Carl Zeiss, Compendium

MICROSCOPY / COMPENDIUM

Glossary
Term Explanation
Resolution The resolving power of the microscope is determined by the ability to make points or lines which are closely adjacent in an object distinguishable in an image. The distance between these distinguishable points or lines is designated as d_o.
This distance can be calculated using the following formula:

           1.22 x Lambda
d_o    =    ----------------------------------
             NA_Objektiv + NA_Kondensor

Lambda =
Mean wavelength of white light, e.g. 550 nm (green)
NA =
numerical aperture

Refractive index The refractive index of the medium between the front lens of the objective and the cover glass plays a very important role in determining which portion of the numerical aperture of the objective is used.
These are the refractive indices for some sample media:

air                       n = 1
immersion oil    n = 1.5180 ± 0.0005
cover glass       n = 1.525 ± 0.0015
glycerin             n = 1.455
water                  n = 1.33

Fluorescence Phenomenon which occurs when high-energy radiation is absorbed by a molecule (fluorochrome) and when this molecule emits light of a longer wavelength than that absorbed (Stoke's shift).
In microscopy, fluorescence is an important technique, since most fluorochromes do not damage cells and can therefore be used in the microscopy of living specimens. Furthermore, they can be bound to antibodies and other specific molecules, permitting the exact localization, observation and measurement of changes. The phenomenon of autofluorescence also exists, where already fluorescent molecules are present in the material to be examined Fluorescence filter sets , consisting of exciter filter, beam splitter and barrier filter, are required, as well as a high-intensity lamp (usually super-pressure mercury or xenon lamps).The decisive factor for good visualization (high contrast of the fluorescent areas against a dark background), especially in the case of weakly dyed specimens, is the high numerical aperture of the objectives used. Doubling of the objective aperture allows four times more fluorescence light to be detected.
Confocal microscopy Unlike "normal" microscopy, only a spot of a minimum size in the specimen is illuminated in confocal microscopy. To enable an image to be formed from this, the specimen must be scanned.
The image of the spot is directed through a pinhole stop in an intermediate image plane. As a result, only light from the focal plane can reach the detector (a photomultiplier). All other (out-of-focus) planes are blocked out. This results in an "optical section". The images are stored electronically and displayed on a monitor. A series of optical sections can be recorded by moving a motor a slight distance along the z-axis each time an image has been recorded, after which the next image is then also recorded. Such a z-series permits the electronic reconstruction of the three-dimensional structure using suitable computer programs. The procedure, which is restricted to incident-light techniques, has completely revolutionized fluorescence microscopy in biology in particular.
The Confocal Scan Module is used to produce confocal images in the eyepiece, with the height of the reflecting specimen being color-coded in colors using a color-converting optical system. This enables even minute defects and contamination on wafers to be detected quickly and reliably.
Objective with correction ring Why cover glass correction for an oil objective?
Oil objectives should function equally well, regardless of whether they are used with or without a cover glass because the cover glasses and the oil should display an almost identical refractive index (homogeneous immersion). Unfortunately, however, theory and practice are not always compatible. Manufacturers often provide cover glasses with a refractive index other than the 1.525 ± 0.0015, and a thickness other than the 0.17 (+0/-0.02) mm required especially for high-aperture objectives.
Deviations of only 0.01 mm in the cover glass thickness can result in unsatisfactory image quality. The refractive index of the immersion medium must also be kept precisely to ensure a perfect image. The Zeiss immersion oil with the refractive index 1.518 is therefore ideal. It is particularly important to compensate for these deviations in the case of high-aperture dry objectives.
All objectives with numerical apertures from 0.85 upwards therefore feature a correction ring.
Numerical aperture
Abbreviation: NA This is the value designating the sine of half of the aperture angle [alpha] of the objective.
This only applies if there is air between the objective and the specimen. To put it more precisely: the refractive index index of the immersion medium must also be taken into account.
The formula therefore is:

NA    =    n x sin (½ x alpha)

n =
refractive index of the medium between the objective front lens and the specimen or cover glass

