Microscopy

What is Fluorescence microscopy?

Fluorescent molecules absorb light at one wavelength and emits light at a different wavelength.

When would you use phase-contrast microscopy (PC)?

For THIN specimens (see a dark/light outline)

What is a transmission EM?

Gives fine detail on INTERNAL CELLULAR COMPONENTS (electron beam shoots through a thin specimen--some electrons get through other are scattered-- image is created by pattern of electron transmitted)

How does its magnification/resolution compare to that of light microscopes?

Has much greater magnification/resolution than light microscopes (100-200X better)

Advantages of scanning electron microscopes

High resolution and magnification

Oil immersion lens

(100x) has a white ring

Low power lens

(10x) has a yellow ring

High power lens

(40x) has a blue ring

Scanning lens

(4x) has a red ring

What is the diagram of the radiation pathway in a light microscope?

-

What is the diagram of the radiation pathway in an electron microscope?

-

What is the resolving power of a TEM?

- 0.1 nm

When was the electron microscope invented?

- 1930s

How many times better is the resolution of an electron microscope than a light microscope?

- 2000 times better

What has to be fitted in an electron microscope and where?

- A near-vacuum within chamber of electron micropscope

What are the limitations of the SEM?

- All same as limitations of TEM - Except species need not be extremely thin as electrons not penetrate

Why does the vacuum have to be created in an electron microscope?

- B/c electrons are absorbed by molecules in air

How can a 3D image be built up from a SEM?

- By computer analysis of pattern of scattered electrons and secondary electrons produced

What is the process within a TEM?

- Consists of electron gun, produces beam of electrons that's focused onto specimen by a condenser electromagnet - Beam passes through thin section of specimen - Parts of specimen absorb electrons (appear dark) others allow electrons to pass through (light) - Image produced on screen, can be photographed to produce a photomicrograph

Limitations Transmission Electron Microscope

Expensive Extensive training required Samples must be dead (Vacuum, Stains) Black and white/ false colour

Limitations Electron Microscopes

Expensive Extensive training required Samples must be dead (Vacuum,Stains) Black and white/ false colour

Mechanical stage

Platform on which the slide is viewed. The stage can be raised or lowered using the course and fine focus knobs

Distinguishing factors between eukaryotic cells and prokaryotic cells

Prokaryotic cells are very small with no nucleus or other membrane bound organelles, while eukaryotic cells are ten times larger contain membrane bound organelles

Which one has limits? (resolution or magnification)

RESOLUTION - depends on light being uses. (as wavelength goes down--more frequency--more power--better vision)

Condenser focus knob

Raises and lowers the condenser lens to vary light delivery. The condenser is most often used in the highest position, just under the stage. If the observed field of view is not uniform enough, it may be improved by lowering the condenser slightly

Disadvantages of light microscopes

Requires staining, magnification limited to 2000x, resolution limited to 200nm

Revolving nosepiece

Rotating mechanism that houses 4 objective lenses Scanning Low power High power Oil immersion

Iris diaphragm

Small device that is inferior to the substage condenser. It serves to control the angle of the cone of light emerging from the top of the condenser. There is no set rule regarding which setting to use for a particular power. Most individuals find that they need to increase contrast (close the diaphragm) when viewing transparent specimens

Condenser

Small substage lens that is designed to collect, control, and concentrate light from the lamp onto the specimen

Microscope lenses must be cleaned with?

Special lens paper

Slide holder assembly

Spring-loaded device that holds the slide in position for viewing

Base and arm

Support structures; used to transport the microscope

Formaldehyde/methanol

Used to TREAT CELLS. Permanently links all cellular proteins together (and protein to nucleic acid) and creates giant network)

What are heavy metals used for in electron microscopes?

Used to fix and stain the samples. (U,Pb,Os,Au)

Mechanical stage controls

Used to position the specimen above the opening in the stage

What is an electron microscope?

Uses concentrated beam of electrons to visualize specimen.

Confocal Microscopy?

