Microscope

Dr. Leena Barhate

Department of Microbiology

M.J.College, Jalgaon

Acknowledgement

• http://www.cerebromente.org.br/n17/histor

y/neurons1_i.htm

• Google Images

• http://science.howstuffworks.com/light-

microscope1.htm

• www.worldofteaching.com

• OU NanoLab/NSF NUE/Bumm & Johnson

The History

• Hans and Zacharias Janssen of Holland

in the 1590’s created the “first”

compound microscope

• Anthony van Leeuwenhoek and Robert

Hooke made improvements by working

on the lenses

Anthony van Leeuwenhoek

1632-1723

Robert Hooke

1635-1703 Hooke Microscope

Light Microscope

• Microscope utilizing light as a source of

illumination is called as light microscopes.

• Type of light microscopes

– Bright field microscope

– Dark field microscope

– Phase contrast microscope

– Fluorescence microscope

Bright field microscope

• Microscope that forms dark image against

bright background is called as bright field

microscope

• Widely used bright field microscope for

basic study of microbes is the compound

microscope

How a Microscope Works

Convex Lenses are

curved glass used to

make microscopes

(and glasses etc.)

Convex Lenses bend

light and focus it in

one spot.

The History

Zacharias Jansen

1588-1631

The “First” Microscope

How a Microscope Works

Ocular Lens

(Magnifies Image)Objective Lens

(Gathers Light,

Magnifies

And Focuses Image

Inside Body Tube)Body Tube

(Image Focuses)

•Bending Light: The objective (bottom) convex lens magnifies and focuses (bends) the image inside the body tube and the ocular convex (top) lens of a microscope magnifies it (again).

Body Tube

Nose Piece

Objective

Lenses

Stage

Clips

Diaphragm

Light Source

Ocular Lens

Arm

Stage

Coarse Adj.

Fine Adjustment

Base

Body Tube

• The body tube holds the objective lenses

and the ocular lens at the proper distance

Diagram

• It holds occular and objectives are lenses.

• It also provides sufficient space for image

formation

Nose Piece

• The Nose Piece holds the objective lenses

and can be turned to increase the

magnification

Diagram

• A base in which objectives are fixed

• Simply rotating the nosepiece can rotate

each objective into place

Objective Lenses

• The Objective Lenses increase

magnification (usually from 10x to 100x)

Diagram

• Second lens system of a microscope

• Mounted on nosepiece and can be rotated into

the place

• There are usually three objective lenses on a

microscope.

• Objective lenses are generally equipped with

microscope having low power, high power and

oil immersion lens and magnification of 10x, 45x

and 100x respectively

• Function: to make real image

• Working distance: it is the distance

between objective and object under

observation

Stage Clips

• These 2 clips hold the slide/specimen in

place on the stage.

Diagram

Iris Diaphragm

• The Diaphragm controls the amount of

light on the slide/specimen

Turn to let more light in or to

make dimmer.

Diagram

• It is equipped with condenser

• Control intensity of light condenser and

therefore controls the amount of light

intensity.

• Lever is equipped with it to adjust the light

intensity.

• Blue colour filter is also equipped below

the condenser

Condenser

• It is third lens system of microscope

• It is located below the stage

• It is responsible for focusing the light on

the specimen

• There are several different types of

condensers depending upon the type of

microscope to be employed

• Abbe’s condenser is most commonly used

Light Source (Illuminator)

• Projects light upwards through the

diaphragm, the specimen and the lenses

• Some have lights, others have mirrors

where you must move the mirror to reflect

light

Diagram

illumination

• Abbe and Nelson

– In this system light source, such as sun light

through a window, or an open lamp flame is

placed before the microscope mirror.

– Any structure or irregularity of the source is

seen directly in the field of view.

– It creates a problem to some extent during

examination of the specimen

• Koelhler’s type:

– It is second ,method of illumination

– Prepared by Dr. August Koehler.

– It eliminated field of view

– The koehler form of illumination is mostly

used today

– Here parallel rays of light generated usually

by a tungsten filament lamp are used to

illuminated the specimen

Ocular Lens/Eyepiece

• Magnifies the specimen image

Diagram

• It is the first lens system of microscope.

