Transmitted Light Microscopes Maintenance, Cleaning and Setup
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Maintenance
A microscope in use, or one 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. It is important to keep the microscope covered when not in use.
The crucial areas of the microscope are:
The external surface of the front lens of the objective
The surface of the camera sensor and its protective glass cover
Both surfaces of the cover slip
The surface of the microscope slide
The surface of the camera adapter optics
Surface of the upper lens of the condenser
The outer and inner surfaces of the eyepiece as well as the upper surface of the reticule
The outer surface of any protective glass covering openings through which light exits
Other glass surfaces in the light path such as halogen lamps, high pressure arc-discharge lamps, fluorescence filters and beam splitters, collector lenses, or contrast and heat filters
Cleaning
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:
Carefully rotate objectives and cameras a small amount within their thread.
Check the slide and cover slip by moving the specimen while focusing initially on the upper and then the lower surfaces.
Check the condenser while moving it up and down and if applicable, by swiveling the front lens slightly.
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, residue from improper cleaning attempts, fingerprints and grease).
Optical Surfaces
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:
Long, thin wooden sticks
High purity cotton (the Siem Medic should be able to spare you some)
Absorbent polyester swabs for cleaning optical components
Soft cosmetic cellulose tissue specifically designed for optical surfaces
Dust blower or ear wash bulb
Distilled water
Solvent for the removal of greasy or oily dirt, such as the Optical Cleaning Solution
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. 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. 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.
Preparation of Cotton Swabs
Wash hands (powdered, latex gloves are not suitable).
Dip the stick into the cleaning solution (aqueous or organic solvent). The cotton fibers attach better to the stick as a result.
Dab the stick onto the wad of 100 percent cotton and loosen some fibers. Do not compact the cotton otherwise the fibers will not separate easily.
Turn the stick so that an even, elliptical cotton bud forms at the tip.
To protect the cotton tip from dirt, the stick should be stored in a plastic sandwich bag. It should not be handled as perspiration and grease (from fingers) will significantly affect its ability to clean.
Remove the cotton tip after every wipe and replace it with a fresh cotton bud.
The stick can be used for a long period of time. Use separate sticks for water-based solutions and organic solvents.
If the use of a high purity cotton lens cleaning tissue is preferred, fold the sheet around the stick so that a sharp point is generated. The point should not be handled. Use the tissue only once and then replace it. Polyester swabs can be used until they no longer clean well.
Cleaning Procedure
Place the objectives, eyepieces, and cameras on a dust-free surface (fresh aluminum foil, for example). Before any cleaning is attempted, the optical surface should be inspected with magnification under reflected light to determine the condition of the component. Particular attention should be given to the presence of any particulate material, which must be assumed to be abrasive, and removed before any other cleaning is done. A magnifying lens of 2x to 3x (such as a loupe) is appropriate for examining larger optics such as oculars and condensers, while the smaller lens elements of objectives require approximately 5x to 10x magnification for proper inspection. It is crucial that particulate matter be removed from a lens surface as the first step in cleaning, because any particle can be abrasive and result in scratches if it is brushed across the surface with even the most gentle lens tissue. All other optical components to be cleaned should be as accessible as possible.
Blow all loose dust particles away with a compressed gas duster, air blower, or a clean camelhair brush. Never blow dust off lens surfaces with a strong breath, and make sure to gently blow air across (not perpendicular to) the lens surface. The safest method of air dusting is to use a rubber bulb or balloon, such as models intended for use as ear and enema syringes for infants. Care is required to avoid touching the tip of the syringe to the lens surface. The best advice is to avoid any use of compressed air cans for lens cleaning. It is difficult with these to control the pressure of air impinging on the surface being cleaned, and there is always the risk of either extremely cold air or freezing liquid being expelled onto a lens surface and causing irreparable damage.
