Light Microscopy

1. By: ShefaaHejazy.
Umm Al-QuraUniversity, Makkah.
Faculty of Medical Sciences. Hematology Dept.
1
st
Semester 1433/2012

2. Introduction
Objectives:
Recognize parts of a compound lightmicroscope (LM)
Learn how to focus an object using Objective Lenses
How to adjust the light source by using the condenser
and iris diaphragm

3. LM in Haematology laboratory
TheMicroscope can magnify an object 100-1000 times of
it’s original size
A microscopic object can be visible by using a system of
lenses and illumination source
Introduction

4. LM use a system of lenses (objectives and oculars) to
manage the path of light beam that travel b/w the object and
eyes in order to magnify that object
A Light compound microscope have two ocular lenses
Objective lenses
Principle and function

5. Parts of Light Microscope
1.Eye piece(ocular lenses):
Magnifying lenses with a magnification power (10x).

6. 2.Body tube:
Contains mirrors and prisms that transmit the image from
the objective lens to the ocular lens.

7. 3.Objective lenses:
Primary lenses that magnify specimen
a)Low power 10x
b)High power 40x
c)Oil immersion 100x

8. 4.Stage:
Holds slide in position.

9. 5.Condenser:
Contains lens system that condenses light before it passes
through the specimen.

10. 6.Iris diaphragm:
Control the amount of light entering the condenser.

11. 7.Coarse and fine adjustment knobs:
Used for focusing.

12. 8.Light:
Source of illumination, bulb.

13. Types of Light Microscopy
A.Bright field microscopy.
B.Dark field microscopy.
C.Fluorescence microscopy.
D.Phase contrast microscopy.

14. A.Bright field microscopy
Stained preparation/ slides

15. B. Dark filed microscopy

16. This method use to examine unstained preparation
e.g. Treponemaspecies.

17. D. Phase contrast microscopy

18. Contrasti.e. structures with differing degrees of brightness or darkness
Denser parts of cell appear Bright
While those have density close to water will appear Dark

19. C. Fluorescence microscopy

20. Most commonly used fluorescent dyes
Acridineorange Auramine/rhodamine

21. “Cryptococcusneoformans”stained blue with Calcoflourwhite

22. Materials
Compound light microscope.
Lens cleaning paper/ cloth.
Immersion oil.
Wet preparation.
Stained preparation.

23. Wet preparation method

24. Examination of a Wet Preparation Using 10x
and 40x Objectives:
First select the 10x objective and focus the slide using coarse
adjustment knob, by moving it up or downward. Then make it clearly
visible using the fine adjustment knob.
Turn to 40x objective and again focus using fine adjustment knob until
object is clearly visible.

25. 1.Focus the smear at 10x then rotate to 100x and add a drop of oil on the
slide.
2.Upward the condenser and open iris diaphragm at maximum level.
3.Slowly upward the stage by coarse knob until the picture appear and the
objective is immersed with oil.
4.Use fine adjustment to gain a clear picture of the slide.
Examination of stained preparation with 100x objective

28. What is the total magnification power?
Power of the objective lens XMagnification of ocular lens
Resolving power of the microscope:
It is a measure of its ability to distinguish b/w two adjacent objects.
The absolute limit of resolving power is the light wavelength
= (400-800nm).
10x

29. Maintenance of microscope
Microscope is a delicate instrument; thus it should be …
Handled gently.
Keep in a clean environment.
Stage if contaminated should be cleaned immediately
with saline to avoid corrosion.

30. A. Optics:
The low and high power should be cleaned after and prior using with
lens paper tissue.
The Oil immersion objective lens can be cleaned with lens paper
tissue and few drops of Xyelen.
B. Non-optics:
Ocular lenses:using a soft camel-hair brush.
Condenser and iris diaphragm: using a soft cloth or tissue moistened
with toluene; and the mirror with 5% alcohol.
Other parts cleaned with mild detergent. Grease/oil removed with
petroleum ether followed by 45% ethanol in water or 5% alcohol.
How to clean a microscope

32. Ocular lenses
Body
Arm
Objective Lenses
Stage
Condenser
Iris Diaphragm
Coarse adjustment
Fine adjustment
Light source