compound microscopy

UNITS OF MEASUREMENT

1m = 103 mm (millimetres)

1m = 106 µm (micrometres)

1m = 109 nm (nanometres)

Sometimes in old texts Angstroms (Å) are used (the

diameter of a hydrogen atom)

1m = 1010 Å

History

• In 14th century spectacles & lenses were used to magnify objects.

• During the later part of the 15th century, da Vinci stressed the important of lenses for studying small object

• The earliest recorded use of a magnifying lens goes back to Conrad Gesber (1558), a swiss biologist---- observed protozoan

• It was Zacharias Janssen, a Dutch astronomer in 1650 added another lens to the telescope & thus provided 1st time prototype of present day telescope & compound microscope.

• The microscope constructed by early scientist had a magnifying power between 10X & 80X

• Galileo had also constructed a microscope at the same time (1610) similar to that of Janssen & it was employed for the study of arrangement of the compound eye of insects

• He improved earlier type of telescope by adding focusing.

• Between 1590 & middle of 17th century, several improvement were made in terms of magnification & refinement of lenses

• Italian scientist- showed that glasses could be ground to any curvature for increasing the magnification.

• 1673 - Antioni van Leeuwenhoek – father of biology – 1st to use microscope for biological studies

• A simple microscope, high power magnification with some microscope having magnification 0f 300 X

• He studied everything he could think of e.g stagnant water, saliva, blood, sperm, insect etc

• He made 247 microscopes & published 450 letters describing protozoa, bacteria, sperms, RBC & nuclei of fish RBCs

• He made many observation of biological importance.• Leeuwenhoek’s contributions have been recorded in a

series of reports of the royal society of London

• Compound microscope was constructed by Robert Hooke (1665) & is forerunner of present day compound microscope.

• Many of Leeuwenhoek,s reports were confirmed by Robert Hooke

• book Micrographia, published in 1665, devised the compound microscope, most famous microscopical observation was his study of thin slices of cork.

• Improve & refinement added—more detail of cell available

• 1830 Achromatic lens were made• 1870 good quality of lens with oil immersion lens• Abbe is credited with design of 1st modern

microscope• Addition of condenser was last imp contribution

to development of compound microscope

Early Compound Microscopes

• Like a telescope in reverse

• Could magnify about 30 times– A magnifying glass

can magnify about 10 times

Microscope

Microbiology

Cytology

Histology

Embryology

Our journey begins here.

Simple to Complex – Life’s Levels of Organization

Properties of light1) Amplitude: minimum displacement of wave from an

equilibrium position• When a light wave decrease or increases in amplitude, it is

observed by our eye in the form of a difference in the intensity of light i.e darkness or brightness.

• Light ray passing through medium– there is decrease in amplitude

• The decrease – greater/lesser depending on refracting index of media

• Light ray can pass through a cell but various parts of cell have different refractive indices (RI).

• Cytoplasm – causes little or no decrease in amplitude- transparent

• Nucleus has higher RI- decrease in amplitude – darker in microscope

2) Frequency: It refers to the number of times a wave crest passes a particular point in one second.

• Frequency remains constant for a light wave.• Light waves or identical frequency of coherent rays

can combine or interfere with each other.

3) Wavelength: it is the distance between two wave crests

• Our eyes are sensitive to different wavelengths of light in the visible range (380-740 nm) & register them in the form of colour

Fig. 3-4

The light spectrumWavelength ---- Frequency

Blue light

488 nm

short wavelength

high frequency

high energy (2 times the red)

Red light

650 nm

long wavelength

low frequency

low energy

Photon as a wave packet of energy

Properties of Light

Reflection:- light bounces off an object• We see colors of objects based on the

wavelengths of light reflected by the surfaces of the object

Absorption:- black object absorbs light than reflect light will gain heat more rapidly than white objects which reflect than absorb light

• Refraction–Bending of light as it passes

through one medium to another of different density–Such as from air in to glass

microscope lens

•Degree of bending depends on relative refractive indices of the media

Principles of Microscopy• Refractive Index:- bending of light from one

medium to another of different densities• Given by RI= speed of light in a given medium speed of light in vacuum• Light is also bent as it passes through a glass

microscope lens• So shape of lens determines how exactly light is

bent• A convex-convex lens (one that is curved outward

on both on both sides) will bent parallel light rays so that light theoretically is focused at a single (focal) point.

