Basic Microscopy

DMSFI Histology Department

 O
L B 1. Explain the significance of each part of the
E J microscope;
A E
2. Describe how each observation procedure is
R C done correctly; and
N T
I I 3. Demonstrate efficiently and effectively the
N V proper way of handling and manipulating the
G E microscope
S
I
N
T
R - Comes from the Greek words “Mikros” and
O “Skopein” which mean “small” and “to look”
D respectively.
U
C
T
- It is an instrument used to see objects that
are too small for the naked eye
I
O
N
P
To magnify a small
U object or to magnify
R the fine details of a
larger object in order to
P examine minute
O specimens that cannot
be seen by the naked
S eye.
E
COMPOUND, BRIGHTFIELD MICROSCOPE
PARTS
PARTS

On/Off switch
• Turns the illuminator
on or off
Brightness adjustment:
• Adjusts the intensity
of light emitted by
the illuminator
PARTS

Microscopic
illuminator
• Light source of the
microscope

• Captures light from

an external source
of a low voltage at
about 100v
PARTS

Base
• Acts as the support
of the microscope

• Carries the
microscopic
illuminators
PARTS

Head

• Carries the
optical parts in
the upper part of
the microscope
PARTS

Arm
• Connects the Head
and the Base
• Supports the head of
the microscope
• Used when carrying
the instrument
PARTS

Eyepiece
• Also known as the
“Oculars” which is the
part used to look
through the
microscope
• Found at the top
• Standard magnification
= 10x
(But can have 5x - 30x)
PARTS

Diopter
Adjustment
• Means to change
focus on one
eyepiece so as to
correct for any
difference in vision
between your two
eyes
PARTS

Nose piece or Turret

• Also known as
revolving nose piece
• Holds the objective
lenses
• Movable hence it can
revolve the objective
lens as you change the
magnification power
PARTS

Objective
Lenses
• Major Lenses used for
specimen
visualization

• Magnification power
of 4x – 100x
 MAGNIFICATION: Ability to produce an image of an
object at a larger scale than its actual size
T RESOLUTION/RESOLVING POWER: Ability to
E distinguish between two closely spaced points on a
specimen; smallest distance between two
R structures at which they can be seen as separate
M objects
- Light microscope: 0.2 um
S
DEPTH OF FIELD: The distance through which you
can move the specimen and still have it in focus
Field of View
view through a microscope.
 Field of View: the area a person can _____
circle
 It is represented by a _______.
increases field of view __________.
 RELATIONSHIP: As magnification __________, decreases
 As you switch from scanning (40X) to high power (100X), the area you see
through the microscope gets smaller.
 We can see better detail with higher powers of magnification, but we cannot
see as much of the image.
PARTS
PARTS
Scanner Objective
4x Magnification
power
PARTS
Low Power
Objective
10x Magnification
power
PARTS
High Power
Objective
40x Magnification
power
PARTS
Oil Immersion
Objective
100x Magnification
power
PARTS
Oil Immersion
Objective
100x Magnification
power
Total Magnification Power
- This is the final magnified view of the image brought about by
the product of the magnification power of the oculars and the
objective lens used.

- Objective lens Magnification x Ocular lens magnification = TMP

PARTS

Stage
• Where the specimen is
placed for viewing

• Contains Stage clips that

hold the specimen slides in
place

• Allows the control of the

slides by using the
mechanical knobs instead
of moving it manually
 The lens inverts the image of the object inside the
Inverted means that the image appears
microscope. _________
upside down and backward compared with the actual
object.

• This means that the slide must be moved in the

opposite direction that you want the image to move.
• Ex. If you move the slide to the right on stage, as
you look through the microscope the slide appears
to move ____.
left
PARTS

Aperture
• Hole on the microscope
stage through which the
transmitted light from
the light source reaches
the stage
PARTS

Condenser
• Used to collect and focus
light from the illuminator
into the specimen
• Plays a major role in
ensuring clear sharp images
that are produced with a
high magnification of 400x
and above
• Found under the stage next
to the diaphragm
PARTS

Diaphragm
• Also known as the Iris
• Controls the amount of
light that reaches the
specimen
• Adjustable apparatus hence
the intensity and size of the
beam of light can be
adjusted
• Found under the stage
PARTS

Adjustment knobs
• Knobs used to focus the
microscope
• Two types:
• Coarse adjustment:
Brings the specimen
into general focus
• Fine adjustment: Fine
tunes the focus and
increases the detail of
the specimen
How does a Microscope work?

This straight-line path is disturbed by

any material of different refractive
index.

Condenser (1.515) -> Air (1.00) ->

Slide (1.515) -> Mounting media
(1.515) -> Cover slip (1.515) -> Air
(1.00) -> Objective (1.515)

Adding immersion oil (1.515) fills in the

air gaps!
 - Always carry the microscope upright (otherwise
the eyepieces may fall out)
H
A
R - Use both hands, one grasping the arm or back
N slot and the other supporting the base
U
D
L
L
E
- Position it on a flat and solid surface where it
I will not be easily knocked off
S
N
G
- Lenses must be clean for resolution. Only use
lens paper or clean tissue. Do not remove any
parts for cleaning
H
A
R
N
U
D
L
L
E
I
S
N
G
 - Always start with the lowest power objective
H (Scanner – 4x) to get oriented and locate an
A area of interest
R
N
D
U - Switch to a higher power to examine field views
L more closely
L
E
I
S - To change magnification, simply rotate the
N nosepiece to bring one of the objectives into
G the light path; you will hear/feel a “click” if the
objective is in place
 - Turn off the light source

H - Center the mechanical stage

A
N
R - Position the nosepiece at the Scanner objective

D
U
L
- Remove the slide from the stage
L
I
E - Clean the stage and lenses, wipe off any oil

N
S
- Wrap the cord around/inside the arm
G
- Carefully carry with two hands and gently place the
microscope in the proper cabinet
Troubleshooting
Occasionally you may have trouble with working your microscope. Here are some
common problems and solutions.

1. Image is too dark!

Adjust the diaphragm, make sure your light is on.

2. There's a spot in my viewing field, even when I move the slide the spot stays in
the same place!
Your lens is dirty. Use lens paper, and only lens paper to carefully clean the
objective and ocular lens. The ocular lens can be removed to clean the
inside. The spot is probably a spec of dust.

3. I can't see anything under high power!

Remember the steps, if you can't focus under scanning and then low power, you
won't be able to focus anything under high power. Start at scanning and walk
through the steps again.

4. Only half of my viewing field is lit, it looks like there's a half-moon in there!
You probably don't have your objective fully clicked into place. .
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Thank you!

DMSFI Histology Department