectives, and should be avoided.

back to top ^Optical Surfaces

Concave or convex surfaces (for example, the front lens of dry objectives and condensers or the eye-lens of some eyepieces) should be distinguished from planar-parallel or flat surfaces like the front lens of most of the immersion objectives, as well as condensers, filters, and the protective glass covering camera sensors or the opening through which light exits. Concave or convex surfaces are cleaned using either the cotton or the new polyester swabs as described in the next section. Easily accessible flat surfaces may be similarly cleaned with soft disposable cellulose wipes. Microscope optics can be composed either of optical glass, quartz, or polymers. The upper surface of almost all components will be coated with an antireflection layer to minimize stray light. Some of these coatings may be wipeable or not, due to their softness. Generally, antireflection coatings are composed of magnesium fluoride and should only be cleaned with agents free from ammonia and acid. Several alternatives to hazardous solvents have been found to be effective in microscope cleaning, and a variety of cleaning agents, as well as cleaning materials, are recommended by different microscopists and manufacturers. Some optical components are surrounded by black antireflection surfaces, which are sensitive to organic solvents. The plastic and rubber parts of the eyepiece will likewise be attacked by organic solvents, including acetone, methylene chloride, and chloroform.

One of the most significant dangers with many of the solvents proven effective for cleaning microscope optics is that they have the potential to dissolve the cements utilized in lens assembly (as do the immersion oils themselves if allowed to remain on the optics). In older microscopes, lenses were cemented using an alcohol-soluble cement such as Canada balsam. Benzene was regarded as a highly effective lens cleaning solvent at the time, but always required great caution to limit contact with the lens for no more than a second or two, due to the high solubility in benzene of balsam and some other cements used for lens mounting (and for mounting coverslips on specimen slides). It is known that benzene is readily absorbed through the skin, (this as well as inhalation of the vapors can cause liver damage) and the most common lens cements are generally composed of synthetic polyacrylic resins, which are not alcohol soluble. Xylene has been widely utilized to clean optical surfaces for years, and though it is highly flammable, toxic, carcinogenic, and may cause skin contact sensitivity, xylene is still less aggressive than benzene. Both solvents are harmful to the mechanical and optical components of many microscopes even though they are recommended as lens cleaning solvents.

The proprietary solvents Histolene and Histoclear are gaining popularity as replacements for xylene in microscopy laboratories, and have been found to be effective for cleaning other instruments as well. Illustrated in Figure 4 are several commercial products designed for cleaning microscopes. Pure distilled water is the safest cleaning fluid for any contamination that is water soluble, but if that is inadequate, commercial photographic lens cleaning liquids are very effective and are safe for precision optics when used sparingly. Sometimes household glass cleaning agents, which contain dilute ammonia, are recommended but they should not be used routinely. The internal workings of the microscope, including the optical surfaces, components of the fluorescent filter sets, cameras, and camera adapters should never be cleaned by the user, but by experienced customer service representatives from the original manufacturer. The user should only clean the external surface of the objective front lens, the condenser front lens, the eyepiece eyelens, glass color and conversion filters, and the external surface of protective glass covering any opening through which light exits.

back to top ^Cleaning Agents and Procedures

The goal in cleaning microscope optical surfaces is to completely remove dust and dirt without leaving any residue of the cleaning agent or damaging the surfaces. The following equipment is required:

For the easy cleaning of flat surfaces, such as the removal of immersion media from cover slips or the removal of immersion media from the front lenses of immersion objectives, soft tissue soaked in diluted dishwashing liquid is suitable. The smooth lens paper (so-called Joseph paper), which is often readily available in research laboratories, is not for cleaning but is intended only for the dust-free storage and protection of optical components. For cleaning purposes, this lens paper is too harsh; it also doesn't absorb the dirt effectively or quickly enough. High purity cotton cleaning tissue represents the single exception. For cleaning of all other optical surfaces, either freshly made cotton swabs, lens tissue, or polyester swabs are used.

Preparation of Cotton Swabs

Cotton swabs that are untreated are appropriate for microscope cleaning, and are commercially available, or may be assembled according to the directions below. Cotton wool, sold by pharmacies in rolls, typically contains some proportion of synthetic fibers, and is not as suitable for delicate surfaces as is 100 percent cotton wool.

Cleaning Procedure

Cleaning is achieved using a spiral motion from the center to the rim (see Figure 5 for details about cleaning procedures). Never wipe using zigzag movements as this will only spread the dirt. Direct pressure from the fingers should never be applied to the glass lens surface in order to minimize the possibility of scratching the lens if any particulates are present on the tissue. With larger optical surfaces such as tube lenses, the spiral motion starts initially at the rim before moving to the middle and is only then followed by a center to rim cleaning motion. In most cases, several wipes are recommended, using a fresh tip each time.

Not all solutions can be used to clean the optics of a microscope. Some clean very effectively, but are either toxic (chloroform and acetone) or environmentally unfriendly (carbon tetrachloride), while others will leave surface residue (xylene, toluene, and diethylether). Residue forms quite easily with the use of xylene and absolute ethanol, as well as when the dirt contains water-soluble components. Acetone is recommended when oil and grease must be removed from cover slips. Acetone attacks most types of plastic as well as rubber, and as a consequence, its use to clean eyepieces can be problematic. In addition, acetone may attack cemented optical components such as objectives, camera adapter lenses, or eyepieces when used frequently. Acetone can also dissolve specialized organic coatings.

