quick review on use and importance of compound microscope

1. MICROSCOPE
&
ITS USES
-Dipesh Tamrakar

2. HISTORY
 Circa 1284 - Italian, Salvino D'Armate is credited with
inventing the first wearable eye glasses.
 1590 – Two Dutch eye glass makers, Zaccharias Janssen and
son Hans Janssen experimented with multiple lenses placed in
a tube creating both the forerunner of the compound
microscope and the telescope.
 1674 – Anton van Leeuwenhoek built a simple microscope
with only one lens to examine blood, yeast, insects and many
other tiny objects. Leeuwenhoek was the first person to
describe bacteria.
 1931 – Ernst Ruska co-invented the electron microscope for
which he won the Nobel Prize in Physics in 1986.

3. INTRODUCTION

Micro: small and Scope: to view
 A microscope is an optical instrument that uses a lens or a
combination of lenses to magnify and resolve the fine details of
an object.
 The earliest methods for examining physical evidence relied
solely on the microscope.
 The magnified image seen by looking through a lens is known as
a virtual image, whereas an image viewed directly is known as a
real image.
 The object to be magnified is placed under the lower lens, called
the objective and viewed through the upper lens, called the
eyepiece.

4.  The human eye can resolve objects down to about 0.2 mm
 The Microscope is used to magnify small objects, those below the 0.2 mm range
 Bacteria and cells are measured in mm or 1 x 10-6
meters
 Viruses are even smaller, measured in nm or 1 x 10 –9
meters
 There are several different types of microscopes

5. COMPOUND MICROSCOPE
 Principle
A real, inverted and magnified image of the object is
first produced by the objective lens. This collects the
light from the specimen and forms the primary image.
The eyepiece enlarges the primary image thus an erect,
virtual and magnified image is formed. This image
again acts as virtual object for the eye (lens of eye)
finally an image is formed on retina thus enabling to see
magnified image.

6. The principle of the compound microscope. The passage of light
through two lenses forms the virtual image of the object seen by the
eye.

7. THE COMPOUND MICROSCOPE
PARTS:
1. The Mechanical System
Base: the support.
Arm: the C-shaped upright structure.
Stage: the plate on which the specimens are placed.
Sub-stage: holding condenser with its iris diaphragm
Body Tube: the hollow tube on which the objectives and
eyepiece lenses are mounted.
Coarse Adjustment: the knob used to focus the microscope
lenses by moving the body tube.
Fine Adjustment: the knob also used to focus the lenses by
moving the body tube, but by a much smaller magnitude.
Condenser Adjustment: the knob moving condenser lens

8. PARTS CONTD…
2. The Optical System
1. Objective Lens: the lens closest to the specimen; usually
several objectives are mounted on a revolving nosepiece.
 Parafocal: when the microscope is focused with one objective in place,
another objective can be rotated into place and the specimen remains
very nearly in correct focus.
1. Eyepiece or Ocular Lens: the lens closest to the eye.
 Monocular: a microscope having only one eyepiece
 Binocular: a microscope having two eyepieces.
1. Condenser: lens system under the microscope stage that
focuses light onto the specimen.

9. PARTS CONTD…
3. The light source:
i. Day light
ii. Electric light:
a. 60 W electric bulb placed 45 cm from the microscope
b. Built in source of light (halogen bulb)
iii. Battery lamp: 6 W bulb connected to 6 Volt
battery.

10.  Condenser/ Iris-Diaphragm
 Fine-focus Adjustment Knob
 Coarse-focus Adjustment Knob

11. • ArmArm
• Mechanical Stage Adjustment knobsMechanical Stage Adjustment knobs
• Fine-focus Adjustment KnobFine-focus Adjustment Knob

12. • Ocular lensesOcular lenses
• Nose pieceNose piece
• Slide ClipSlide Clip
• Mechanical stageMechanical stage
• Light sourceLight source
• BaseBase

13. FINE-FOCUS VS. COARSE-FOCUS
 Condenser/Iris-Diaphragm
 Course-focus knob
 Fine-focus knob

14. OBJECTIVE LENSES
 Objective lenses
 4x or Scanning lens
 10x or Low Power lens
 40x or High Power lens
 100x or Oil immersion lens
 Lens Power
 Lens Numerical Aperture