Remove all water-soluble dirt with distilled water. If this step is unsuccessful, repeat using a solution of diluted washing-up liquid. Remove any remaining residue with a dry cotton swab, but breathe with an open mouth on the surface first to generate a film of moisture. In so doing, be careful to not spray droplets of saliva. An effective method of preparing lens paper for this cleaning method is to fold all four corners of a piece of tissue together, leaving the untouched center of the tissue bulging out. The corners can be twisted together slightly to form a stem for handling the tissue. When the tissue is held by this stem, and wiping performed with the puffed-out tissue center, the force that can be applied to the objective is limited by the springiness of the tissue. Circular wiping motion can be applied in this manner, with very little direct force on the lens surface.
To remove oily dirt, use a solution of dilute washing-up liquid initially. If this action does not produce a satisfactory result, repeat the cleaning using a solvent (Optical Cleaning Solution L or petroleum ether). Greasy dirt must always be removed using a solvent. Dip the cotton or polyester swab into the cleaning solution and shake off excess liquid. An excess of liquid in a cotton bud will flow over the rim of the lens and attack the lens cement. This may consequently lead to the removal of the cement between bonded components. The solvent should remove as much dirt as possible. In order to increase the retention time of volatile organic solvents in the cotton bud, some users chill the solvent (-10 degrees Celsius to -20 degrees Celsius). Chilled solvents have a disadvantage. Due to their low temperature, condensation may form on the lens surface and leave a residue. A more suitable method to improve the retention time of a solvent is to add isopropanol.
Cleaning is achieved using a spiral motion from the center to the rim . 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.
Objectives
Catalog: Objectives & Reticles
Microscope objectives are perhaps the most important components of an optical microscope because they are responsible for primary image formation and play a central role in determining the quality of images that the microscope is capable of producing. Objectives are also instrumental in determining the magnification of a particular specimen and the resolution under which fine specimen detail can be observed and recorded using the microscope.
Objectives are generally classified as achromats, fluorites, and apochromats, with a plan designation added to lenses with low curvature of field. Furthermore, objectives can be specifically classified into transmitted light and reflected light versions.
Most of our high-performance objectives feature spring mounts to protect the specimen, and many of the immersion objectives have nosepieces that can be snapped into the top position of their spring mount to enable the easy application of immersion fluids.
At the start of the expedition, ask your scientist what objectives they want placed on their scopes. There is no need to change out objectives at the end of the expedition. Need to add general microscope objective set up for different fossil groups/core describers/thin-section/microbiol.
Achromats are produced in versions designed for polarized light and phase contrast, but not fluorescence or differential interference contrast (DIC). Plan and epi-plan objectives are improved achromat versions with excellent flatness of field up to diameters of 24 millimeters or more. In addition, reflected light achromat objectives exhibit excellent contrast and a variety of working distances. The specifications required to identify objectives (see Figure above) are usually inscribed on the decorative barrel protecting the internal lens elements. Apochromatic objectives usually contain two lens doublets and a lens triplet for advanced correction of both chromatic and spherical aberrations. With apochromat and fluorite objectives, the diffraction-inducing spreading of the intensity distribution can be virtually eliminated. An achromat objective still has substantial intensity in the first fringe, while the apochromat approaches the theoretical resolution limit where the longitudinal chromatic aberration is greater than the wave-optical depth of field. Need to add more information about each objective type and how and when to use.
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.
Alignment
You will need to regularly check the microscope's alignment with six steps:
Lamp Alignment
Zeroing Eyepieces
Köhler Illumination
Centering Stage Rotation and objectives
Centering Microscope Cameras
Lamp Alignment
To ensure even illumination, it is important to periodically check the alignment of the transmitted light bulb in your microscope. This is especially true after the bulb has been replaced. To align, turn the brightness knob down to a fairly low setting, then remove the frosted glass filter from the light path. Place a piece of lens paper over the field diaphragm to see the image of the filament. Use the adjustment knobs on the lamp to center the image of the filament.
Eye pieces
The eyepieces should be adjusted for each uses, where the distance between the eyepieces (the interpupillary distance) can be adjusted so that a user can see the image with both eyes without difficulty. Once you have finished maintenance, you should zero the eyepieces. If you wear your glasses to use the microscope, the diopter setting should be at 0 since your vision is already corrected. To adjust, try gently pushing together or pulling apart the eyepieces to decrease/increase the distance between them.
