The microphotography on this website was produced using a stereomicroscope or a trinocular biological microscope.

Stereo Microscope

The stereomicroscope has 20X (twenty times) magnification with both incident and in-base illumination. A photograph identified as 20X would be taken with the stereomicroscope.

Trinocular Biologicalmicroscope

The biological microscope has four objective lenses 4X,10X,40X and 100X. The eyepieces for the biological microscope are 10X or 20X. The two 10X eyepieces (one of each eye) produce magnifications of 40X, 100X, 400X and 1,000X. The 20X eyepieces produce magnifications of 80X, 200X, 800X and 2,000X. However, the microphotographs are taken through the third optical port and are not influenced by the eyepieces in use. The digital camera used has a frame size of 2,048 x 1536 pixels (picture elements). This frame size is reduced to 640x480 pixels for use on this website. The photographs will be identified by the magnification of the objective lenses with 10X eyepieces. Photographs identified as 40X, 100X, 400X and 1,000X are taken with the trinocular biologicalmicroscope. A photograph identified as 20X would be taken with the stereomicroscope. The fields  of view of the trinocular-biologicalmicroscope photographs are considerably smaller than the fields of view seen through the eyepieces, that is, the magnification is greater. The size of the field of view for each magnification is described below.

Specimen sizes in microbiology are measured in units of micrometers (μm), that is, one millionth of a meter (10-6 meter). The international spelling of micrometer is "micrometre." However, this website uses the spelling common in the United States.

Characteristics of the Stereomicroscope

The stereomicroscope is set at 20X magnification with 10X eyepieces and 2X objectives. There is one objective for each eyepiece permitting a three-dimensional view of the specimen. The specimen may also be illuminated from below and/or above. The field of view remains relatively constant but varies somewhat with focusing. The following photograph shows a 1,000 micrometer (μm) target on a slide and demonstrates the approximate field of view produced by the digital camera used with this microscope. This field of view is with the focus on a standard slide on which the target is etched. The stereomicroscope is not designed to photograph etched slides optimally. Therefore, the target is not as clear as it would appear under a biologicalmicroscope. The field of view under these conditions is about 5,300μm x 3,975μm. The diameter of the outer circle in the microphotograph is 4,000μm. This information allows for fairly accurate measurements from the stereomicroscope microphotographs using a scale based upon the following microphotograph. However, the scaling microphotograph and the specimen microphotograph must be printed the same size.

Stereomicroscope View of Calibration Slide

The following stereomicroscopic microphotograph shows a 2,000μm black dot on the calibration slide. One may print out the microphotograph above and the following microphotograph to verify the size of the dot. Ensure that both microphotographs are printed the same size.

Stereomicroscope Dot Image

Characteristics of the Trinocular Biologicalmicroscope

The Condenser 

The biological microscope is both a bright-field and dark-field microscope depending upon the condenser in use. The condenser is the device that directs the light from the light source onto the specimen and is beneath the microscope stage on which the specimen rests.. The bright-field condenser focuses the light onto the specimen directly causing the background to be bright. The dark-field condenser scatters the light around the specimen causing the background in the field of view to appear dark. Dark-field condensers are useful for specimens that are so transparent that they cannot be seen against a bright background. The following microphotograph shows a bright-field examination of saliva at 40X magnification.

Bright-field Image of Saliva 

The following microphotograph shows the same specimen under dark-field illumination.

Dark-field Image of Saliva 

The blue to black color of the specimen is due to the way the white light is refracted in the camera system, The flatter parts of this mostly transparent specimen fade from blue to white because the light is not refracted as much. The following microphotograph shows the same specimen at a higher magnification, 100X, where the white light is refracted towards the yellow. These color differences are less apparent when viewing the specimen through the eyepieces. Color differences due to refraction will be less apparent when the specimen is flat. Most microphotographs on this site will be adjusted to show colors appropriately.

Saliva at 100X 

The Filters 

Many specimens are seen better in colored light. The biological microscope has three filters that can be mounted on the condenser, blue, green and yellow. The following 100X magnification microphotograph shows a saliva smear under blue light.