The painted external surfaces of modern microscopes are powder-coated and extremely durable. They can be cleaned well with a very lightly moistened microfiber cloth. Loose dust and other dirt can be removed using a brush of soft hair used exclusively for this purpose. Following complete cleaning of the mechanical components and carefully wiping up any liquid spills in the vicinity of the instrument, a small vacuum cleaner with a flexible hose and soft brush attachment can be employed to remove any loose material on the stand and table area around it. Extreme care should be taken to avoid touching any optical surfaces with the vacuum brush.

back to top ^Proper Use and Removal of Immersion Oil

Adhering to the following procedures in the use of immersion oil will significantly ease the task of removing the oil from microscope components before it causes damage. It is important to recognize that immersion oils are not inert with respect to either optical or mechanical microscope components, and if left in contact with the instrument, oil will penetrate into gears and sliding mechanisms and into crevices between lens elements and their mounting structures with the potential to cause irreversible damage.

The full utilization of the microscope optical system numerical aperture when immersion objectives are used requires a double oiling technique in which a single drop of immersion oil is applied to the top lens surface of the substage condenser and another single drop on the top of the specimen slide. Only a single drop of oil at each specimen-optical interface can be accommodated without producing contamination that may be impossible to remove without complete disassembly or factory servicing of the instrument. The condenser is then raised just to the point that the oil drop contacts the lower surface of the slide, and the objective front lens is brought into contact with the oil drop on top of the slide. It should be stressed that the oil immersion technique is only to be used with a condenser equipped with an immersion-type top lens, and with immersion objectives. Any attempt to improve the performance of a dry objective by application of immersion oil will likely result in its destruction, as such objectives are optimized optically for use in air, and are not sealed against the intrusion of fluids into the lens barrel.

After each specimen has been examined, the immersion oil should be completely removed, even if additional slides are going to be observed. Immersion oil is most safely removed using only lens tissue, without employing any solvents. Lens cleaning paper that is designed specifically for use on high quality optics must be employed, and it should be stored in a covered container to prevent contamination with airborne particulates. A folded piece of lens tissue (held under light tension with two hands) is drawn across the objective front lens to absorb the oil, and repeated with a new area of the tissue. This gentle wiping of the lens surface should be repeated with as many tissues as required until no oil streaks are seen on the tissue, and each tissue should be discarded immediately to avoid inadvertently re-using contaminated tissues on the objective. Using plenty of fresh lens tissues is essential to the success of this procedure. The natural tendency to minimize waste is misdirected, considering the relative cost of lens tissue compared to the potential of damaging an expensive objective.

When working with inverted (tissue culture) microscopes, the front lens of every objective will be more exposed to dust than that in an upright microscope; all long working distance dry objectives with magnifications between 10x and 100x need to be regularly checked. Inverted microscopes present special problems with regard to the use of oil-immersion objectives because spilled or migrating oil can very easily intrude into the interior of the objective at the juncture between the body and the telescoping spring-mounted front lens barrel. If oil is allowed to accumulate, it can conceivably flow, under the force of gravity, into the objective turret or nosepiece.

When there is no residual trace of immersion oil apparent on the final tissue paper, another tissue should be used to wipe the lens with moisture from the breath. As previously mentioned, one must not blow through closed lips onto the lens, but should breath gently on it with the mouth open so that no saliva droplets are expelled. This procedure should be repeated several times with a new tissue each time. Following cleaning of the optics, immersion oil should be cleaned from both surfaces of the specimen slide using laboratory tissues. All areas on the instrument, such as portions of the base or stand, should be routinely checked for any traces of immersion oil, which if found, may be removed with laboratory towels or soft cotton cloth.

Practical Considerations for Cleaning Microscope Optics

back to top ^How to Avoid Contamination

The opening of the binocular tubes should always be protected either with the eyepieces or with dust covers. If no dust covers from the manufacturer are available, aluminum foil is a suitable substitute. The best fundamental method to prevent dust accumulation is to first cover the microscope with two additional plastic bags and then the dust cover supplied by the manufacturer. The microscope should never be located in a position where it could be affected by acidic or alkaline vapors, such as in or near a wet chemical photographic laboratory.

In tropical regions, microscopes frequently experience a buildup of fungus. Although there are over 100,000 fungus species, two members of the genus Aspergillus are believed responsible for most lens deterioration. Optimum growth conditions for these fungi are relatively high temperature and high humidity, but they are more adaptable to lower humidity levels than most other fungi. Fungal contamination can best be minimized by reducing the humidity of the room either by air conditioning or by installing an infrared lamp above the microscope (at a minimum distance of 150 centimeters or 5 feet). Fungi growing on glass surfaces are not attached by roots and can be wiped off, but unfortunately, residual corrosion marks remain and the original lens performance cannot be recovered and the lens must be replaced. The only effective means to avoid fungal damage to optical components is to prevent its growth in the first place.

Conclusions

The ideal microscopy room would be designed specifically for that purpose, and incorporate every mechanism available for limiting contamination by dust, chemical vapors, and other airborne contaminants, as well as isolating the instrument from acoustic and mechanical vibration and temperature variations. This ideal situation is seldom realized, and most microscopes are located in areas subject to a considerable number of environmental deficiencies. Some contamination is unavoidable, due to the rigors of daily use, but at the very least, the microscope should be protected as well as possible during periods of non-use by covering the entire instrument with a suitable cover. Instrument manufacturers and aftermarket suppliers offer a variety of specially designed dust covers. Of the several types of plastic covers available, those made of softer more flexible material are probably less prone to attraction of dust. Lint-free fabric covers are also available, and provide an effective dust barrier that can minimize the need for cleaning.

While the cost of a modern research grade microscope can range from approximately a few tens of thousands to several hundred thousand dollars, if properly used and maintained, the basic optical and mechanical components of the instrument can easily outlive several generations of microscopists. Only if the instrument is used correctly and maintained regularly is it capable of producing the best image data possible. Careless, incorrect operation and maintenance techniques not only result in unreliable and poor quality images, but cause productivity to suffer and the instrument's useful lifetime to be greatly reduced.

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