W2 (LEC): MICROSCOPY AND HISTOTECH

A microscope (Greek: _________= small and _______ = aim)

1.) micron
2.) scopos

An instrument for viewing objects that are too small to be seen by the naked or unaided eye

microscope

The science of investigating small objects using such an instrument is called ___________

microscopy

First compound microscope invented.

1590

Who and when is the first microscope developed.

1.) Hans Janssen and Zacharias Janssen

2.) 1590

Who and when did compound microscope was invented?

1.) Galileo Galilei
2.) 1609

Who and when did a simple 2-lens ocular system that was chromatically corrected was developed?

1.) Christian Huygens
2.) 1620

Is generally credited with bringing the microscope to the attention of biologists.

Anton Van Leeuwenhoek

He discovered bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells, microscopic nematodes etc.

Anton Van Leeuwenhoek, 1661

• Degree of enlargement

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

Magnification

• Ability to reveal closely adjacent structural details as separate and distinct

• The greater it is, the smaller limit of resolution

Resolution power

• The min distance between two visible bodies at which they can be seen as separate and not in contact with each other

• The smaller the limit of resolution the higher resolving capacity

Limit of Resolution

Limit of resolution of compound microscope

0.2 micrometers

Limit resolution of scanning electron microscope

0.020 micrometers

Limit resolution of transmission electron microscope

0.0025

This microscope cannot resolve structure smaller than 0.2 micrometers

Compound microscope

This microscope can resolve structure smaller than 0.2 micrometers

Scanning and transmission microscope

Magnification of ocular lens

10x

Who and when invented the first usable compound microscope with 2 sets of lens.

John Cuff, 1750

Magnification power and total magnification of scanning objective lens

1.) 4×
2.) 40x

Magnification power and total magnification of low power objective lens

1.) 10x|
2.) 100x

Magnification power and total magnification of high-power objective lens

1.) 40×
2.) 400x

Magnification power and total magnification of oil immersion objective lens

1.) 100x

2.) 1000x

How to get the total magnification?

multiply the objective and ocular lens magnification

Color of:
1.) scanner
2.) LPO
3.) HPO

1.) Red
2.) Yellow
3.) Silver/White

• Highest magnification

• It uses ___ to prevent prevents refraction of light outwards and allows it to pass straight into objective

1.) Oil immersion Objective
2.) Oil

Enumerate the 6 types of eye piece:

1.) Monocular
2.) Binocular
3.) Trinocular
4.) Huygenian
5.) Ramsden
6.) Compensating

Eye piece used in lab

Huygenian eye piece

Eyepiece used in astronomy

Ramsden eye piece

Eyepiece used in lab and is usually called correction lenses

Compensating eye piece

Eye piece that strains the eyes

Monocular eye piece

Give examples of illuminator

1.) Lamp
2.) Sunlight
3.) Battery
4.) Battery operated lamp
5.) 60 W bulb (most commonly used)
6.) Quartz halogen light

• Produces high-contrast images of transparent specimens.

• No need to stain the sample (unstained sample)

Phase Contrast Microscope

Phase contrast microscope was first described in ___ by Dutch physicist ___________

1.) 1934
2.) Frits Zernike

Produces high contrast and 2D images of transparent specimens.

Phase contrast microscope

Advantage of Phase contrast microscope is that living cells can be examined in their _________

natural state

No need to stain the sample

Phase contrast microscope

3 most common uses of Phase contrast microscope

1.) Enables visualization of internal cellular components
2.) Diagnosis of tumor cells
3.) Examination of growth and behavior of wide variety of living cells in cell culture.

• Specimen appears gleaming bright against dark background

• not considered fluorescence

Dark Ground Microscope

• Specimen stained with fluorescent dye, when examined under microscope with ultraviolet light is seen as bright object against dark background.

Fluorescence Microscope

Fluorescence Microscope Principle: Absorbs light at a _____ wavelength (UV Light) and emits light at a _____ wavelength (visible light).

1.) shorter
2.) longer

What is used to dye organisms in fluorescence microscope.

Fluorochrome

• Yellow Fluorescent dye

• Stain bacteria such as _____________

• Used in microbiology, mycobacterium tuberculosis

1.) Auramine Rhodamine
2.) Tubercle Bacilli

Where does auramine rhodamine dye was commonly used?

1.) microbiology
2.) mycobacterium tuberculosis

______________ dye:

• Fluorescent dye that gives _________ with RNA

• Fluorescent dye that gives __________ with DNA

• Used in histology

1.) Acridine Orange R
2.) orange red stain
3.) yellow green stain

• Fluorescence dye used in diagnostic application.

• to identify antibodies present

Immunofluorescence

• use a beam of highly energetic electrons to examine objects on a very fine scale.

Electron microscope

Examination using electron microscope yields info about organisms ________ and _________

1) Morphology
2.) Composition

This type of electron microscope produces 2D image where the electrons pass through the specimen. This also requires ultra-thin samples.

Transmission electron microscope

Advantages of using TEM?

examines tissue at different layers

This scan to give a 3D view of the surface of an object which is black and white. Electrons reflects on the surface of the sample. Sample is coated w/ metal commonly gold.

Scanning electron microscope

The most common fixative is buffered isotonic solution of ______________ (formalin).

4% formaldehyde

In this technique the sample are immersed in multiple baths of progressively more concentrated _______ to dehydrate the tissue, followed by a clearing agent such as, _____ or ________.

1.) Dehydration
2.) ethanol
3.) xylene
4.) Histoclear

The removal of water from the tissue and replacement with ethanol (stronger bond with water)

Clearing

During this ____ hour process, paraffin wax will replace the water: soft, moist tissue are turn into paraffin block, which is then placed in a mold containing more molten wax and allowed to cool and harden.

1.) Embedding
2.) 12-16

Much faster embedding technique than paraffin wax, usually used in surgery. This medium is liquid at room temperature but when cooled will solidify.

non-fixed tissue in a freezing medium

The tissue then sectioned into very thin (___ micrometer) sections using a microtome.

1.) Sectioning
2.) 2-8

Is a built-in knife; come up to tissue ribbons

Microtome

Transferring of tissue ribbon into storage

mounting

Serves as adhesive

Albumin

To see the tissue under the microscope, the sections are stained with one or more pigments. Done to give contrast to tissues. Without this, it is very difficult to see differences in cell morphology.

Staining

Most commonly used stains in histology and histopathology, abbreviated H&E.

1.) Hematoxylin
2.) Eosin

Hematoxylin colors _______, eosin colors _________.

1.) nuclei blue
2.) cytoplasm pink

Types of stain:

• Nuclear components; hematoxylin

Basophilic

Types of stain:

• Cytoplasmic components; eosin

Acidophilic