light microscope

LIGHT MICROSCOPEDr. Vaani Lalitya

Objectives

Main topics addressed: The basics properties of light. Image formation. The light microscope and it’s

components. Advantages and limitations of light

microscope

Light

Visible light occupies a very narrow portion of 400 – 700nm between UV and Infrared radiation in the electromagnetic spectrum.

Electromagnetic energy is complex, which is both wave like and particle like.

The natural light we see is a complex mixture of lights with different wavelengths.

Therefore almost all light sources provide a mixture of wavelengths of light.

Electromagnetic spectrum.

Light

Natural light which is a mixture of lights of different wavelengths.

Monochromatic Laser

Properties of Light

Shorter wavelengths have higher energy for a given brightness of light.

Energy content of light is expressed as energy level or amplitude based electron volts per photon.

Visible light has an energy of one electron volt per photon and the energy increases as one moves towards the violet and ultraviolet range of the spectrum.

Properties of Light

Approaching the soft X- Ray spectrum, the energy levels reach 50-100eV per photon.

It is this higher energy in the shorter wavelengths of that is exploited to elicit fluorescence in some materials.

Emission spectrum. Color temperature.

Properties of light

Some of the light is absorbed by the medium through which it passes.

This is seen as reduction in amplitude and energy level.

The medium through which the light passes can also have an effect in the speed of light which is known as Retardation.

Retardation

The media through which the light passes will be able to slow down or retard the speed of the light in proportion to the density of the medium.

Higher the density, greater the retardation.

Light rays entering a sheet of glass at right angle are retarded but their direction is unchanged.

Refraction

If the light enters the glass at any angle other than right angle, a deviation in the direction will occur in addition to retardation, known as Refraction.

A curved lens will exhibit both refraction and retardation.

The extent of which is determined by angle of incidence, refractive index and curvature of the lens.

Refraction

Refractive index

Refractive index = Sin I Sin RWhere I is the angle of incidence and R is angle of

refraction.Higher the density of the medium, greater the RI. The RI of most transparent substances is known

and is of great value in the computation and design of lenses, microscope slides and coverslips, and mounting media.

Air has a refractive index of 1.00, water 1.30, and glass a range of values depending on type but averaging 1.50.

Image formation

Focal point. Focal length. Conjugate foci. Real image. Virtual image.

Focal point and focal length

Conjugate foci

There are conjugate foci on either side of the lens such that the object placed at one focus will produce a clear image on the screen placed at the other focus.

The conjugate foci vary in position. As the object is moved closer to the lens, image will be formed further away, at a greater magnification and inverted. This is real image.

This is formed by the objective lens of the microscope.

Real image formation

Conjugate foci

If the object is placed yet nearer the lens, within the principle focus, the image is formed on the same side as the object, is enlarged, the right way up, cannot be projected on to a screen and can be seen through lens. This is called virtual image.

This is formed by the eye piece of the microscope and it appears to be at a distance of approximately 25cm from the eye, around the object stage level.

Virtual image

Ray path through microscope

Chromatic aberration

Light is composed of spectrum of colors, each having a different wavelength, will be refracted to a different extent, with blue being brought to a shorter focus than red.

This lens defect is known as chromatic aberration and results in an unsharp image and distorted edges.

This is known as chromatic aberration and it’s correction is known as achromatism.


It is possible to construct compound lenses of different glass elements to correct this.

An achromat is corrected for two colors, red and blue, producing a secondary spectrum of yellow/green, which is in turn corrected by adding more lens components like fluorospar, three colors can be brought into focus – the more expensive – Apochromat.

Spherical aberration

Spherical aberration is caused by the virtue of its curvature, where the light rays entering the lens at the periphery are refracted more than the light rays entering at the center of the lens, and thus not brought to a common focus.

These defects are also corrected by using a combination of lens elements of different glass and of different shape.

Spherical aberration

LIGHT MICROSCOPE

A brief history…. 1590- Hans Lippershey and Zacharias Janssen have

made the first compound microscope. 1609- Galelio Galilie developed a microscope with a

convex and concave lens. 1665- Robert Hooke’s book called micrographia

documented a wide range of observations through microscope.

1674- Anton Van Leeuwenhoek 1826- Joseph Jackson Lister developed achromatic

lens 1860- Ernst Abbe discovers the Abbe sine condition. 1931- Ernst Ruska starts to build the first electron

microscope.

Zacharias Janssen

Anton van Leeuwenhoek’s microscope

Light microscopes

Two basic configurations of light microscope:

1. simple microscope 2. compound microscope

Simple microscope

Simple microscope is where a single lens or a closely placed set of lens is used to magnify the object.

The magnification is usually limited. Eg. Magnifying glass and eyepiece of

a compound microscope.

Compound microscope

Light microscope - Introduction

Also known as Optical microscope. The light microscope, so called

because it employs visible light and a system of lenses to visualize small objects.

Optical microscopes are the oldest design of microscope and were probably invented in their present compound form in early 17th century.

Light Microscope

Probably the most well-used and well-known research tool in biology.

The biggest challenge lies in A. obtaining sufficient contrast. B. finding the focal plane. C. obtaining good resolution. D. recognizing the subject when one

sees it.

Light microscope

Standard light microscope

Principally has two components: The microscope proper and The stand/supporting system.

