ZOO101 Final Term Highlighted Handouts 2023

BIOLOGICAL TECHNIQUES

Lesson 1: Microscopy

Microscopy is the technical field of using microscopes to view samples & objects that cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye).

MICROSCOPE: Is an instrument for viewing objects that are too small to be seen by the naked or unaided eye.

What is microscopy and what is it used for?

Light microscopy is a general term used for any type of microscopy where light is being transmitted from a source which is on the opposite side of the sample, to the objective lens. Generally, the light is passed through a condenser to focus it on the sample to have maximum brightness. After the light has passed through the sample, it goes through the objective lens to magnify the image of the sample & then to the oculars, where the enlarged image is viewed.

Types of Microscopes

A good microscope should have three properties:

1. Good resolution: Resolution power refers to the ability to produce separate images of closely placed objects so that they can be distinguished as two separate entities. The resolution power of: The unaided human eye is about 0.2 mm (200 μm) The light microscope is about 0.2 μm An electron is about 0.5 nm The resolution depends on refractive index. Oil has a higher refractive index than air.
2. Good contrast: This can be further improved by staining the specimen.
3. Good magnification: This is achieved by the use of concave lenses.

History of microscopy

MICROSCOPE: Is an instrument for viewing objects that are too small to be seen by the naked or unaided eye.

A microscope (Greek: mikron = small and skopein = to look).

MICROSCOPY: The science of investigating small objects using a microscope

1590 - Hans Janssen and his son Zacharias Janssen, developed first microscope.

• 1609 - Galileo Galilei

1625 – Giovanni Faber coined the name microscope for the compound microscope Galileo submitted to the Accademia dei Lincei

1590 - Hans Janssen and his son Zacharias Janssen, developed first microscope.

Parts of microscope

An eyepiece is that part of an optical system, which is directed to the viewer. It is a construction of at least one or more lenses. The function of the eyepiece in a microscope is to convert the realenlarged-intermediate-image from the objective into an enlarged-virtualimage. It contains the ocular lens, which the user looks through to see the magnified specimen. The ocular lens has a magnification that can range from 5x to 30x, but 10x or 15x is the most common setting.

Lens tube The lens tube is connected with the eyepiece and it´s main task is to hold it.

The diopter adjustment

It is a control knob on your binocular. It is designed to let you compensate for differences between your own two eyes. Once you set the diopter, then the two barrels should stay in proper relation.

The objective lenses

It combines with the eyepiece lens to increase magnification levels. Microscopes generally feature three or four objective lenses, with magnification levels ranging 4x to 100x.

Objective revolvers are used in microscopes with multiple objective lenses, that have different magnification factors. An objective (lens) is that part of an optical system, which is directed to the object. It´s task is to collect the light rays, that are reflected from the observed item. The objective generates a real-optical image.

The Microscope Illuminator

Microscopes require a light source for viewing. This can come in the form of a built-in, lowvoltage illuminator light, or a mirror that reflects an external light source like sunlight.

Stage and Stage Clips

The stage is a platform for the slides, which hold the specimen. The stage typically has a stage clip on either side to hold the slide firmly in place. Some microscopes have a mechanical stage, with adjustment knobs that allow for more precise positioning of slides.

Clip

The clip serves as a holder for the object plate and makes sure, that it doesn´t get out of its place unintentionally

Types

Depending on the number of lenses, there are two i. e

1. Simple light microscopes: use a single lens to magnify an object and cannot reach high
2. Compound light microscope (it has a higher magnification compared to the simple microscope because it uses at least two sets of lenses, an objective lens, and an eyepiece).

The lenses are aligned in that, they can be able to bend light for efficient magnification of the image.

The simplest microscope of all is a magnifying glass made from a single convex lens, which typically magnifies by about 5–10 times. Microscopes used in homes, schools, and professional laboratories are actually compound microscopes and use at least two lenses to produce a magnified image.

Most compound microscopes can magnify by 10, 20, 40, or 100 times

Magnification on a microscope refers to the amount or degree of visual enlargement of an observed object. Magnification is measured by multiples, such as 2x, 4x and 10x, indicating that the object is enlarged to twice as big, four times as big or 10 times as big, respectively.

Magnification Limits

For a standard light-based microscope, the maximum magnification extends up to 1,500x; beyond this, objects under view become excessively fuzzy because the wavelengths of light limit the clarity of images. Electrons, on the other hand, have much shorter wavelengths. According to Auburn University, electron microscopes produce useful images with magnifications up to about 200,000x.

Magnification and Distance on a Microscope

The magnification on a microscope must be adjusted carefully in proportion to distance. For optical microscopes, the higher the magnification, the closer the lens must be positioned to the object being observed. If the lens gets too close, it may crash into the specimen, destroying the slide or specimen and possibly damaging the lens, so exercise great care when using magnifications over 100x. Most microscopes allow for adjustment of the lens-object distance, as well as providing preset default positions that place the higher magnification lenses closer to the slide.

Degree of enlargement.

No of times the length, breadth or diameter, of an object is multiplied.

depends upon 3 factors

1. Optical tube length · 2. Focal length of objective · 3. Magnifying power of eye piece

4. Total magnification=magnification of the eyepiece x magnification of the objective.

=10x40 = 400

=10x100 = 1000

Principle of microscope

5. Generally.. microscopes visualize an image by using a glass lens and magnification is determined by, the lens’s ability to bend light and focus it on the specimen, which forms an image.
6. When a ray of light passes through one medium into another, the ray bends at the interface causing refraction.
7. The bending of light is determined by the refractive index, which is a measure of how great a substance slows the speed of light. The direction and magnitude of the bending of the light are determined by the refractive indexes of the two mediums that form the interface.