Magnification• In light microscopy, visible light is bent by a

series of ground glass microscope lens to achieve magnification

• Magnification- enlargement of object• Two lenses magnify images – ocular lens &

objective lens• Image of specimen much larger than object

itself• Total magnification is the product of

magnification of individual lens

Resolution: degree to which detail in specimen is retained in magnified image

Resolving power:• Capacity of microscope or any other

instrument to distinguish between images of two pointed objects lying very close together

• Unaided eye – 0.1 mm apart• Microscope - 0.2 µm apart• Limit of resolutionLimit of resolution:• Minimum distance at which two objects

appears as two distinct object

ResolutionResolution

Actual What We Might See

Even if we magnify an image of two objects, we can not distinguish them unless we have adequate resolution.

Limit of resolution = 0.61λ NA

NA is light gathering capacity of objective

NA (Numerical aperture) = n sinα

constant

Wavelength of illumination

Numerical aperture

Refractive index of air or liquid between specimen & lens

Sine of semi-angle of the aperture

• Limit of resolution inversely proportional to resolving power (N.A)

• So higher N.A lower limit of resolution• Sinα value cannot exceed 1 & R.A of instrument is constant• 40X objective RA= 1.6, 100X objective RA= 1.6

• So limit of resolution = 0.61 X wavelength (λ) 1 X 1.4

• So limit of resolution directly dependent on wavelength

• In case of red light with a wavelength of 6000 ---- limit of resolution = 0.25 µm

• But if violet light which has wavelength of 4000 is used, then limit of resolution = 0.17 µm

• Thus NA is constant & only way to decrease the wavelength of the light

• For this purpose UV light can be used• But UV cannot pass through glass lenses thus

necessitating the use of quartz

Resolution vs. Magnification

Light MicroscopeLight Microscopelight as source light as source of of

illuminationilluminationglass lensesglass lenseslimited limited resolution resolution (loses resolving (loses resolving power at power at magnifications magnifications above 2000X)above 2000X)

THE MICROSCOPE• EYEPIECEYEPIEC

EE

THE MICROSCOPE• ARMARM

THE MICROSCOPE• BASEBASE

THE MICROSCOPE

• BINOCULAR BINOCULAR TUBETUBE

THE MICROSCOPE

• REVOLVINGREVOLVING NOSEPIECENOSEPIECE

THE MICROSCOPE

• OBJECTIVEOBJECTIVELENSLENS

THE MICROSCOPE

• MECHANICALMECHANICALSTAGESTAGE

THE MICROSCOPE

• STAGE STAGE CLIPSCLIPS

THE MICROSCOPE

• IRIS IRIS DIAPHRAGMDIAPHRAGM

THE MICROSCOPE

• COARSE COARSE ADJUSTMENT ADJUSTMENT KNOBKNOB

THE MICROSCOPE

• FINEFINE

ADJUSTMENTADJUSTMENT

KNOBKNOB

THE MICROSCOPE

• LAMPLAMP

THE MICROSCOPE

• BULBBULB

THE MICROSCOPE

• ON/OFFON/OFFSWITCHSWITCH

Reading an objective

How Does a Microscope Work?

the eye

the image

the specimen

Each lens magnifies theimage, increasing its overall size

A lens is a bi-convex disk that bends light

The farther the light rays are bent, the largerthe image appears

The bent rays produces an image

How Does a Microscope Work?

the eye

the image

the specimen

The image is always seen upside down and backwards from its actual position

Know and be able to use the Know and be able to use the vocabulary of the microscopevocabulary of the microscope

• Magnification• Resolution• Numerical

aperture

• image larger• image clearer• ability of lens to

gather light

Know and be able to use the Know and be able to use the vocabulary of the microscope vocabulary of the microscope

contd…contd…• Working

distance• Depth of field • Parfocality

• between lens & stage• bottom to top of

slide• all objectives in

reasonable focus at the same time

Care of microscope

• Carrying a microscope: one hand grabs the arm and the other hand supports the bottom of the base.

• Lens care– DO NOT TOUCH THE LENS! The oil from your hands can

etch the glass.– CLEAN THE LENS WITH LENS PAPER ONLY! Other paper

has fiber that can scratch the lens.• Putting away the microscope: rotate to the 4X

objective and roll the nosepiece away from the stage so that the space between the stage and nosepiece is at a maximum.

ThAnk YoU