Components of Compound microscope

Light source Condenser Objective stage Objectives Body tube Eye piece Magnification Supporting stand

Light source- Illumination Initially it used to be sunlight. Later oil lamps were used. Simple pearl bulb. Or high intensity

lamp. Built-in light source for the recent

microscopes. Halogen bulbs. Neutral density filters to reduce

excess brightness.

Condensers

The first major optical component. The main purpose of the condenser is to

focus the available light into the plane of the object.

Within comfortable limits, the more the light at the specimen, the better is the resolution of the image.

Capable of vertical adjustments Adjusting the Iris diaphragm/aperture

diaphragm.

Iris diaphragm

The correct adjustment is when the numerical aperture of the condenser is matched with the numerical aperture of the objective in use.

This is achieved, approximately, by removing the eye piece, viewing the sub-stage iris diaphragm in the back focal plane of the objective, and closing it down to two-thirds of the field of view.

Objective stage

Above the condenser is the rigid stage with an aperture(1-1 ½ inches) through which light may pass. It has metal spring clips.

The stage supports glass slide bearing the specimen and therefore should be sturdy and perpendicular to the optical path.

In order to hold the slide firmly and to move the slide easily and smoothly, a mechanical stage is either attached or built in.

Vernier scales are incorporated in most cases. Spherical stages.

The objective

The objective screws into the lower end of the body tube by means of a standard thread, thus all objectives are interchangeable.

They are usually designated by their focal length rather than their magnifying power because their actual magnifying power depends on the tube length at which they are used.

Some manufacturers label objectives with the magnifying power of the lens where there is no draw tube.

Objectives

The type and quality of the objective has the greatest influence on the microscope as a whole.

The main task of the objective is to collect the maximum amount of light possible from the object, unite it, and form a high quality magnified real image somewhere above.

Fixed tube length systems(160mm-DIN Std; And 170mm- Leitz only) have now been replaced by infinity corrected objectives.

Objectives

The ability of an objective to resolve detail is indicated by its numerical aperture and not by its magnifying power.

Resolution is restricted by two factors: the numerical aperture and wavelength of the light employed.

NA = n Sin u

Numerical aperture

N = n x Sin u, where n is the refractive index of the medium

between the coverglass over the object and the front lens of the objective, for example air, water, or immersion oil, and u is the angle included between the optical axis of the lens and the outermost ray that can enter the front lens

Body tube

Above the nose piece is the body tube Three main forms are available : monocular,

binocular, and the combined photo-binocular. Provision is made for the binoculars to adjust

the interpupillary distance. Altering the interpupillary angle may alter the

mechanical tube length thus altering the optical path.

This can be corrected by adjusting the individual eye piece tubes or by a compensating mechanism built into the tube.

Eye piece

Final stage in the path of the optical path.

Two most commonly used ones are huygenian (achromatic objective) and compensating eyepiece (apochromatic objective).

It is basically a simple microscope to observe image formed by the objectives.

It has two lens. Field lens(lower) and upper lens.

Eye piece

Their function is to magnify the image formed by the objective within the body tube, and present the eye with a virtual image, apparently in the plane of the object being observed; usually this is an optical distance of 250 mm from the eye

Magnification

Total magnification is the product of the magnification values of the objective and eyepiece, provided the system is standardized to an optical tube length of 160nm.

The formula for magnification is (Optical tube length/Focal length of

objective) × Magnification of eyepiece.

Micrometry

The standard unit of measurement in microscopy is a micron, which is 0.001mm.

An eyepiece micrometer scale is used in conjunction with a stage micrometer.

Eyepiece micrometer scale is divided into 1/10 and 1/100 graduations, is placed inside Huygenian eyepiece, resting on a field stop.

Kellner eyepieces have scale permanently in position.

Micrometry

The stage micrometer has a 3 x 1 inch slide on which a millimeter scale is engraved in 1/10 to 1/100 graduations.

Insert eyepiece and stage micrometer scales. Select an objective to be used and focus on

stage micrometer scale. Determine the no. of divisions on the

eyepiece scale equal to the no. of divisions on the stage micrometer scale. Remove the stage micrometer and focus on the object to be measured.

Micrometry

Determine the no. of eyepiece divisions exactly covered by the object.

Assuming 100 eyepiece divisions were equal to 10 stage divisions, and say for eg. the object has covered 15 eyepiece divisions,

100 stage divisions = 1mm = 1000 microns 10 stage divisions = 100microns 1 eyepiece division is equal to 1 micron, Then 15 eyepiece divisions = 15 microns.

Advantages of light microscope Most widely used tool to study

organic and inorganic research. Cost effective. Simple setup with very little

preparation required.

Disadvantages

Biological samples are often low contrast with little natural pigmentation, so samples usually need to be stained.

Staining may destroy or introduce artefacts Resolution is restricted to ~0.2 μm.

TLC!! Proper care of microscope! Cleaning. Daily cleaning routine. Microscope – dusted daily, outer

surface of the lens should be wiped with lens paper or well washed silk.

Top lens of the eyepiece polished to remove dust or fingermarks.

Clean the substage and mirror in a similar way

Cover it when not in use.

References

Bankroft’s theory and practice of Histological techniques, chapter 3.

Susan C. Lester’s manual of Surgical Pathology, Part I, Chapter 9.

Handbook of Histopathological technique by C. F. A. Culling.

https://en.wikipedia.org/wiki/Optical_microscope

http://www.nobelprize.org/educational/physics/microscopes/timeline/

http://www.nature.com/nprot/journal/v7/n9/fig_tab/nprot.2012.096_T1.html







Thank you…

light microscope

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