Version of fluorescence microscopy that uses lasers to observe thin section of a thick sample. (laser shoots into the middle of the sample and only illuminates a thin section- doesn't get fluorescence in the entire cell + sharper images)

How should microscopes be put away?

With the lowest power objective (4x scanning lens)

How much magnification do light microscopes use?

magnifies 1000x

How scanning electron microscopes work

A beam of electrons hit the surface of the specimen creating a 3D image

At the end of lab how should your cord be?

Unplug the cord, fold it up and secure with velcro

Course focus knob

Used for initial focusing of specimens at 4x and 10x

What objective should be used when beginning to focus?

4x

What is the process within a SEM?

- Directs beam of electrons onto surface of specimen from above, rather than penetrating from below - Beam then passed back and forth across portion of specimen in regular pattern - Electrons scattered by specimen and pattern of scattering depends on contours of specimen surface

What are the two main advantages of an electron microscope?

- Electron beam has very short wavelength and microscope can t/f resolve objects well, high resolving power - As electrons are negatively charged, beam can be focused using electromagnets

What is the resulting image from a TEM and how can this be overcome?

- Flat, 2D image - Taking series of sections through specimen, building up 3D image by looking at series of photomicrographs

What is the issue with creating a 3D image from a series of photomicrographs from a TEM and how has it been overcome?

- Long process - Invention of scanning electron micrograph (SEM)

What is the resolution of a light microscope like and why?

- Poor - Relatively long wavelength of light

What can harm the resolving power of a TEM?

- Problems with the specimen

What is an artefact and where may they appear?

- Things that result from way specimen is prepared - On finished photomicrograph but not part of natural specimen

Why must a specimen be thin in a TEM?

- To allow electrons to penetrate

What are the two types of electron microscope?

- Transmission electron microscope (TEM) - Scanning electron microscope (SEM)

What are the limitations of the TEM?

- Whole system must be in a vacuum, t/f living specimens cannot be observed - Complex 'staining' process required, even then image only in black and white - Image may contain artefacts, not always easy to be sure if true image on photomicrograph

What is the benefit of Phase contrast and DIC microscopy over bright-field scopes?

- can be used to OBSERVE UNSTAINED/LIVE CELLS. (Uses differences in thickness and refractive indices between objects within the cell and between the cell and its surroundings to enhance contrast.)

What type of light is used bright-field microscopy? What happens to that light (e.g. what do our eyes detect) How is contrast usually obtained,

-Bright-field uses visible light. -The light passes through a series of lenses prior to passing through the sample.. the eyes detect what gets through and what doesn't. -Contrast is obtained by staining (fixing) the cells.

What is a Scanning EM?

-Gives 3-D image of the sample SURFACE TOPOGRAPHY. (Specimen is coated w/ heavy metals--electron beam shoots surface--excites electrons in the metal and electrons are released from the sample-- time it takes for electrons to reach detector is detected as light or dark area)

What are the applications?

1. Add fluorescent dyes that bind to specific molecules in the cell (DNA,H+, Ca2+) 2. Link a fluorescent protein to a protein of interest (ex. GFP- Green Fluorescent protein) --attach GFP protein to actin protein to make fusion protein [LIVE CELLS] 3. Immunofluorescence- add a fluorescently-labeled antibody specific for a protein of interest. [FIXED CELLS]

Types of microscopes that use visible light?

1. Bright-field microscopy 2. Phase-contrast microscopy 3. Differential Interference contrast (DIC) microscopy

Types of microscopes that use UV-light?

1. Fluorescence microscopy 2. Confocal microscopy

Types of microscopes?

1. LIGHT MICROSCOPE 2. ELECTRON MICROSCOPE

What issues need to be considered when choosing a microscope?

1. OBSERVING LIVE OR DEAD CELLS 2. CONTRAST (stain or fluorescent dye) 3.THIN OR THICK SAMPLE 4. MAGNIFICATION/RESOLUTION

How many points are deducted from your prelab grade for each microscope infraction?

10 points

What should always be used when making a wet mount?