• It is present at top of the microscope

• In the microscope, the occular is capable

of 10x magnifications

• Eyepieces of 5x, 15x and 20x

magnification potential are also available.

• One eyepiece can be replaced by another

• Function: to make virtual image of

specimen

Arm

• Used to support the microscope when

carried. Holds the body tube, nose piece

and objective lenses

Diagram

Stage

• Supports the slide/specimen

Diagram

• It is the platform on which the specimen to

be viewed is placed.

• Some stages have clips to hold the glass

slide in place.

• Others have a mechanical stage, which

make it possible to move the slide across

the stage in both horizontal and vertical

directions

Coarse Adjustment Knob

• Moves the stage up and down (quickly) for

focusing your image

Diagram

• They are used to move the body

tube/stage relative to the objectives and

occular, making it possible to focus the

image

Fine Adjustment Knob

• This knob moves the stage SLIGHTLY to

sharpen the image

Diagram

Base

• Supports and stabilizes the microscope

Diagram

Types of Microscopes:

1. Compound Light Microscope (what we

use most often)

2. Stereoscopes – also known as dissecting

scopes

3. Electron Microscopes

Parts of the Microscope

Arm

Parts of the Microscope

Light Source

Diaphragm

Parts of the Microscope

Stage

Stage Clips

Parts of the Microscope

Revolving

Nosepiece

Objective

Lenses

Parts of the Microscope

Ocular Lens

Parts of the Microscope

Coarse adjustment knob

Used only when low power objective is used!!

Parts of the Microscope

Fine adjustment knob

Carrying a Microscope

Steps to Use:

1. Rotate the low power objective into place and make sure the

stage is all the way down.

2. Place slide on stage making sure object to be viewed is

centered over the hole in the stage. Use the stage clips to

hold the slide in place.

3. Turn light on.

4. Focus first with the coarse adjustment knob. Once in focus on

low power, turn the nosepiece until the next higher lens is in

place.

5. Use FINE adjustment knob ONLY and focus the object.

Remember:

1. If you are seeing perfectly round, clear circles then you

just may be looking at air bubbles. Check your slide and

try again.

2. Microscopes must always be properly put away.

3. Slides and cover-slips should be washed, dried, and

returned to their proper place.

Important Vocabulary :

magnification \mag-ne-fe-'ka-shen\ n 1.

apparent enlargement of an object 2.

the ratio of image size to actual size

A magnification of "100x" means

that the image is 100 times bigger

than the actual object.

resolution \rez-e-loo-shen\ n 1. clarity,

sharpness 2. the ability of a

microscope to show two very close

points separately

Highest Typical Resolution

Optical Microscope ~200 nm

Electron Microscope ~0.1 nm

Factor affecting magnification

• Optical tube length

• Focal length of objective

• Magnifying power of eye piece and

objective

Formula to determine magnification

• Magnification of microscope

= Objective Magnification x eye piece magnification

Magnification

ocular power = 10x

low power objective = 20x

high power objective = 50x

a) What is the highest magnification you

could get using this microscope ?

500x

Ocular x high power = 10 x 50 = 500. (We

can only use 2 lenses at a time, not all

three.)

b) If the diameter of the low power field is 2

mm, what is the diameter of the high

power field of view in mm ?

.8 mm

The ratio of low to high power is 20/50. So at high

power you will see 2/5 of the low power field of

view (2 mm). 2/5 x 2 = 4/5 = .8 mm

c) in micrometers ? 800 micrometers

To convert mm to micrometers, move the decimal

3 places to the right (multiply by 1000). .8 mm x

1000 = 800 micrometers

d) If 10 cells can fit end to end in the low power field

of view, how many of those cells would you see

under high power ? 4 cells.

We can answer this question the same way we go

about "b" above. At high power we would see 2/5

of the low field. 2/5 x 10 cells = 4 cells would be

seen under high power.

