bio10-exercise-1-microscopy.pdf

General Biology

First Sem. A.Y. 2015-2016

Instrument used to make fine details visible

Device used to observe/study small and/or

detailed objects that are too small to be seen by

the naked eye

The microscope must accomplish three tasks:

– Create a magnified image of the specimen (magnify)

– Separate details in the image (resolve)

– Render the details visible to the eye, camera, or other imaging device (contrast)

Uses light

Uses electron beams

LIGHT MICROSCOPE

ELECTRON MICROSCOPE

o Refers to the use of transmitted light to observe specimens

SIMPLE COMPOUND

Simple glass magnifiers

Convex lenses

Uses a single lens to enlarge the object in one step

Two-stage magnification

The objective projects a magnified image into the draw tube of the microscope and the eyepiece further magnifies the image projected by the objective.

Virtual Image -

“Floating” in space about about 10 in below the top of the observation tube where the eyepiece is inserted.

Accurate representation of the specimen (detail, shape, color/intensity)

1. Optical Parts

2. Mechanical Parts

Mirror or Electric Lamps

Source of illumination

Provides light which is needed to view the specimen

Mirror has concave and plane side

Objectives / Objective Lenses

Gathers the light passing through the specimen and then projects an accurate, real, inverted IMAGE of the specimen up into the body of the microscope.

Scanner

Low power objective (LPO)

High power objective (HPO)

Oil immersion objective (OIO)

Eyepiece / Ocular lens

“Looks at” the focused, magnified image projected by the objective and magnify that image a second time (usually 10x) as a virtual image seen as if 10 inches from the eye.

Can be fitted with scales or markers or pointers or crosshairs that will be in simultaneous focus with the focused image.

Condenser

Substage component that gathers light from the light source and concentrates it into a cone of light that illuminates the specimen with uniform intensity over the entire field of view.

Iris diaphragm

Substage component that controls the amount of light that would enter the condenser

Closing the iris diaphragm will increase contrast at the cost of resolution

Numerical aperture

Resolving power and resolution

Magnifying power

Working distance

Numerical Aperture: width of the cone of light that can enter the objective lens.

A property of the lens, usually written on it.

The measure of the light gathering power of the lens

𝐍𝐀 = n (sin µ)

n = refractive index of the mediumµ = half of the angular aperture

MEDIUM n

Air 1.00

Water 1.33

Glycerine 1.47

Oil 1.515

The ability of the microscope to distinguish between two close objects as separate and distinct

Resolution: The smallest distance between two points

on a specimen that can still be distinguished as two separate entities.

Factors that affect resolving power and resolution

Numerical aperture of the objective

Wavelength of illuminator

Where:

R = Resolution or resolving distance

𝝀 = wavelength of illuminator

NA = numerical aperture

R = 𝝀

𝟐 𝑵𝑨

OBJECTIVE TYPE

Plan Achromat Plan Fluorite Plan Apochromat

Magnification

N.AResolution

(µm)N.A

Resolution(µm)

N.AResolution

(µm)

4x 0.10 2.75 0.13 2.12 0.20 1.375

10x 0.25 1.10 0.30 0.92 0.45 0.61

20x 0.40 0.69 0.50 0.55 0.75 0.37

40x 0.65 0.42 0.75 0.37 0.95 0.29

60x 0.75 0.37 0.85 0.32 0.95 0.29

100x 1.25 0.22 1.30 0.21 1.40 0.20

N.A. = Numerical ApertureWavelength

(nanometers)Resolution

(micrometers)

360 .19

400 .21

450 .24

500 .26

550 .29

600 .32

650 .34

700 .37

The ability of a lens to magnify an object

The degree to which an object is enlarged/reduced in size

2 types

Linear Magnification

Magnification of illustration

𝒍𝒊𝒏𝒆𝒂𝒓𝒎𝒂𝒈𝒏𝒊𝒇𝒊𝒄𝒂𝒕𝒊𝒐𝒏 = 𝒎𝒂𝒈𝒏𝒊𝒇𝒊𝒄𝒂𝒕𝒊𝒐𝒏𝒐𝒃𝒋𝒆𝒄𝒕𝒊𝒗𝒆 ×𝒎𝒂𝒈𝒏𝒊𝒇𝒊𝒄𝒂𝒕𝒊𝒐𝒏𝒐𝒄𝒖𝒍𝒂𝒓

Objective Mobj Mocu * LM

Scanner 4x 10x 40x

LPO 10x 10x 100x

HPO 40x 10x 400x

OIO 100x 10x 1,000x

*varies per ocular used. Some oculars have 15-20x magnification.

𝒎𝒂𝒈𝒏𝒊𝒇𝒊𝒄𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒊𝒍𝒍𝒖𝒔𝒕𝒓𝒂𝒕𝒊𝒐𝒏 =𝒔𝒊𝒛𝒆𝒅𝒓𝒂𝒘𝒊𝒏𝒈

𝒔𝒊𝒛𝒆𝒂𝒄𝒕𝒖𝒂𝒍

8 µmDrawing scale!

