1. Microscopy Flashcards by Riya .

Biological drawing checklist

sharp pencil
• take up at least half the page
• lines need to be clear and continuous (no shading/colouring)
• label lines in pencil
• label lines touch the actual part your labelling
• label lines don’t cross over each other
• ensure proportions are correct
• label all areas that you have shown
• no arrow heads
• LOW POWER TISSUE PLAN

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Define magnification

How many times bigger image size is than actual size of a specimen

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Define resolution

The ability to distinguish between two points in an image - detail

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Resolution of light microscope

200nm

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What does it mean if something is closer together than 200m on a light microscope

They will be seen as one object

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Max magnification of a light microscope

1500x

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Type of samples for a light microscope

Thin, transparent samples
Living or dead

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What stains DNA

Acetic Orcein

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What colour does acetic orcein stain dna

Dark red

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Why may some samples be sectioned (embedded in wax)

To help preserve the structure while cutting

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How does a light microscope work

Has two convex glass lenses: objective (near specimen) and eyepiece lens.
Mirror/light source directs light through condenser (focuses light), diaphragm and through sample.
Image is magnified by the objective lens (usually 4x, 10x or 40x).

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Pros of light microscopes

• Inexpensive to buy and operate
• Small and portable
• Sample preparation does not usually lead to distortion
• Vacuum not required
• Natural colour is seen - unless stained
• Specimens can be living or dead

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Cons of light microscope

Lower magnification
• Lower resolution
• Bubbles in cover slips - artefacts

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What are artefacts

damage caused in specimen preparation

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Resolution of TEM

0.02nm

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Resolution of SEM

0.2 nm

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Resolution of LSCM

200nm

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Magnification of SEM

X100,000

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Magnification of TEM

× 500,000

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Magnification of LSCM

× 2000

SEM and TEM samples

dead
•dries and coated with heavy metals

Why do electron microscope samples need to be coated with heavy metals

to increase the level of contrast in the final image.

Types of samples in LSCM

Different layers at different depths
Living or dead

How does TEM work

A beam of electrons is passed through a vacuum to ensure electrons are traveling in a straight line,

with a wavelength less than 1mm is transmitted through the specimen and focused to produce and image.

How does an SEM work

A beam of electrons is sent across the surface of a specimen and the reflected electrons are collected

Pros of TEM

High magnification • High resolution

TEM cons

Specimen must be fixed in plastic • Must be dead • Expensive • Complex sample preparation • Vacuum required • Sample preparation often distorts image • Black and white images produced - but can be coloured digitally

SEM pros

They can be used on thick or 3-D specimens • They allow the external, 3-D structure of specimens to be observed

SEM cons

Lower resolution than TEMS • Samples must be dead • They don't produce a colour image

How does an LSCM work

Uses lasers • Cells are stained with a fluorescent dye • A thick section of tissue, or a living organism, can then be scanned with a laser beam which can be reflected by the dyes • The laser beam is scanned at different depths

LSCM advantages

- can be used on thick or 3d specimens - allow external, 3d structure to be observed - depth selectivity + very clear images produced

LSCM uses

A use in optometry Eg. Looking at scratches in the cornea

What does the pinhole do in LSCM

Prevents scattered light from being detected - would blur the image

What type of microscope produces these

TEM - can see Golgi apparatus + mitochondria

What type of microscope produced these

SEM

What do the magnets do in an electron microscope

The condenser lenses are magnets which can focus and direct the electron beam

Dry mount

Solid specimens are viewed whole or cut into thin slices (sectioning). • Specimen placed on centre of slide, cover slip ontop

Dry mount examples

dust, insect parts -whole, muscle tissue or plants - sectioned.

Wet mount

Specimens suspended in liquid (water or immersion oil). Cover slip placed at angle, aquatic samples and other living organisms viewed.

Purpose of stains

- contrast is higher - more structures visible - clearer image obtained

Differential staining

Makes cells + structures visible/ distinguish between components Increase contrast Identify differences between cells and identify differences between organelles

Types of staining techniques

Gram stain + acid fast technique

How to calculate the length of a sample

- use eyepiece graticule + calibrate using stage micrometer - measure diameter of sample in graticule units - take repeat measurements + calculate mean length - use calibrated epu to calculate length

How to improve on sample taking

Sharp blade = slide thin enough that individual cells are visible Wet mount = prevents dehydration Squash slide = easier to see individual cells

2nd part - Using Fig. 2, and the information provided, suggest and explain why the cytoplasm of cell C and cell D reacted differently to the stain.

If a question asks you to draw cells based of a microscope image what do you usually need to include