Numerical Aperture

• It is a ratio of diameter of lens to its focal

length

• Formula:

Numerical aperture(NA) = ηsinθ

η = Refractive Index

θ = Half angel of aperture

Numerical aperture

• BAC is cone of light

• θ is the half the angle of cone light formed at objective aperture

• Theoretical limit of BAC is 180°

(2θ = 180° )

• So θ = 90 °

• NA of dry lens cannot be greater that 1, since the refractive index of air is 1 and value of sin θ = 1

θ

θ

Resolution (Resolving power)

• It is the abililty to reveal closely adjacent

points as separate and distinct

• Formula:d = 0.5 x λ

NA

• Where

λ = Wavelength of light

NA = Numerical Aperture

• d= become small as resolution increase

• Hence resolution is inversely proportional to the wavelength and directly proportional to the numerical aperture.

• Maximum resolution = lowest wavelength light

• Compound microscope with blue filter below condenser help to resolve the image.

Example

• Green light wavelength =550nm

• Objective with NA = 1.4 then what will be the resolution?

d = 0.5 x λ

NA

d = 0.5 x 550

1.4

d = 196 nm

• Microscope can reveal two closely associated points by 196 nm.

Immersion oil and its use in

compound Microscope

• Observation by compound microscope,

using the 100x objective needs special oil.

• It is also known as immersion oil in

microscopy

• Immersion oil is cedar wood oil obtained

from gymnospermic juniperous vergiana

• It is colourless liquid and has refractive

index 1.55 (same as glass)

Advantage

• Use of oil avoids diffraction of rays.

• If air is present between specimen and

objective, some light is lost due to

diffraction of ray. Thus the image observed

is fuzzy and the finer detail may lost.

• Thus oil help to get sharper image.

• For Maximum resolution NA value must be high.

• The value of θ cannot exceed 90°.

• Hence by increasing refractive index NA value can be

increased

• Refractive index is function of the bending of light

from air though glass and back again.

• It can be made possible by filling medium which has

refractive index larger than refractive index of air

• Refractive index of oil is larger than air

OPTICAL MICROSCOPES

Image construction for a simple biconvex lens

Rayleigh criterion for resolution

www.microscopy.fsu.edu ; www.imb-jena.de

See more interactive tutorials at www.microscopy.fsu.edu

Numerical Aperature Resolution Rayleigh Criterion

ComparisonBright-

FieldDark-

Field• Full

aperture

is

illuminated

• A central

obstruction blocks

the central cone.

www.microscopy.fsu.edu

Dark-Field

Optical Microscopy•A central obstruction

blocks the central cone.

•The sample is only

illuminated by the

marginal rays.

•These marginal rays must

be at angles too large for

the objective lens to

collect.

•Only light scattered by the

object is collected by the

lens.

www.microscopy.fsu.edu

Dark-Field

Optical

Microscopy

THE ELECTRON MICROSCOPE

The wavelength of the electron can be tuned by changing the accelerating voltage.

de Broglie : λ = h/mv

λ: wavelength associated with the particle

h: Plank’s constant 6.63×10-34 Js;

mv: momentum of the particle

me= 9.1×10-31 kg; e = 1.6×10-19 coulomb

P.E eV = ½mv2 λ = h/(2meV) = 12.3/V (for V in KV, λ in Å)

V of 60 kV, λ = 0.05 Å Δx ~ 2.5 Å

Microscopes using electrons as illuminating radiation

TEM & SEM

Components of the TEM

1. Electron Gun: Filament, Anode/Cathode

2. Condenser lens system and its apertures

3. Specimen chamber

4. Objective lens and apertures

5. Projective lens system and apertures

6. Correctional facilities (Chromatic, Spherical, Astigmatism)

7. Desk consol with CRTs and camera

Transformers: 20-100 kV; Vacuum pumps: 10-6 – 10-10 Torr

Schematic of E Gun & EM lens

Magnification: 10,000 – 100,000; Resolution: 1 - 0.2 nm

www.udel.edu

TEM IMAGES

www.udel.edu ; www.nano-lab. com ; www.thermo.com