Recommendations for cleaning of optical components Accessible optical surfaces (front lenses, eyepiece back lenses, condenser front lenses) should normally be cleaned using mild cleaners. Optics cleaning paper or a white linen cloth (both fluff-free) can be used. A wooden stick wrapped in medical-grade cotton wool is also suitable. Slight moistening with distilled water may also be of assistance in the cleaning procedure. Cleaning is always performed in circular movements, starting at the center and working toward the edge. Fluff or dust can be blown off with the bellow-like devices obtainable from camera stores
Petroleum ether used for medical purposes should be used occasionally to remove stubborn dirt or oil. The benefit of this agent is that it evaporates easily and therefore does not enter gaps or joints, i.e. the lacquers are not damaged during the short reaction time of the agent.
The dust cover supplied protects the microscope from dirt when it is not in use.
In the event of severe contamination, please contact Zeiss Service personnel.
Field of view number The field of view number designates the diameter of the visible intermediate image in millimeters.
Therefore, the diaphragm limiting the intermediate image in the eyepiece is of decisive importance. Furthermore, the microscope must permit the bundle of rays to pass through the entire beam path.
The Axioplan 2, Axiophot 2 and Axiotron 2 microscopes have been designed for the field of view number 25, while the Axioskop, Axiovert and Axiolab microscopes have been designed for 20 mm fields.
Plan APOCHROMAT, Plan-NEOFLUAR and Epiplan-NEOFLUAR objectives have been flattened for the 25 mm field of view, and ACHROPLAN objectives for the 23 mm field.
Fields of view are decisive for the area of the object imaged. The object field therefore increases with the field of view number.
Examples:

Plan-NEOFLUAR objective 40x and eyepiece 10x/25
object field = 0.625 mm

ACHROPLAN objective 40x and eyepiece 10/20
object field = 0.5 mm

Plan-NEOFLUAR objective 1.25 and eyepiece 10/25x
object field = 20 mm

Plan-NEOFLUAR objective 63x and eyepiece 10x/20 and Optovar 1.25x
object field = 0.254 mm

Depth of field The depth of field (mm) is the area above and below the focal plane in the object which is still perceived as sharply imaged. The overall imaging depth is calculated using the following formula:

D = D_g + D_w
where D_g is the geometric imaging depth based on a visual acuity of 2º and D_w is the wave-optical component of the imaging depth.

    0.143
D_g = -------------------
    M_total

    Lamda
D_w = -------------------
    2(NA)²

Lambda =
mean wavelength of white light = 0.00055 mm (green)

NA =
numerical aperture of the objective

M_total =
overall magnification in the microscope

Useful magnification This is obtained when the magnification is between 500 and 1000 times the numerical aperture of the objective. Since the eye has a limited resolving power, the magnification should be selected so that image details can still be resolved by the eye. If the overall magnification is below this range, details can no longer be recognized by the eye
If the overall magnification is above this range, this is called empty magnification. The objective is no longer able to resolve the structures. The image therefore seems out of focus.
Examples:

Plan-NEOFLUAR objective 40x/0.75 + eyepiece 10x = 400x
375x ... 750x       OK

ACHROPLAN objective 100x/1.25 + eyepiece 16x = 1600x
625x ... 1250x     not OK

Plan-APOCHROMAT objective 20x/0.75 + photo eyepiece

10x + camera factor 0.25 + additional magnification 10x = 500x
375x ... 750x       OK

Magnification and lateral magnification The total magnification of the microscope is calculated from the magnifying power of the objective multiplied by the magnification of the eyepiece and, where applicable, multiplied by intermediate magnifications
A distinction is made between magnification and lateral magnification.
While the magnification always refers to the impression of the eye, the lateral magnification is always a measurable value. If an object is viewed with the eye from a distance of 250 mm, the magnification is 1x. If the distance is 500 mm, the size of the object seen is halved - the magnification is reduced to 0.5x. The viewing angle under which something is seen is a decisive factor. When looking into the microscope, the viewing angle under which the object is seen is increased precisely by the factor resulting from the above calculation of the total magnification. This does not mean that the entire viewing angle is also imaged in the eye! The viewing angle is limited by the field of view.
The term lateral magnification is always used if an image is produced which is to be measured using a scale. For example, a photo is taken using a microscope camera and the size of an object detail in the specimen is then to be determined. For this, the detail on the photo is measured using a scale. If this length is divided by the total magnification, the original size is obtained.
Example: Object size 10 mm on the paper photo, taken with objective 40x, photo eyepiece 10x, camera magnification 0.25x and additionally magnified 4x from the negative.

10 mm
-------------------------
40 x 10 x 0.25 x 4

The result is 0.025 mm or 25 µm. The size of the object detail therefore was exactly 25 µm. The lateral magnification is 400:1. If the photo is viewed from a distance of 250 mm, it appears to be 400 times as large, as if the object detail on the microscope slide were viewed with the naked eye.

