This section will discuss of some elements of the microscope as well as their adjustment. Optical physic principles of image formation are not discussed here, we suggest that the reader consult specific literature on the subject. This text is for persons who have experience with the microscope. Our discussion is on some particularities that are perhaps forgotten.
The microscope elements
The microscope's principal parts
The microscope is an optical instrument made of several elements. The total magnification of the microscope is the product of the objective magnification multiplied by the ocular magnification. Thus, an 40x objective with an 10x ocular provides a total magnification of 400.Oculars
There are several types of oculars that distinguish themselves by the degree of chromatic correction. The most current ocular is the 10x type C. This type is used with achromatic objectives. Oculars are also available with magnifications of 8x to 12,5x. Some oculars are constructed to allow examination with corrective glasses. The pupillary distance, that is the distance between the eye and the oculars, is larger. These oculars are uncomfortable to those that do not wear corrective glasses because, the correct position is somewhere in front of the lens. A solution to this problem is to install a rubber bonnet. This will allow adjustment with the best distances and cut the ambient light.Another characteristic of the oculars is the field coefficient. This coefficient determines the diameter of the observed field. Thus, a 10x objective and an ocular having a field coefficient of 16 gives a field of 1,6 mm of diameter (1600 m) which represents a surface of grossly 2 square mm. The diameter of the field is calculated by dividing the field coefficient by the magnification of the objective. If the sediment volume is 20 ul with a coverslip of 22x22 mm and the objective is a 10x ignoring the volume that exceeds these edges of the coverslip, one arrives to a volume of 80 nl / field with a 16 field coefficient.
Objectives
The standard objectives for the routine urinary sediment are the 10x (lpf) and the 40x (hpf). The 25x is an intermediate efficient objective but is rarely used mainly because results are reported at a 100x magnification for casts and 400x for cells.The majority of microscopes will come equiped with a 4x and a 100x objectives. The immersion 100x is used for special examination like eosinophil count or PAP stain. The 4x is very useful to retrieve a viewed element or to rapidly scan the slide for a large size element.
On each objective one can see
inscriptions that describe it. Unfortunately, there are no
absolute standards. In our example, the word Plan describes the type of objective. The former is planachromatic
meaning a flat image with a chromatic correction. The term Ph
would describe a phase contrast objective.The 40/0,65 numbers indicate the magnification 40x and the numerical aperture 0,65. The number 160 is for the microscope tube length and 0,17 is the coverslip thickness to be used with this objective.
Immersion objectives have a colored ring near the front lens. The color of the ring indicates the type of immersion fluid to use. Black is for oil while orange is for glycerine.
Numerical aperture
The numerical aperture of an objective is the refractive index of the space between the front lens and the slide, multiplied by the sinus of half the opening angle. (O.N. = n * sin (q/2)
The maximum magnification
giving a clear image can hardly be greater than 500 times the
numerical aperture. With greater magnification one starts to see
artifacts, generated by diffraction. Air has a refractive index
of 1,0 while immersion oil as an index of 1,515. Immersion with
oil allows a magnification 1,5 times greater than air. Very thin
sediments can be examinated under immersion oil. The images are
clearer but the operation is messy and not very popular. Objective care
The major efect of a dirty or scratched objective is a loss of image clarity. In this condition, the microscopical field is seen with an impression of fog. Many solutions especially formulated for objective cleaning are sold by scientific goods companies. It is necessary however, to wipe rapidly and completely the cleaning liquid because these solutions can, in the long run, damage the cement that retains the front lens. Some use a mixture of ethanol and ether (50 / 50) that has the advantage to dry out very rapidly. When the front lens is scratched, there is no other than to replace it. A frequent and often ignored cause of scratches is scraping the front lens on the slide holder lever. The lever is usually thicker than the glass slide. It is therefore necessary to avoid working at the displacement limits ( for those who put two 22x22 coverslips on the same glass slide).Condenser
The function of the condenser
is to concentrate the light on the object. The field illuminated
by the condenser has to be uniform. The normal position of the
condenser is almost completely up with the front lens of the
condenser near the slide but not touching it. Adjustment of the
condenser to its optimal position is explained below. Some
condensers possess a removable front lens. This particularity is
useful for examination at low power field. Removing the front
lens allows a more smooth uniform lighting with the 2,5x and 10x
objectives. With the front lens up, an optimal adjustment at high
power fields can be attained and this without compromise. In each condenser, there is an iris diaphragm. The role of this diaphragm is to select the light rays that will pass at the center of the collecting lens. This iris must not be used to adjust the light intensity; one uses the lamp's rheostat for this adjustment. Closing down the diaphragm increases the field depth and the contrast but decreases the resolution and the luminosities. A diaphragm that is to closed can also give ghost images.
