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FREQUENTLY ASKED MICROSCOPE QUESTIONS
What is the difference between resolving power of an objective and resolution?
How do N.A. and magnification relate to the brightness of an image?
What is dioptric correction or adjustment?
Explain what field number means.
What is interpupillary distance?
What does working distance mean?
How is total magnification calculated?
What is meant by the term useful magnification?
Why do some objectives require immersion oil or water?
Why do some objectives have an iris?
Do I need special objectives for darkfield microscopy?
What does the inscription "0.17" on the objective mean?
What does the objective inscription "160" mean?
What is an infinity-corrected objective?
Why do some objectives have the inscription "Plan"?
Why do objectives usually have a coloured ring on them?
What do the inscriptions "LWD" or "ULWD" mean on an objective?
What do the objectives with the inscriptions "NIC" or "DIC" mean?
Why do some objectives have a spring-loaded front lens?
Why am I getting a worse image with a 40x than with a 20x objective?
Can I use phase contrast objectives for other types of observation?
Can I use an infinity corrected objective on a finite tube length microscope?
What does "C" or "K" or "WF" or "H" mean when printed on the eyepieces?
What does a neutral density filter do?
What is a daylight blue filter, and why do I need one?
How is a daylight blue filter used?
Why place a green filter in the light path?
What is the difference between achromat and plan achromat objectives?
What does "DIN" standard mean?
What is brightfield microscopy?
What is the difference between resolving power of an objective and resolution?
Resolving power refers to the clarity with which an objective can clearly separate two points or lines lying close together. The shorter the distance between the points or lines, the greater the resolving power. Also, the higher the N.A. (numerical aperture) number of the objective, the greater its resolving power. Resolution is the ability to distinguish two points as two points. One may need to compromise between resolving power and resolution to achieve the desired image.
How do N.A. and magnification relate to the brightness of an image?
The higher the N.A. value for a given magnification, the brighter the image. A higher magnification reduces the brightness of an image.
The height difference within features of an object that appear acceptably sharp when viewed with an optical instrument. Depth of field depends on the objectives, eyepieces and tube factor. Depth of field decreases when increasing magnification.
What is dioptric correction or adjustment?
Dioptric correction is the compensation of long or short sightedness by the adjustment of the eyepieces.
Light rays from all points in the field of view converge together at this point. This is where the user's eye must be positioned.
Explain what field number means.
The field number is the diameter of the eyepiece lens usually expressed in millimeters.
Field of view of FOV is the amount of the object that can be seen with a particular optics combination. It is the circular area which can be seen when looking into a microscope. It is combination of objective and eyepiece characteristics and field of view decreases with increasing magnification.
What is interpupillary distance?
Interpupillary distance is the distance between the centres of the pupils of your two eyes.
A stereomicroscope can be said to be parfocal when an object can be observed from the lowest magnification to the highest without having to refocus.
What does working distance mean?
The working distance is the distance from the object plane to the front of the objective. Working distance decreases each time a higher power objective is used.
How is total magnification calculated?
The total magnification of a microscope configuration is calculated from the magnifying power of the objective multiplied by the magnification of the eyepiece, and, if installed, multiplied by the auxiliary lens.
What is meant by the term useful magnification?
Useful magnification is obtained when the magnification is between 500 and 1000 times the numerical aperature of the objective. Since the human eye has limited resolving power, the magnification should be selected so that the image details can still be distinguished by the eye. If the magnification falls below this range, details can no longer be recognised by the eye. If the magnification is above this range, this is simply referred to as "empty magnification" as the objective is no longer able to resolve structures. The image therefore appears out of focus.
Why do some objectives require immersion oil or water?
The resolving power of an objective depends on its numerical aperture, which in turn depends on the refractive index of the medium between the specimen and the objective lens. A higher refractive index means the lens can gather more light and deliver a better image intensity. Air has a relatively low refractive index, and when it is the medium between specimen and the lens, lower N.A. objectives perform at their best capacity. Higher N.A. objectives need a higher refractve index to operate and immersion oil provides that higher index. For optimum performance, you will need to oil the top lens of the condenser to the bottom of the specimen slide. Immersion objectives are marked "oil" or "oel". Objectives marked "wi" require water as the immersion contact medium.
Why do some objectives have an iris?
In order to preserve darkness of the background for darkfield microscopy, the objective cannot have an N.A. higher than the lowest N.A. marked on the darkfield condenser. An iris that can reduce an objective's N.A. can allow you to use higher N.A. objectives for darkfield work. Objectives with an N.A. above 1.2 require an iris for darkfield. For ordinary brightfield observation, the iris can simply stay wide open.
Do I need special objectives for darkfield microscopy?
In most cases, from a transmitted light observation, you will only need a darkfield stop in the condenser. At higher magnifications, you will need an objective with an iris as well as a darkfield condenser.
What does the inscription "0.17" on the objective mean?
The "0.17" refers to the thickness in millimeters of the coverglass that was assumed by the lens designer in computing the corrections for the objective. For objectives with a numerical aperture higher than 0.45, departing from this thickness (or using no coverglass at all) may result in a less than satisfactory image.
What does the objective inscription "160" mean?
The 160 identifies a finite tube length objective, with a distance of 160mm from the nosepiece (where the objective screws in) to the top of the observation tube (where the eyepiece inserts). When you lengthen this distance by inserting accessories in the light path above the nosepiece, spherical aberrations will occur unless the accessories include the proper optical corrections.
What is an infinity-corrected objective?