Glossary
Term	Explanation
Resolution	The resolving power of the microscope is determined by the ability to make points or lines which are closely adjacent in an object distinguishable in an image. The distance between these distinguishable points or lines is designated as d_o. This distance can be calculated using the following formula: 1.22 x Lambda d_o = ---------------------------------- NA_Objektiv + NA_Kondensor Lambda = Mean wavelength of white light, e.g. 550 nm (green) NA = numerical aperture
Refractive index	The refractive index of the medium between the front lens of the objective and the cover glass plays a very important role in determining which portion of the numerical aperture of the objective is used. These are the refractive indices for some sample media: air n = 1 immersion oil n = 1.5180 ± 0.0005 cover glass n = 1.525 ± 0.0015 glycerin n = 1.455 water n = 1.33
Fluorescence	Phenomenon which occurs when high-energy radiation is absorbed by a molecule (fluorochrome) and when this molecule emits light of a longer wavelength than that absorbed (Stoke's shift). In microscopy, fluorescence is an important technique, since most fluorochromes do not damage cells and can therefore be used in the microscopy of living specimens. Furthermore, they can be bound to antibodies and other specific molecules, permitting the exact localization, observation and measurement of changes. The phenomenon of autofluorescence also exists, where already fluorescent molecules are present in the material to be examined Fluorescence filter sets , consisting of exciter filter, beam splitter and barrier filter, are required, as well as a high-intensity lamp (usually super-pressure mercury or xenon lamps).The decisive factor for good visualization (high contrast of the fluorescent areas against a dark background), especially in the case of weakly dyed specimens, is the high numerical aperture of the objectives used. Doubling of the objective aperture allows four times more fluorescence light to be detected.
Confocal microscopy	Unlike "normal" microscopy, only a spot of a minimum size in the specimen is illuminated in confocal microscopy. To enable an image to be formed from this, the specimen must be scanned. The image of the spot is directed through a pinhole stop in an intermediate image plane. As a result, only light from the focal plane can reach the detector (a photomultiplier). All other (out-of-focus) planes are blocked out. This results in an "optical section". The images are stored electronically and displayed on a monitor. A series of optical sections can be recorded by moving a motor a slight distance along the z-axis each time an image has been recorded, after which the next image is then also recorded. Such a z-series permits the electronic reconstruction of the three-dimensional structure using suitable computer programs. The procedure, which is restricted to incident-light techniques, has completely revolutionized fluorescence microscopy in biology in particular. The Confocal Scan Module is used to produce confocal images in the eyepiece, with the height of the reflecting specimen being color-coded in colors using a color-converting optical system. This enables even minute defects and contamination on wafers to be detected quickly and reliably.
Objective with correction ring	Why cover glass correction for an oil objective? Oil objectives should function equally well, regardless of whether they are used with or without a cover glass because the cover glasses and the oil should display an almost identical refractive index (homogeneous immersion). Unfortunately, however, theory and practice are not always compatible. Manufacturers often provide cover glasses with a refractive index other than the 1.525 ± 0.0015, and a thickness other than the 0.17 (+0/-0.02) mm required especially for high-aperture objectives. Deviations of only 0.01 mm in the cover glass thickness can result in unsatisfactory image quality. The refractive index of the immersion medium must also be kept precisely to ensure a perfect image. The Zeiss immersion oil with the refractive index 1.518 is therefore ideal. It is particularly important to compensate for these deviations in the case of high-aperture dry objectives. All objectives with numerical apertures from 0.85 upwards therefore feature a correction ring.
Numerical aperture Abbreviation: NA	This is the value designating the sine of half of the aperture angle [alpha] of the objective. This only applies if there is air between the objective and the specimen. To put it more precisely: the refractive index index of the immersion medium must also be taken into account. The formula therefore is: NA = n x sin (½ x alpha) n = refractive index of the medium between the objective front lens and the specimen or cover glass
