MICROSCOPY

1. Definition:
Microscopy is the technique of obversing and recording images of samples and
objects that cannot be seen with naked eye (objects that are not inside the
resolution range of normal eyes) by using microscopes.
Micropscopy is divided into 3 well – known branches: optical, electron, and
scanning probe microscopy, along with the emerging field of X – ray
microscopy.

2. Structure:
All microscopes have a coordinated system of lens organized to magnify
images of the specimen. The main differences among the microscopes are the
power that the microscopes need to produce images, nature and to arrange the
lens system.

Fig. 1: Principle of compound light microscope

and phase – contrast microscopy
 Fig 2: Structure of the compound light microscopes
 Ocular lens (eyepiece) : Viewers look through to see the specimen. The eyepiece
normally has the magnification power of 10x or 15x.
 Diopter Adjustment: Uses as a means to change focus on one eyepiece so as to
correct for any difference in vision between your two eyes.
 Head (body tube): Containing a prism beding the light so that the light travel
through magnifiyng lenses from objectives to eyepieces.
 Objective lens: Gathering the light of the specimen and helping to magnify the
image of the specimen. The objective lens can magnify the images up to 4x, 10x,
40x, and 100x.
 Mechanical stage: Where to put the specimen and using stage clips of stage
holder to clamp the specimen. In the light microscope, the stage is movable and it
can moves horizontal and vertical by using X – axis knob and Y – axis knob.
 Consender: The lens system under the stage used to gather and focus light from
the light source onto the specimen. There is a consender adjustment control used to
adjust the height of the consender.
 Aperture iris diaphragm: The hole in the middle of the stage which allows light
from the source to reach the specimen.
 Light source (Illumination): Providing the light to the specimen. Being controlled
by the light switch (On/Off) or the voltage of the transfomer attached to the
illuminator.
 Coarse Focus Knob and Fine Focus Knob: Bring the specimen into the general
focus and fine – tuning the focus to increase the detail of specimen.
Therefore, to caculate the total magnification of an image of the specimen, take the
power magnification of the objective lens multiply with the power of the eyepiece.
 MAGNIFICATION = EYEPIECE LENS MAGNIFICATION × OBJECTIVE LENS MAGNIFICATION

3. Usage:

3.1 General instruction:

- Advoid dropping the microscope, hitting it on a lab bench, or having the
eyepieces fall out.
- Use both hands to hold the microscope erect. Keep one hand on the arm and the
other on the microscope's bottom.
- Keep the microscope away from the bench's edge.
- When not in use, turn off the illuminator and disconnect the power cables from
the power supply.
- While focusing, be careful not to break the coverslip, microscope slide, or even
the objective lens.
- Set the stage to the lowest position first.
- Use the lowest power objective lens to locate the ready specimen, then switch to
the higher power objective lenses.
- Never use the coarse adjustment knob to focus the high power lenses, and never
use these lenses to examine thick specimens.

3.2 Preparing the specimen:

Material : a microscope glass slide, a microscope cover glass, observed specimen

Step 1: Put the specimen in the middle of the glass slide.

Step 2: Add a drop of water or alcohol or stain onto a specimen to make the
specimen can be seen more clearly (this step is unnecessary if the specimen is
already wet).

Step 3: Lower slowly the coverslip starting from the edge until the two glasses
slides stick together. Advoiding the forming of air buddle.
 3.2 Using the light microscope:
- Use lens-cleaning sheets, clean the eyepieces and objective lens (if necessary)
- Put the prepared microscope glass slide into the clip
- Lower the stage of the microscope and turn the objective lens to the lowest power
objective (4x)
- Turn on the illumination and diagram and adjust the consender level
- Use the X and Y-axis knobs to move the specimen into the light region of the
stage
- Use the coarse focus (clockwise direction), raise the stage near to the objective
lens while observing through the eyepieces until the specimen comes into focus.
When focusing the microscope, be careful that the objective lens doesn’t touch
the slide, as it could break the slide and destroy the specimen.
- Adjust the light intensity as needed with the diaphragm, and center the specimen
by sliding the slide with the X and Y-axis knobs.
- Change the scanning objective (4x) to the high-power objective (10x), (40x), and
(100x). Adjust the focus using the fine focus knob solely to fine-tune it.