L5 Optical Microscope

8/16/2019 L5 Optical Microscope

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OPTICALMICROSCOPY

ELECTRON MICROSCOPY

CHARACTERISATIONS OFMATERIALS



CONTENTS

3.0 Instrumentation

• 3.1 Sample Prep

• 3.2 Measurement Systems and Types

4.0 Examples

5.0 Correct Presentation of Results

• 5.1 Publication

• 5.2 Presentation





Let us watch video on parts of compoundmicroscope



Let us watch video on light sources inmicroscope



Let us watch video on lenses and eyepiecesystems



Anatomy of a Modern Light Microscopes

Illumination systemsProvides visible light by which the

specimen is observed.

Three main types:

1. Low voltage tungsten filament

bulbs

2. Tungsten halogen bulb

3. Gas discharge bulbs



Let us watch video on How does theMicroscope Work



Light Microscopy: Basics in opticsCompound Microscope

Sample is placed in front of

objective focal plane. Intermediate

imagei s formed by objective and is

observed through eyepiece.



Two Sets Of Conjugate Focal Planes In The Kohler SystemIllustrated In A Transmitted Light Microscopes



Type of Optical Microscope

Reflected-light microscopy

is used for a range of materials, including metals, ceramics and composites.

Contrast between different regions when viewed in reflected light can arisefrom variations in surface topography and differences in reflectivity (e.g. of

different phases, different grain orientations, or boundary regions).

These features are revealed by a series of specimen preparation techniques which, when carried out with care, can produce useful, high quality images.



Type of Optical Microscope

Transmission mode

can be used when the specimen is transparent.

The specimen is usually in the form of a thin slice (e.g. tens of microns thick).Contrast arises from differences in the absorption of light through differentregions.

This method is used for the examination of minerals and rocks, as well asglasses, ceramics and polymers. In addition, the transmission mode can often be further enhanced with use of polarised light.

Polarised light microscopy

is a specialised use of the transmission mode, and contrast is due to differencesin birefringence and thickness of the specimen.

This can allow the observation of grains, grain orientation and thickness.



Type of Light Microscope

Brightfield Microscope

Brightfield microscopes are the most common type. A brightfield microscope

focuses light through a condenser lens and passes it through the specimen. Above

the specimen is a tube containing an objective lens and an eyepiece lens that

together focus the light to form an enlarged image. Images seen through a

brightfield microscope generally have poor contrast unless the specimen has been

stained with dyes or chemicals that adhere to certain types of structures in the cell

Dark Field Microscopes

Dark field microscopes are similar to brightfield microscopes but are equipped with

a dark field patch stop and a direct illumination block. Together, these twocomponents ensure that only light scattered by the specimen passes through the

objective lens. When viewed through a dark field microscope, a specimen appears

as if it were on a black background. Dark field microscopy can provide enhanced

contrast, although some of the features that are visible with bright field microscopy

may not be visible with the dark field technique.



Fluorescence MicroscopesFluorescent materials absorb light or electromagnetic radiation of one wavelength

and re-radiate electromagnetic radiation of another wavelength (typically a longer

wavelength). In a fluorescence microscope, the specimen is first stained with

fluorescent dyes or molecules that adhere to specific molecules or structures in the

cell. Light from the light source shines on a dichroic mirror that reflects light of some

wavelengths and transmits light of other wavelengths. The light from the source isreflected onto the sample, where the fluorescent dyes absorb it and emit light that

passes through the dichroic mirror into the objective lens. A fluorescent microscope

produces an image in which only the cell components tagged with fluorescent dyes

are visible.

Confocal Microscope A confocal microscope is similar to a fluorescence microscope but incorporates

some additional features, most notably a pinhole aperture that excludes out-of-focus

light. Consequently, the confocal microscope can form a very sharp image of a

single plane in the sample. Confocal microscopes are powerful tools but require long

exposure times to form a good image.

