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MICROSCOPEBASICS

Dr.T.V.Rao MD

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THE HISTORY Many people experimented with

making microscopes

Was the microscope originally made by accident? (Most people were creating telescopes)

The first microscope was 6 feet long!!!

The Greeks & Romans used “lenses” to magnify objects over 1000 years ago.

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THE HISTORYHans 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 Leeuwenhoek1632-1723

Robert Hooke 1635-1703

Hooke Microscope

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ANTIONI VAN LEEUWENHOEK

• Leeuwenhoek is called "the inventor of the microscope"

• Created a “simple” microscope that could magnify to about 275x, and published drawings of microorganisms in 1683

• Could reach magnifications of over 200x with simple ground lenses

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DHOW A MICROSCOPE

WORKS

Convex Lenses arecurved glass used to make microscopes(and glasses etc.)

Convex Lenses bendlight and focus it inone spot.

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DHOW A MICROSCOPE

WORKS WITH..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).

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INTRODUCTIONA 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 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.

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D DEFINITIONS

Absorption When light passes through an object the intensity is reduced

depending upon the color absorbed. Thus the selective absorption of white light produces colored light.

Refraction Direction change of a ray of light passing from one transparent

medium to another with different optical density. A ray from less to more dense medium is bent perpendicular to the surface, with greater deviation for shorter wavelengths

Diffraction Light rays bend around edges - new wavefronts are generated

at sharp edges - the smaller the aperture the lower the definition

Dispersion Separation of light into its constituent wavelengths when

entering a transparent medium - the change of refractive index with wavelength, such as the spectrum produced by a prism or a rainbow

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REFRACTION

Light is “bent” and the resultant colors separate (dispersion).Red is least refracted, violet most refracted.

dispersion

Short wavelengths are “bent” more than long wavelengths

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DREFRACTION & DISPERSION

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Light is “bent” and the resultant colors separate (dispersion).Red is least refracted, violet most refracted.

dispersion

Short wavelengths are “bent” more than long wavelengths

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DBECAUSE OF REFRACTION WE CANNOT

SHOOT SOMETHING IN WATER0

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.But it is really here!!

He sees the fish here….

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LENSES AND THE BENDING OF LIGHT

light is refracted (bent) when passing from one medium to another

refractive indexa measure of how greatly a substance

slows the velocity of lightdirection and magnitude of bending

is determined by the refractive indexes of the two media forming the interface

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LENSES focus light rays at a

specific place called the focal point

distance between center of lens and focal point is the focal length

strength of lens related to focal length short focal length

more magnification

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DPRINCIPLES IN

MAGNIFICATION

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THE LIGHT MICROSCOPE

many typesbright-field microscopedark-field microscopephase-contrast microscopefluorescence microscopes

compound microscopesimage formed by action of 2 lenses

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COMPOUND MICROSCOPES

In compound microscopesimage formed by action of 2 lenses

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THE COMPOUND MICROSCOPE

The Optical SystemObjective 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.

Eyepiece or Ocular Lens: the lens closest to the eye.Monocular: a microscope having only one eyepiece

Binocular: a microscope having two eyepieces.

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THE COMPOUND MICROSCOPE

The Optical SystemObjective 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.

Eyepiece or Ocular Lens: the lens closest to the eye.Monocular: a microscope having only one eyepiece

Binocular: a microscope having two eyepieces.

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COMPOUND MICROSCOPE

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DARK FIELD MICROSCOPE

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DPRINCIPLES OF PHASE

CONTRAST MICROSCOPE

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FLUORESCENT MICROSCOPE

Dichroic Filter

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter

EPI-Illumination

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ELECTRON MICROSCOPE

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THE BRIGHT-FIELD MICROSCOPEproduces a dark image against a brighter background

has several objective lensesparfocal microscopes remain in

focus when objectives are changed

total magnification product of the magnifications

of the ocular lens and the objective lens

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THE CONVENTIONAL MICROSCOPE

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Focal lengthof objective= 45 mm

Object toImage Distance = 195 mm

Mechanicaltube length= 160 mm

Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press

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Eyepiece

Body Tube

Revolving NosepieceArm

Objective Lens

StageStage Clips

Coarse Focus

Fine Focus

Base

Diaphragm

Light

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Body Tube

Nose Piece

ObjectiveLenses

Stage Clips

Diaphragm

Light Source

Ocular Lens

Arm

Stage

Coarse Adj.