Saliva Smear with Blue Filter 100X Magnification 

This saliva specimen is smeared on the slide as opposed to the other photographs above that show saliva bubbles deposited on the slide. The prominent cells in this blue-light microphotograph are squamous epithelial cells that line human cheeks. The following microphotograph shows the same specimen under green light.

Saliva Smear with Green Filter 100X Magnification 

Finally, the same specimen under yellow light.

Saliva Smear with Yellow Filter 100X Magnification 

Note that the blue filter did not produce expected results. However, the cell outlines are more prominent. Various color filters along with other adjustments of the microscope can bring out details not readily apparent. Details can be made more apparent by staining the specimen with various dyes. Iodine, for example, will stain the starch in cells while not affecting the color of other parts of the cell. A very common procedure using various stains including iodine is called "Gram staining." This protocol will stain various parts of a cell different colors. The iodine will stain only starch in the wall of a cell, specifically a starchy polymer called "peptidoglycan." Cell walls containing peptidoglycan are said to be Gram positive. Those without peptidoglycan are Gram negative. This procedure helps to differentiate cells. When staining protocols are used for this website, the procedures will be described.

The following microphotograph shows the saliva smear at 400X magnification under yellow light.

Saliva Smear withYellow Filter 400X Magnification 

The next microphotograph shows a human blood smear at 400X magnification in white light. Note that the background appears blue.

Human Blood Smear 400X Magnification 

The Microphotograph's Field of View 

The microphotographs produced by the biologicalmicroscope have fields of view that are determined by the objective lenses. The following microphotograph was taken with the 4X objective lens (40X magnification).

Calibration Slide 40X Magnification 

Each division on this calibration slide is 0.01 millimeters or 10 micrometers (μm). Therefore, the field of view at 40X is 3,000μm wide x 2,250μm high. The field of view is determined on the flat surface of a standard slide. Three dimensional specimens may required focus adjustments that affect the field of view slightly.

The next microphotograph shows the same calibration side at 100X magnification, that is, with the 10X objective lens. The microphotographic field of view at 100X is 1,200μm x 900μm.

Calibration Slide 100X Magnification 

The following microphotograph shows the same calibration slide at 400X magnification, that is, with the 40X objective lens. The microphotographic field of view at 400X is 300μm x 225μm. 

Calibration Slide 400X Magnification 

The following photograph shows the 400X microphotograph above overlaid on another 400X microphotograph of a spatterdock leaf. Overlays of the calibration slide images can be printed on transparent sheets by most printers. The transparencies can then be used to estimate the size of items in a microphotograph on this website. Ensure that the microphotographs to be examined are printed in exactly the same way you printed the transparencies. However, for most purposes, the scale on the microphotograph alone is adequate for estimations of sizes.

400X Overlay

The next photograph shows an overlay of the blood smear microphotograph above. Note that a blood cell is slightly less than ten micrometers in diameter.  Red blood cells (erythrocytes) have a diameter of about 6 to 8 micrometers.

Blood Smear with Overlay

A measurement of the squamous epithelial cells in the yellow 100X microphotograph above indicates that these irregular, flat cells can be about 90μm long as shown in the following photograph.

Saliva Smear with Overlay

The next microphotograph of the calibration slide is at 1,000X magnification, that is, with the 100X objective lens. This microphotograph required the use of immersion oil. The microphotographic field of view at 1,000X is 120μm x 90μm.

 

Calibration Slide 1000X Magnification 

The following microphotograph shows a calibration slide dot precisely 2,000μm in diameter at 40X magnification. If this dot is measured with the scale in the 40X microphotograph above, the diameter will measure 2,000μm. You can print out both microphotographs and verify the diameter. Ensure that the two microphotographs are printed exactly the same size. The microphotographs produced by the biological microscope also have a scale, usually in the lower right hand corner, that may be used to approximate the size of objects in the microphotograph. These scales are approximate because the displayed or printed pictures on computer devices may be distorted.

Calibration Slide with 2000 micrometer Dot 

 

 

 

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