Type of Light Microscope



Reflected Microscope Anatomy



Principle Of Bright Field Microscopy

Light from an incandescent source is aimed toward a lens beneath the stage

called the condenser, through the specimen, through an objective lens, and to

the eye through a second magnifying lens, the ocular or eyepiece.

The condenser is used to focus light on the specimen through an opening inthe stage.

After passing through the specimen, the light is displayed to the eye with an

apparent field that is much larger than the area illuminated.

Typically used on thinly sectioned materials



Principle Of Dark Field Viewing

To view a specimen in dark field, an opaque disc is placed underneath

the condenser lens, so that only light that is scattered by objects on the

slide can reach the eye.

Instead of coming up through the specimen, the light is reflected by

particles on the slide.

Everything is visible regardless of color, usually bright white against a

dark background



Principle Of Dark Field Viewing



Mirror Blocks For Reflected Light



Let us watch video on Darkfield Setup



How to calculate microscope image size



Quality of Image

When you look at a specimen using a microscope, the quality of the image

you see is assessed by the following:

Brightness

Resolution

Contrast



Quality of Image with different brightness

Brightness - How light or dark is the image? Brightness is related to the

illumination system and can be changed by changing the voltage to the lamp

(rheostat) and adjusting the condenser and diaphragm/pinhole apertures.

Brightness is also related to the numerical aperture of the objective lens (the

larger the numerical aperture, the brighter the image).

Image of pollen grain under good brightness (left) and poor brightness (right)



Quality of Image with different resolution

Resolution - How close can two points in the image be before they are no longerseen as two separate points? Resolution is related to the numerical aperture of

the objective lens (the higher the numerical aperture, the better the resolution)

and the wavelength of light passing through the lens (the shorter the wavelength,

the better the

Image of pollen grain with good resolution (left) and poor resolution (right)

http://science.howstuffworks.com/light2.htm

http://science.howstuffworks.com/light2.htm



Quality of Image with different contrast

Contrast - What is the difference in lighting between adjacent areas of

the specimen? Contrast is related to the illumination system and canbe adjusted by changing the intensity of the light and the

diaphragm/pinhole aperture. Also, chemical stains applied to the

specimen can enhance contrast.

Image of pollen grain with good contrast (left) and poor contrast (right)



Example image from dark field and bright fieldmicroscope



Measuring Image with Light Microscope

Estimating Viewing Field with a RulerStep 1: Use a clear ruler with a cm/mm scale to measure the diameter of your viewing field

at scanning (40x). On our scopes, we estimated the viewing field to be about 4 mm across.




Step 2: Repeat the process on low power (100x). We estimate our low power viewing field tobe about 2 mm across. Convert mm to microns for both the scanning and low power. There

are 1000 microns in a millimeter. So… Scanning = 4000 microns; Low Power = 2000

microns.

The measurement is an estimation though, and probably not very accurate. For instance, if

you have a slide with the word “are” across it, use your measurement of the viewing field to

estimate the distance of each of the letters. Based on the ruler marks, the letter “r” is about1100 microns, and the entire word, which takes up most of the viewing field is about 4000

microns across. In reality, you cannot place your slide over the ruler, so you have to make a

guess based on how large your microscope’s viewing field is.




Calculating High Power Field of View

Measurements on High Power can be a little more complicated. If you try to use the clearruler technique, you’ll find that you cannot see the individual ruler marks. This is wheremath comes in, the values you estimated above can be used to solve a ratio problem anddetermine the size of your viewing field on high power.

Solving for “High Power Field of View” ….

X / 2000 = 100/400

X (high power field of view) = 500 microns

Now that you have 500 microns as an estimate of your viewing field, any object you areviewing under high power can be estimated based on that. I tell my students to look at aparamecium and guess how many paramecia can fit end to end on their slide. They guessthey can fit two, then the length of their paramecium is about 250 microns.






Measuring grain Size through ASTM grain size number



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L5 Optical Microscope