Recommendations for cleaning of optical components	Accessible optical surfaces (front lenses, eyepiece back lenses, condenser front lenses) should normally be cleaned using mild cleaners. Optics cleaning paper or a white linen cloth (both fluff-free) can be used. A wooden stick wrapped in medical-grade cotton wool is also suitable. Slight moistening with distilled water may also be of assistance in the cleaning procedure. Cleaning is always performed in circular movements, starting at the center and working toward the edge. Fluff or dust can be blown off with the bellow-like devices obtainable from camera stores Petroleum ether used for medical purposes should be used occasionally to remove stubborn dirt or oil. The benefit of this agent is that it evaporates easily and therefore does not enter gaps or joints, i.e. the lacquers are not damaged during the short reaction time of the agent. The dust cover supplied protects the microscope from dirt when it is not in use. In the event of severe contamination, please contact Zeiss Service personnel.
Field of view number	The field of view number designates the diameter of the visible intermediate image in millimeters. Therefore, the diaphragm limiting the intermediate image in the eyepiece is of decisive importance. Furthermore, the microscope must permit the bundle of rays to pass through the entire beam path. The Axioplan 2, Axiophot 2 and Axiotron 2 microscopes have been designed for the field of view number 25, while the Axioskop, Axiovert and Axiolab microscopes have been designed for 20 mm fields. Plan APOCHROMAT, Plan-NEOFLUAR and Epiplan-NEOFLUAR objectives have been flattened for the 25 mm field of view, and ACHROPLAN objectives for the 23 mm field. Fields of view are decisive for the area of the object imaged. The object field therefore increases with the field of view number. Examples: Plan-NEOFLUAR objective 40x and eyepiece 10x/25 object field = 0.625 mm ACHROPLAN objective 40x and eyepiece 10/20 object field = 0.5 mm Plan-NEOFLUAR objective 1.25 and eyepiece 10/25x object field = 20 mm Plan-NEOFLUAR objective 63x and eyepiece 10x/20 and Optovar 1.25x object field = 0.254 mm
Depth of field	The depth of field (mm) is the area above and below the focal plane in the object which is still perceived as sharply imaged. The overall imaging depth is calculated using the following formula: D = D_g + D_w where D_g is the geometric imaging depth based on a visual acuity of 2º and D_w is the wave-optical component of the imaging depth. 0.143 D_g = ------------------- M_total Lamda D_w = ------------------- 2(NA)² Lambda = mean wavelength of white light = 0.00055 mm (green) NA = numerical aperture of the objective M_total = overall magnification in the microscope
*Useful magnification*	This is obtained when the magnification is between 500 and 1000 times the numerical aperture of the objective. Since the eye has a limited resolving power, the magnification should be selected so that image details can still be resolved by the eye. If the overall magnification is below this range, details can no longer be recognized by the eye If the overall magnification is above this range, this is called empty magnification. The objective is no longer able to resolve the structures. The image therefore seems out of focus. Examples: Plan-NEOFLUAR objective 40x/0.75 + eyepiece 10x = 400x 375x ... 750x OK ACHROPLAN objective 100x/1.25 + eyepiece 16x = 1600x 625x ... 1250x not OK Plan-APOCHROMAT objective 20x/0.75 + photo eyepiece 10x + camera factor 0.25 + additional magnification 10x = 500x 375x ... 750x OK
Magnification and lateral magnification	The total magnification of the microscope is calculated from the magnifying power of the objective multiplied by the magnification of the eyepiece and, where applicable, multiplied by intermediate magnifications A distinction is made between magnification and lateral magnification. While the magnification always refers to the impression of the eye, the lateral magnification is always a measurable value. If an object is viewed with the eye from a distance of 250 mm, the magnification is 1x. If the distance is 500 mm, the size of the object seen is halved - the magnification is reduced to 0.5x. The viewing angle under which something is seen is a decisive factor. When looking into the microscope, the viewing angle under which the object is seen is increased precisely by the factor resulting from the above calculation of the total magnification. This does not mean that the entire viewing angle is also imaged in the eye! The viewing angle is limited by the field of view. The term lateral magnification is always used if an image is produced which is to be measured using a scale. For example, a photo is taken using a microscope camera and the size of an object detail in the specimen is then to be determined. For this, the detail on the photo is measured using a scale. If this length is divided by the total magnification, the original size is obtained. Example: Object size 10 mm on the paper photo, taken with objective 40x, photo eyepiece 10x, camera magnification 0.25x and additionally magnified 4x from the negative. 10 mm ------------------------- 40 x 10 x 0.25 x 4 The result is 0.025 mm or 25 µm. The size of the object detail therefore was exactly 25 µm. The lateral magnification is 400:1. If the photo is viewed from a distance of 250 mm, it appears to be 400 times as large, as if the object detail on the microscope slide were viewed with the naked eye.

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