15. MAGNIFICATION:
 The degree of enlargement is the magnification of the
instrument.
 The magnification of an objective is obtained as follows:
magn = size of image / size of object
Or
= (mechanical tube length/ focal length
of objective) X mag of eyepiece
Or
= obj mag X eye piece mag

16. RESOLVING POWER
 Resolving Power – the ability of an optical system to distinguish or separate two small,
distinct objects or points from one another
Resolving power is a function of the wavelength of light being used (λ), in nm, and
another factor called “the numerical aperture” (NA) of the objective lens
 Numerical Aperture – a mathematical constant that describes the relative efficiency of
a lens in bending light rays
 In general, the shorter the wavelength of light being used (λ), the better the resolving
power
Depth of Focus – The range in distance between the lens and the specimen that can
be changed without upsetting the sharpness or resolution of the image. Depth of
focus decreases as magnification increases.
 Diameter of Field – The diameter of the total field of view through a microscope at a
given magnification. Diameter of field decreases as magnification increases.
N.A.x2
nmin
.)P.R(powersolvingRe
λ
=

17. OIL IMMERSION LENSES
 To achieve the maximum possible magnification and resolution with the light
microscope immersion oil must be used
 The light rays do not bend when passing from glass to immersion oil
 Allows the maximum amount of light to enter the objective lens
 Effectively increases the Numerical Aperture (NA) of the lens
 Can achieve magnification up to 1000 x with the light microscope
 Immersion oil must always be used with an oil immersion lens (100x)!
 Conversely, immersion oil should never be used with the other objective lenses!

18. OIL IMMERSIONS:
Oil immersionOil immersion Refractive indexRefractive index
1.1. Canada balsamCanada balsam
2.2. Xylol balsamXylol balsam
3.3. Cedar wood oilCedar wood oil
4.4. EuparolEuparol
5.5. GlycerolGlycerol
6.6. AnisolAnisol
7.7. WaterWater
1.5351.535
1.5241.524
1.5101.510
1.4831.483
1.4601.460
--
1.01.0
Note: good oil immersion of NA approx 1.4Note: good oil immersion of NA approx 1.4

19. MICROSCOPES
 Expensive instruments
 Require care while using and handling
 Always carry using two hands, one hand supporting the base,
and one hand grasping the arm
Steps using a compound Light Microscope
1. Plug in the power cord .
2. Turn the power on and adjust the light intensity as necessary.
3. Use the Course Adjustment Knob to obtain maximum working
distance.
4. Place your slide on the stage in the slide clip.
5. Adjust the light intensity using the iris-diaphragm.
6. Begin observations using scanning power (4X) and use the
coarse adjustment knob to obtain the minimum working
distance until the specimen comes into focus.

20. 7. Adjust the slide position on the stage until the specimen is
centered in the field of view.
8. You may now use the stage adjustment knobs to scan the
specimen.
9. Use the Fine-focus knob to focus and obtain the sharpest
possible image.
10. You may now rotate the Low Power objective into
position.
11. Since these microscopes are parafocal the image should
be just out of focus with this new objective lens.
12. Use the Fine focus knob, moving in small increments in
both directions until a clear, focused image is obtained.
13. You may now move the High Power Objective lens into
position. Do not use the coarse focus knob for focus
adjustments with high power or oil immersion lenses.
Adjust focus using the fine-focus knob only.

21. USING THE OIL IMMERSION LENS
1. Once a clear focused image is obtained at high power (40X) rotate the
objective lens halfway between the high power and oil immersion
objective lens.
2. Place a drop of immersion oil on the slide where the light passes through
the glass slide.
3. Then rotate the oil immersion lens (100X) into position.
4. The lens should just contact the immersion oil.
5. Moving only the fine focus knob in small increments, and in both
directions, bring the specimen into sharp focus.
6. Adjust the light intensity using the iris-diaphragm as necessary.

22. CLEANING AND STORAGE
1. When finished viewing the slide use the coarse adjustment knob to
maximize the working distance and remove the slide from the stage.
2. If you want to view another slide start the process from the beginning
and follow the steps previously listed.
3. If you are finished with the microscope, clean the stage and lenses
using only lens paper and lens cleaning fluid if necessary. Clean the
ocular lenses, scanning objective lens, low power, high power, and
finally the oil immersion lens, in that order.
4. Rotate the scanning objective lens into position and move the stage to
minimum working distance for storage.
5. Turn the light off and unplug the power cord.
6. Store the power cord as directed and replace the dust cover.
7. Store in the appropriate space as directed.