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