Fine Adjustment

Base

Skip to Magnification Section

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SOME PRINCIPLES 04

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Rule of thumb is is not to exceed 1,000 times the NA of the objective

Modern microscopes magnify both in the objective and the ocular and thus are called “compound microscopes” - Simple microscopes have only a single lens

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BINOCULAR MICROSCOPE Schematic diagram

of a stereoscopic microscope. This microscope is actually two separate monocular microscopes, each with its own set of lenses except for the lowest objective lens, which is common to both microscopes.

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DDIAGRAMMATIC REPRESENTATION

OF MICROSCOPE

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31The principle of the compound microscope. The passage of light through two lenses forms the virtual image of the object seen by the eye.

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MAGNIFICATION An object can be focused generally no

closer than 250 mm from the eye (depending upon how old you are!)

this is considered to be the normal viewing distance for 1x magnification

Young people may be able to focus as close as 125 mm so they can magnify as much as 2x because the image covers a larger part of the retina - that is it is “magnified” at the place where the image is formed

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MAGNIFICATION To determine your magnification…you just

multiply the ocular lens by the objective lens Ocular 10x Objective 40x:10 x 40 = 400

Objective Lens have their magnificationwritten on them.

Ocular lenses usually magnifies by 10x

So the object is 400 times “larger”

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MAGNIFICATION0

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1000mm

35 mm slide24x35 mm

M = 1000 mm35 mm

= 28

The projected image is 28 times larger than we would see it at 250 mm from our eyes.

If we used a 10x magnifier we would have a magnification of 280x, but we would reduce the field of view by a factor of 10x.

There used to be things called “slide Projectors”

p

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MICROSCOPE RESOLUTION Ability of a lens

to separate or distinguish small objects that are close together

Wavelength of light used is major factor in resolutionshorter wavelength

greater resolution

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INFINITY OPTICS0

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Sample being imaged

Primary Image Plane

Objective

Other optics

Ocular

Other optics

Tube Lens

InfiniteImageDistance

The main advantage of infinity corrected lens systems is the relative insensitivity to additional optics within the tube length. Secondly one can focus by moving the objective and not the specimen (stage)

Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press

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OBJECTIVES 04

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Limit for smallest resolvable distance d between 2 points is (Rayleigh criterion):

d = 1.22

Thus high NUMERICAL APERTURE is critical for high magnification

In a medium of refractive index n the wavelength gets shorter:n

This defines a “resel” or “resolution element”

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NUMERICAL APERTURE Resolving power is directly related to numerical

aperture. The higher the NA the greater the resolution Resolving power:

The ability of an objective to resolve two distinct lines very close together

NA = n sin u

(n=the lowest refractive index between the object and first objective element) (hopefully 1)

u is 1/2 the angular aperture of the objective

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NUMERICAL APERTURE0

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m

NA=n(sin m)

Light cone

(n=refractive index)

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DMICROSCOPE OBJECTIVES

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SpecimenCoverslip

Oil

MicroscopeObjective

Stage

60x 1.4 NAPlanApo

Standard Coverglass Thickness

#00 = 0.060 - 0.08#0 = 0.080 - 0.120#1 = 0.130 - 0.170#1.5 = 0.160 - 0.190#2 = 0.170 - 0.250#3 = 0.280 - 0.320#4 = 0.380 - 0.420#5 = 0.500 - 0.60 mm

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Refractive Index

Objective

n=1.52

n = 1.52

n = 1.52

Specimen

Coverslip

Oil

n=1.33

n = 1.52

n = 1.0

n = 1.5

Water

n=1.52

Air

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DOIL IMMERSION INCREASES

MAGNIFICATION

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CARING FOR A MICROSCOPE

Clean only with a soft cloth/tissue

Make sure it’s on a flat surface

Don’t bang it

Carry it with 2 HANDS…one on the arm and the other on the base

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DCARRY A MICROSCOPE

CORRECTLY

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USING A MICROSCOPEStart on the lowest magnificationDon’t use the coarse adjustment

knob on high magnification…you’ll break the slide!!!

Place slide on stage and lock clipsAdjust light source (if it’s a

mirror…don’t stand in front of it!)Use fine adjustment to focus

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DTEACHING MICROSCOPY IS

A ART

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Created by Dr.T.V.Rao MD for “ e “ Learning for

Basic Medical Graduates in Developing countries

email doctortvrao@gmail.com