A coverslip

Explain the difference between a simple microscope and a compound microscope

A single microscope has a single lens, while a compound microscope has one lens at the eyepiece of the microscope and a second lens near the object to be studied

Disadvantages of scanning electron microscopes

All specimens must be dead, uses heavy gold ions (can do damage to specimen), image must be in a vacuum, expensive used mostly in research labs

Who is known as the "father" of microscopy?

Anton van Leeuwenhoek

(TEM) Transmission Electron Microscope

Beam of electrons (directs) specimen Creates image based on electrons that are absorbed Focused using electron magnets

How transmission electric microscopes work

Beam of electrons pass through the specimen and focus on the screen below

What part of the microscope controls light intensity?

Brightness control dial

Advantages of transmission electron microscopes

Can show image up to 500,000 x magnification, much better resolution

Which type of microscope (simple or compound) will you be using in lab?

Compound

What part of the microscope collects, controls, and concentrates light from the lamp onto the specimen?

Condenser

As you move from higher to lower magnification amount of light needed ___________________________

Decreases

Why is it necessary to adjust the amount of light (increase/decrease) after rotating a new objective lens into place?

Different magnifications may require different amounts of light to view the object

To focus an object move the stage __________

Downward

(SEM) Scanning Electron Microscope

Dried beam of electrons at specimen Create image on electrons reflected Electromagnets

Advantages light microscope

Easy to use cheap to purchase True colour but sometimes require staining Could use live specimen

Explain the proper technique for transporting the microscope

Hold it upright with one hand on the arm and other supporting the base. You must also avoid jarring the scope when setting it down on the lab bench, also avoid touching the lens surface

Actual size=

Image size/ magnification

As you move from higher to lower magnification field of view ________________

Increases

As you move from higher to lower magnification working distance __________________-

Increases

Electron microscope

Instead of light electrons are used Shorter wavelength higher resolution Specimens not directly observed: computer form images Based on how many electrons are absorbed by different regions of the specimens

What part of the microscope increases contrast?

Iris diaphragm

Resolving power

Is the ability of a microscope to differentiate between two close together objects Higher resolution means that objects that are closer together can be seen as separate points

Why is an object no longer in view when you switch from low to high power?

It is more magnified, which means you lose it if you do not center and focus object prior

How light microscopes work

Light clarifies image and lense refracts light around the glass to produce image

Light microscopes

Light is focused using glass lens Relies on light being able to pass through the specimen Regions that absorb more light appear darker in the image Calculation (Magnification = Eyepiece lens x objective lens)

Limitations light microscope

Low resolution Low magnification (Max 1250) Specimens thin; may not be a representative

What is the difference between magnification and resolution (in microscopy)?

MAGNIFICATION: increases the apparent size of an object (no real limit) RESOLUTION: Ability to distinguish between two very closely positioned objects. (sharpness/blurriness)

Ocular lens

Magnify objects ten times their apparent size (10x)

Fine focus knob

Makes very slight changes at 40x and 100x

Advantages Transmission Electron Microscope

Much higher resolution than light microscopes (1nm) Provides derailed images, interior structures High magnification (500,000x)

Advantages Electron Microscopes

Much higher resolution then the light microscope Detailed images if surface structures High magnification (200,000x) 3-D image

When and where were the first microscopes built?

The first microscope was built during the final years of the 16th century in England

When would you use Differential Interference microscopy (DIC)?

Thick objects (have a shadow cast to 1 side= 3-D appearance)

Advantages of light microscopes

Thin specimen can be alive, cheapest, common

Disadvantages of transmission electron microscopes

Thin specimens only, heavy positively charged ions( can damage specimen) for staining, expensive used mostly in research, black and white only

What is a microtome used for?

To cut thin sections of tissues. (In transmission EM)

What is cell fixation and what occurs to the cell during it?

Treating the cell such that its structural integrity is maintained in conditions that would normally destroy the cell. (creates artifacts)

Who is credited with inventing the compound microscope?

Zacharias Janssen and Hans Janssen