With an infinity-corrected objective, light rays emerge in parallel projected toward infinity. Such an objective requires a tube lens in the light path to converge the parallel rays so that they come into focus at the eyepiece diaphragm.
Why do some objectives have the inscription "Plan"?
A plan objective projects a flat image of the entire field of view.
Why do objectives usually have a coloured ring on them?
With standard colours for most manufactureres, these rings make it easy to identify the magnification of the objective:
A red ring means 4x or 5x
A yellow ring means 10x
A green ring means 20x
A blue ring means 40x, 50x or 60x
A white ring means 100x
What do the inscriptions "LWD" or "ULWD" mean on an objective?
These letters identify long or ultra-long working distance objectives where the working distance is much longer than standard objectives of similar magnification.
What do objectives with the inscriptions "NIC" or "DIC" mean?
These letters designate an objective designed especially for use in Nomarski or Differential Interference Microscopy.
Why do some objectives have a spring-loaded front lens?
These objectives will have a very short working distance. To protect your microscope objective and your specimen, a spring-loaded front lens assembly retracts upon gentle contact with the stage or specimen. The retractable lens will not, however, protect against rough and continuous contact also also known as "crashing" the objective.
Why am I getting a worse image with a 40x than with a 20x objective?
The specimen may have a thicker cover glass than the standard 0.17mm, or you may have a thicker than normal glass slide. To improve the image, you might try using a dry objective with a correction collar, or you can try using 40x or 50x "oil immersion" objectives, since the immersion objective has less sensitivity to variations in cover glass thickness.
Can I use phase contrast objectives for other types of observation?
Yes, just move the phase condenser to the brightfield or "empty" position and employ the standard Koehler illumination procedure.
Can I use an infinity-corrected objective on a finite tube length microscope?
No, because the finite system does not include a tube lens to focus parallel rays.
What does "C" or "K" or "WF" or "H" mean when printed on the eyepieces?
Microscopes objectives do not include correction for lateral chromatic aberration and require a compensating eyepiece (labeled "C" or "K") to provide correction. "WF" signifies "widefield", meaning you can see more of the specimen at a given time. "H" signifies "high eyepoint" which means you don't have to put your eyes so close to the eyepiece during observation. These are typically meant for users who wear glasses but anyone can use them.
Used for photomicrography rather than observation, a photoeyepiece picks up the image delivered by the objective and projects it onto the film plane inside the camera. Photoeyepieces usually come with low magnification power to lessen the chance of empty magnification when the images they project onto film are magnified.
Why can't I use eyepieces of increasingly higher magnification to achieve higher total magnification?
To maintain useful magnification with satisfactory clarity and resolution, you must avoid empty magnification or making the specimen appear bigger, but not clearer. In general, total magnification should not exceed 750x - 1000x the N.A. of the objective. For example, with a 40x, N.A.0.65 objective, the total magnification should be between 480x and 650x.
What does a neutral density filter do?
Neutral density filters absorb light evenly across the visible spectrum, thus lowering the intensity of light without changing its colour temperature.
What is a daylight blue filter, and why do I need one?
A daylight blue filter absorbs some of the yellow to red light from the microscope lamp, resulting in colouration much closer to natural daylight which is beneficial comfortable viewing.
How is a daylight blue filter used?
Use of a daylight blue filter is intended for observation purposes only, providing a pleasant background to the field of view. Do not use this filter for photomicrography or with daylight colour film.
Why place a green filter in the light path?
Human eyes see the colour green the best. Since monochromatic light eliminates chromatic aberration, a green filter markedly improves the performance of achromats. In addition, phase contrast objectives give their best images with green light.
What is the difference between achromat and plan achromat objectives?
Objectives are corrected for field curvature and colour aberration. The difference between achromats and plan achromats is the degree of the flatness of the field. When the image is in focus from the centre towards its edges, the field is said to be "flat". In general, the flatter the field of an objective, the more lenses it contains and the more expensive the cost.
What does "DIN" standard mean?
"DIN" is an abbreviation for "Deutsche Industrial Normen". This is a German standard that has been adopted internationally as an optical standard used in most quality microscopes. The focal tube length of a DIN objective is 160mm. The former standard was RMS (Royal Microscope Society), which had a longer tube length (170mm). Most DIN optics can be interchangeable. However DIN and RMS objectives are not interchangeable.
A number usually engraved on an eyepiece, which refers to the diameter of a baffle or raised ring inside the eyepiece. The "FN" or "field number" determines the viewing field for the eyepiece.
Coaxial refers to the movement of coincident axes or gears that share one common axis. On the coaxial controls of a graduated mechanical stage, one knob controls the "X" axis movement and the other controls the "Y" axis movement. On a coaxial focusing system the fine focus control is inside of the coarse focus control.
Darkfield microscopy is a method by which the specimen (transparent or semi transparent) is seen as a bright object against a dark, usually black, background.
What is brightfield microscopy?
Brightfield is probably the most common type of micrscopy found in schools, industry and medical fields. In brightfield microscopy, a transparent or translucent specimen either naturally coloured or stained appears dark against a bright background or field.
Phase contrast is a technique for revealing the structural features of microscopic transparent objects that cannot otherwise be accomplished with brightfield microscopy. Phase appears to achieve the same effect as staining a specimen (which would kill a live specimen).
Oil immersion is used with high power objective lens (usually 100x) as a medium between the lens and the coverslip. Because oil has the same light transmitting properties as glass, it cuts down the refraction of light rays. Other requirements include the use of a 1.25 Abbe condenser to be used.