Given: size of illustration = 9cm; actual size of specimen = 5 um

𝒎𝒂𝒈𝒏𝒊𝒇𝒊𝒄𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒊𝒍𝒍𝒖𝒔𝒕𝒓𝒂𝒕𝒊𝒐𝒏 =𝟗 𝒄𝒎

𝟓 𝝁𝒎

8 µm

𝒎𝒂𝒈𝒏𝒊𝒇𝒊𝒄𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒊𝒍𝒍𝒖𝒔𝒕𝒓𝒂𝒕𝒊𝒐𝒏 =𝟗 𝒄𝒎

𝟎. 𝟎𝟎𝟎𝟓 𝒄𝒎

𝒎𝒂𝒈𝒏𝒊𝒇𝒊𝒄𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒊𝒍𝒍𝒖𝒔𝒕𝒓𝒂𝒕𝒊𝒐𝒏 = 𝟏𝟖, 𝟎𝟎𝟎𝐱

Distance between the tip of the objective and the specimen.

↑ magnification - ↓ working distance

↑ NA ↑ magnifying power

↑ NA ↑ resolving power

↑ magnifying power ↓ working distance

Base, Pillar and Arm Provide stability and

mechanical support to the microscope

Houses electrical components for operating and controlling the intensity of the lamp (base)

Supports the structures that hold the objective and ocular lenses (arm)

Supports the stage and substage (pillar)

Inclination joints*

Stage

Supports the specimen to be viewed

Has an opening for passing the light (stage aperture)

Specimens are held in place using the stage clips

Specimens can be moved using the stage knobs

Nosepiece

Holds the objectives of various magnifications

Dust shield

Prevents dirt from settling on the objectives

Body tube

Holds the nosepiece and dust shield

Draw tube

Holds the oculars in place

Adjustment knobs*

These elevate or lower the stage in larger (coarse) or smaller (fine) increments

Coarse adjustment knob is used to bring the specimen into view

Fine adjustment knob used bring specimen into focus

Condenser adjustment knobs

These elevate or lower the condenser to adjust contrast

Iris diaphragm lever

Controls the diameter of the iris diaphragm

OLD NEW

OLD NEW

Objectives 3 4, Spring-loaded

Range of magnification 100-970x 40-1,500x

Adjustment knobs Separate Combined

Coarse adjustment knob Moves the body tube Moves the stage

Illumination Natural or artificial light reflected through a

mirror

Electric lamp

Condenser knob Absent Present

Stage Immovable, can be tilted Movable, parallel

Stage clips Fixed Movable

Inclination joint Present Absent

Number of ocular 1 1-2

Produces 2D image of greater detail

Produces 3D image for examination and dissection of whole specimens

BRIGHTFIELD STEREOSCOPE

BRIGHTFIELD STEREOSCOPE

Magnification 40-1,000x Maximum of 50x

Orientation of image Inverted Upright

Movement of object across field

Opposite Same

Type of image produced

2D, Virtual 3D, Real

• Uses electron beams to magnify objects smaller than about 0.2µm, such as viruses and specific cell organelles

• Can magnify objects up to 100 000 times

• Better resolution is due to shorter wavelengths of electrons

• Images produced are always black and white

LIGHT MICROSCOPE ELECTRON MICROSCOPE

Source of “illumination”

Visible light Electron beams

Lens system Glass Electromagnets

Medium Air/Oil Vacuum

Specimen holder Glass slide Copper mesh grid

Maximum resolution 0.2µm 0.0025nm

Maximummagnification

2,000x 100,000x

Specimen Live or dead Dead

Image produced 2D 2D or 3D

Color of image Colored Black and white

Objects seen Frog’s eggs, cells, nuclei, mitochondria, lysosomes,

chloroplasts, flagella, cilia, cell wall, cell membrane

Ribosomes, endoplasmic reticulum, Golgi body, proteins, lipids, viruses, small molecules

LIGHT MICROSCOPE ELECTRON MICROSCOPE

1. Turn on the lamp and set the intensity for comfortable viewing.

2. Place the prepared slide on the stage and center it over the stage opening.

3. Raise the condenser into its position.

4. Turn the coarse adjustment knob to lower the stage until it stops.

5. While looking down the ocular, slowly focus upward with the coarse adjustment knob until the specimen comes into view.

6. Manipulate the diaphragm lever to reduce or increase the light intensity to produce the clearest, sharpest image.

7. For a higher magnification, center the specimen in the field of view, then rotate the HPO into position while watching from the side.

8. Focus using the fine adjustment knob.

It should take very little movement of the adjustment knob to bring the image into focus (parfocality).

A particle in the image centered in the field of view should remain in the center as objectives are changed (parcentricity).

1. Focus the specimen with the 10x objective. Then switch to the 40x objective and center a desired feature in the field of view.

2. Turn the nosepiece slightly (objectives are not set) and gently place a drop of immersion oil on top of the cover slip.

3. Rotate the immersion oil objective into the light path.

4. Use the fine adjustment knob until the specimen comes into focus.

Ocular Micrometer

Stage Micrometer

10 20 30 400

Calibration constant = Stage Units x 0.01 mm*

Ocular Units

= 4 x 0.01 mm

6

Calibration constant = 0.00667 mm

* 1o micrometers can be used instead of 0.01 mm

Actual size = Ocular units x C.C.

= 17 x 0.00667 mm

= 0.113 mm

10 20 30 400