Figure: zeroing the eye piece
Köhler Illumination
Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and good alignment results in greater resolving power and light transmittance. There are two aperture diaphragms: the condenser and the field stop diaphragm. Opening and closing of the condenser aperture diaphragm controls the angle of the light cone reaching the specimen.
Switch on the light source
Peer into the microscope and bring your slide into focus as sharply as possible. The instrument is ready to be configured for Köhler illumination.
Narrow the size of the field diaphragm. Note that the condenser might not yet be centered, so only one edge of the view-field will contain elements of the field diaphragm leaves. Open the diaphragm a bit if the illumination is very miss-aligned.
Translate the condenser up and down via the adjustment knob until you see a sharp image of the edges from field diaphragm.
t this stage, the field diaphragm is in sharp focus, but the condenser is not yet centered. The centering knobs on the microscope condenser mounting frame are used to center the condenser.
Once the condenser is centered, open the field diaphragm until its leaves move completely out of the viewfield.
The only remaining task is to optimize the contrast. The condenser aperture size must now be corrected in order to improve contrast and to seek a suitable balance between contrast and resolution.
Note, opening the condenser diaphragm too wide increases resolution, however, this also decreases contrast and the image tends to “wash out”. Closing the condenser diaphragm too much decreases resolution and increases contrast to the point that diffraction artifacts start to appear in the image (light halos next to edges, scratches and dirt in the light path, etc).
Centering Stage Rotation
The 360-degree circular rotating stages should be centered to the 10x objective so specimens will remain in the exact center of the viewfield when they are being rotated. A majority of the circular microscope stages pivot on three adjustable pins, one at the front of the stage for correct positioning and the other two equally spaced at 120-degree angles near the rear of the stage.
The stage can be centered with minute adjustments to the rear pivot pins by turning the centering screws.
Start by locating a small circular feature on a relatively simple brightfield specimen in an area devoid of other distracting structures.
Next, relocate the chosen feature to the center of the viewfield in the eyepiece crosshairs
Rotate the stage through 180 degrees until the centered specimen feature is overlapped by one of the vertical eyepiece crosshairs
Using the pivot screws, relocate the specimen feature to a distance approximately half way back to the crosshair center
Finally, move the specimen feature back to the center of the crosshairs and repeat the alignment sequence. After a few cycles, the specimen feature should be centered in the viewfield as the stage is rotated.
After the stage has been centered with respect to the microscope optical axis with the 10x objective installed in the optical train, the other objectives (4x, 20x, 40x, and 100x) should be sequentially centered to the stage. Microscopes having centerable objectives contain a pair of Allen (or a similar drive) setscrews in the nosepiece that translate the objective laterally within its seat. Each objective can be centered using the procedure described above for the circular stage by using the objective centering tools provided by the manufacturer. Accurate centering of the stage and all objectives is paramount to quantitative analysis in polarized light, and avoids the operator losing sight of specific features in the viewfield during stage rotation. After completing the centering procedure, review the steps to ensure all of the objectives on the microscope are accurately centered before continuing.
Microscope Cameras
Lastly, check the alignment and scale bar of the microscope's camera. Your camera attaches to your microscope via a part called the camera mount (c-mount). Make sure that the camera is securely attached to the c-mount.
Verify that the camera is oriented in the proper direction: What appears through the eyepieces should be identical in orientation to what appears on the screen. To adjust the camera orientation, loosen the set screw and rotate the camera. Tighten the set screw when finished.
Bring the camera into alignment with the microscope by using an allan key to adjust the central ring till the image on the lower monitor looks sharp.
Scale Bar Setup in Image Capture
When Scale Bar applied by Image Capture is possibly inaccurate, a camera has been changed or Image Capture has been reinstalled. The following is a document that describes the process step by step to check it and correct it. (Calibrating ImageCapture scale bars.docx)
References
http://zeiss-campus.magnet.fsu.edu/articles/basics/care.html
https://www.igb.illinois.edu/sites/default/files/upload/core/PDF/Basic_microscope_alignment.pdf