The separating power of the microscope is given by:
O.N. is the numerical
aperture, l is
the wavelength, d is the separating power that
is, the smallest distance between two elements that are seen as
separated. Adjustement of the condenser's diaphragm
The optimal opening is when the field is 70 to 80% illuminated when viewed with the ocular removed.Centering the condenser
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This is a five step
operation:
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Kohler adjustement.
The reasons for this adjustment is that the zone illuminated by the field diaphragm has to correspond to the observation field. An illuminated zone larger than the observation field leads to a loss of contrast.This is a multiple step operation:
- Remove any diffusing filter on the lamp or in the filter holder.
- Put a slide on the stage and adjust to see the preparation clearly.
- Completely close the field diaphragm (lamp diaphragm).
- Move the condenser up or down until the border or the iris hexagon is clear and neat.
- Center if necessary.
- Open the field diaphragm until the tip of the hexagon
touches the field limit.
- Install the diffusing filter.
Polarized light microscopy
Polarized light
The light rays of a halogen lamp are made of a great number of waves. Each of these waves is characterized by a propagation direction and a vibration of wavelength lambda perpendicular to the direction. With normal light, the vibrating part is of equal intensity in all directions. The light is said to be unpolarized.Some substances have a property called dichroism. These substances can favor a plane of vibration so that the light emerging from the substance is all vibrating in the same plane. The light is said to be polarized. Selection of the wave is done by eliminating others by: reflection, refraction, transmission, or dispersion.
There are many ways to obtain polarized light. The simplest and most economical option is to use polaroid filters. The filters are made of parallel oriented microcrystals frozen in a plastic matrix. Polaroid can be obtained as a plastic sheet. The working filters are cut out from the sheet to the size and shape needed. A microscope can be "equiped" with these filters for --?--$0,50 and it works!!
The light emerging from the polaroid filter is vibrating in one plane. A second filter (analyzer) placed in front of the polarizer with a plane angle of 90 degrees will extinct all the incoming light. The filters are said to be in a crossed configuration.
Polarized light microscopy
To do polarized light microscopy one
must install two polarizing filters. The first is placed in the
head and the second on the lamp or in the condenser's filter
holder. The latter is rotated to obtain a complete extinction of
light.Elements seen in polarized light microscopy of urine are mostly birefringent crystals.
Birefringency
Birefringency is a crystal property.A light ray passing through a different phase with an angle will be deviated by a property called refraction. An isotropic body will have the same refractive index for all the direction of incident light. Many crystals are said to be anisotropic that is birefringent. The two refracted waves emerging from a birefringent crystal are called the ordinary wave and the extraordinary wave. Each wave is polarized and vibrates in a plane different by 90 degrees to the other. Between crossed polarizing filters, birefringent crystals are visible. Many birefringent crystals produce a characteristic interference pattern.
Uses
Polarized light microscopy is a must for the identification of oval fat bodies. These contain lipid droplets containing cholesterol esters in a liquid crystal state witch shows an easily recognizable maltese cross interference pattern.Polarized light microscopy can also help for the identification of crystals. The birefringent property of a crystal can be strong, moderate, slight, with chromatic dispersion, with a maltese cross pattern...
Phase contrast
Principle
Bright field microscopy is based on color (gray scale) differences of the object with its surrounding media. An uncolored and transparent object of refractive index n with a thickness e in an uncolored and transparent media of refractive index n' will be invisible unless the borders exhibit diffraction. Since the light rays passing through the object follow a different path than the light rays passing only in the media, the object is said to have a phase difference.The phase difference is given by:
The phase difference j is:
- proportional to the path difference delta; e(n-n')
- inversly proportional to the wavelength lambda;
Elements of the urinary microscopy that benefit the most from phase contrast are the hyaline casts specially the "Early" type. Phase contrast is also a superior way to observe cell nuclei as long as the cell is not too vacuolated. Phase contrast superiority is limited with loaded dirty sediments since many elements having a high phase difference produce a halo making the reading difficult.
Phase contrast rings adjustement
To have an optimal image one must adjust the ring's convergence. The operation is relatively simple.- Select a low power phase contrast objective.
- Select the corresponding condenser ring.
- Remove an ocular and replace it with the adjustment telescope.
- Adjust the telescope by sliding until a clear images of the rings.
- With the adjusting screw, overlaps the rings to single.
- Replace the ocular.
