Brightfield observation methods

Microscopes for brightfield observation

The basic operation of a brightfield observation will be explained using as an example an upright microscope provided with an optical system capable of Koehler illumination (see figure below).

Step1 : Turning on the lamp

Turn on the power switch, and the lamp lights up.

Step2 : Adjusting the illumination

Place a color balancing filter above the field lens.
Adjust the illumination to an appropriate level using the brightness adjuster.

Color balancing filter

Step3 : Adjusting the interpupillary distance of the eyepiece

Adjust the interpupillary distance until the left and right viewfields merge into one.

Adjusting the interpupillary distance of the eyepiece

Step4 : Adjusting the diopter

4-1. Align the diopter ring with the reference groove.

Rotate the diopter ring, and align its edge with the reference groove.

Adjusting the diopter

4-2. Place the specimen.

With the coverglass facing upwards, place the specimen on it.

4-3. Focus with the 10X objective.

Change to the 10X objective.

Turn the focus knob and focus the specimen.

Focus with the 10X objective

4-4. Focus with the 40X objective.

4-5. Change to the 10X objective.

4-6. Adjust the diopter ring.

  • 1With the right eye look into the right eyepiece and adjust the right diopter ring until the specimen comes into focus.
  • 2With the left eye look into the left eyepiece and adjust the left diopter adjustment ring until the specimen comes into focus.

4-7. Repeat steps 4-4 to 4-6 again.

Step5 : Field diaphragm focusing and centering

5-1. Turn the 10X objective into the light path.

5-2. Close the field diaphragm as far as possible.

Rotate the field diaphragm ring, till the field diaphragm is as small as possible.

5-3. Focus the field diaphragm image onto the plane of the specimen

Turn the condenser focus knob until the field diaphragm image is focused on the plane of the specimen. (Both the specimen and field diaphragm image are focused.)

5-4. Move the field diaphragm image to the center of the viewfield.

5-5. Change to the 40X objective.

5-6. Adjust the size of the field diaphragm.

Rotate the field diaphragm ring, until the field diaphragm image is circumscribed by the viewfield.

Step6 : Observation

6-1. Selecting the objective

Rotate the nosepiece and switch to the objective with the desired magnification.
After changing the objective adjust the condenser aperture and field diaphragm to suit the objective.

6-2. Adjusting the aperture diaphragm

Normally, if the aperture diaphragm is closed to 70-80% of the numerical aperture of the objective then a clear image with reasonable contrast will be obtained. The scale on the condenser aperture diaphragm ring shows numerical aperture (N.A.), so adjust the condenser aperture diaphragm ring in accordance with the scale.

The numerical aperture of every objective is indicated on the side of the objective.
40X/0.65X magnification 40X, numerical aperture 0.65

Adjusting the aperture diaphragm

6-3. Adjusting the field diaphragm

The field diaphragm restricts the illumination light to the observation area of the specimen. If it is opened more than necessary it will result in reduced contrast due to stray light.
Whenever the objective is changed, rotate the field diaphragm ring and adjust the field diaphragm image so that it is circumscribed by the surrounding field of view.

6-4. Adjusting the illumination

If the magnification of the objective is increased then the field of view becomes darker. Adjust the illumination with the brightness adjuster as necessary. Note that when the illumination changes the color tone also changes at the same time. If the voltage is turned down, the illumination darkened red tones increase. If the voltage is turned up and illumination increased, then blue tones increase. In order to maintain a specific tone an ND filter is used to adjust the illumination.

ND filter

Step7 : Turning off the lamp

Turn off the power switch and the lamp will be turned off.

Oil immersion operation

An objective on which “Oil” is displayed (a black band is also provided on the end) is an oil immersion objective. An oil immersion objective is used with immersion oil filling the space between the tip of the lens and the specimen. For oil immersion objective of the numerical number 1.0 or greater an oil immersion condenser is also used in order to obtain sufficient performance. The space between the tip of the condenser lens and the specimen is filled with immersion oil, the same as for the objective.

Color balancing filter

Effect of a color balancing filter

If the lamp illumination is used as it is the object observed will be bathed in a yellow tone. If a color balancing filter is used then the color tones are natural. A color balancing filter is always used with brightfield observation.

Adjusting the interpupillary distance of the eyepiece

Adjusting the interpupillary distance and diopter

Before starting observation, it is necessary to make adjustments on interpupillary distance and diopter. If users continue observations without making the adjustments, they may suffer eye fatigue. Also, if the adjustments are made, differences of visual acuity between the right and left eyes are corrected, facilitating viewing with both eyes. This also minimizes deviation in focus when the magnification is changed.

Effects of diopter adjustment

Focus with the 10X objective

Let's try it!

Focusing
When you try to focus, simply turning the focus knob at random will not bring the image into focus. When using a high magnification objective, sometimes the specimen or the objective can be damaged by pressing the objective against the specimen slide. The following is an example of how to focus while avoiding these problems.

  • 1.Switch the 10X (or the 4X) objective into the light path.
  • 2.Rotate the coarse focus knob to raise the stage as far as possible. (*1)
  • 3.While looking through the eyepiece, slowly rotate the coarse focus knob to lower the stage. When the focus position is reached, release the coarse focus knob.
  • 4.Slowly rotate the fine focus knob to adjust the focus. Even when making observations with a high magnification lens, first use a 10X (or 4X) objective to focus on the specimen. Then change to the high magnification objective and use the fine focus knob to focus the image.

Hints:

  • When turning the coarse focus knob while looking through the eyepiece, always turn it in the direction that will lower the stage.
  • When using the coarse focus knob to raise the stage, never look into the eyepiece, and do the operation while watching the stage and objectives in order not to damage the specimen.
  • First use a low magnification objective for focusing, and then change to the high magnification lens.
  • *1With a 10X or 4X objective the working distance is long, so when using a standard thickness slide glass and coverglass the specimen and objective will not come into contact even when the stage is at its highest position.
    (The working distance is the distance from the objective to the top surface of the coverglass. The standard thickness is 1.1mm for slide glass and 0.17mm for coverglass.)

Adjusting the aperture diaphragm

Rotate the condenser aperture diaphragm ring and the size of the aperture changes. If the aperture is reduced then the illumination decreases, fine detail becomes difficult to see, contrast is increased, and the area of focus deepens. If the aperture is opened then the illumination increases, fine detail becomes easier to see, contrast is decreased, and the area of focus becomes shallower.

Generally, if the aperture is closed to 70-80% of the numerical aperture of the objective then a clear image with reasonable contrast will be obtained. Also, the aperture diaphragm is used to adjust the numerical aperture of the illumination light, not the brightness. To adjust the brightness, the brightness adjuster is used.

  • It is possible to see the actual aperture condition by removing the eyepiece and looking down the eyepiece tube. (sometimes the eyepiece is fixed to the eyepiece tube with screws. In this case first loosen the screws before removing the eyepiece)

ND filter

This is a filter for adjusting illumination by changing the transmittance of the light. Unlike adjusting illumination by changing the voltage, the color tone does not change.

(Normally several types are available. Nikon ND filters are ND 2, 4, 8, 16, corresponding to light intensity of 1/2, 1/4, 1/8 and 1/16 respectively.