For light microscopy: 1. Describe 2. State resolution and magnification

1. Uses light and lenses to magnify images 2. 200 nm resolution and >2000x magnification

For electron microscopy: 1. Describe 2. State resolution and magnification

1. Uses a beam of electrons as a source of illumination 2. Up to 50 nm resolution and 10,000,000x magnification

Chapter 2: 2.2 Imaging in Cell Biology Flashcards by Lucky Cat

List:

3 considerations when it comes to microscopy

Magnification
Resolution
Clarity

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In microscopy:

Magnification means…

The image size produced by the microscope is much larger than the actual size of the object

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In microscopy:

What does resolution mean?

Ability to distinguish between two adjacent objects

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True or False:

Larger resolution means a more powerful microscope

False, smaller resolution means a more powerful microscope

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How is resolution measured?

Measured as the distance resolved between two points

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State:

The formula for resolution

d = 0.61λ / n(sinα)
* d is distance resolved between two points
* λ is wavelength of the light
* n is refractive index of the medium/solution that the sample is submerged in
* α is half the angle of light entering the objective lens

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In microscopy:

What does clarity mean?

Ability to identify different structures

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What are the two types of microscopy used today?

Light microscopy
Electron microscopy

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For light microscopy:

Describe
State resolution and magnification

Uses light and lenses to magnify images
200 nm resolution and >2000x magnification

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For electron microscopy:

Describe
State resolution and magnification

Uses a beam of electrons as a source of illumination
Up to 50 nm resolution and 10,000,000x magnification

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State resolution required to view:

Tissues

1 mm - 100 μm

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State resolution required to view:

Cells

100 μm - 10 μm

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State resolution required to view:

Organelles

1 μm - 100 nm

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State resolution required to view:

(Macro)molecules

10 nm - 0.1 nm

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What structures can light microscopy be used to view?

Tissues
Cells
Organelles

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What structures can electron tomography be used to view?

Cells
Organelles
(Macro)molecules

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What light wavelengths are used to contribute to the resolving power of each microscopy technique?

Light (400-700 nm)
Electron (<300 nm)

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Define:

Transmission Light Microscopy

An external source of light passes through the specimen, and is observed by the viewer

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List:

Types of Transmission Light Microscopy

Bright field
Dark field
Methods to increase contrast (Phase contrast and Differential interference contrast/DIC)

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Describe:

Bright field Transmission Light Microscopy

Light passes directly through the specimen (simplest)

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Describe:

Dark field Transmission Light Microscopy

Light source is aligned such that directly-transmitted (unscattered) light that passes through the specimen is minimized; only scattered light is observed

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Describe:

Phase contrast

Specimens surrounded by a halo of light

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Describe:

Differential interference contrast (DIC)

Images appear 3-D

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Describe:

Compound Light Microscope

Objective lens magnifies by 100x and ocular lens by 10x for a total of 1000x magnification

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# List: Components of a compound microscope (13)

* Eyepiece lens * Barrel * Objective lenses * Stage * Diaphragm * Lamp * Arm * Coarse and fine knobs focus * Base * Stage clips * Stage control * Power button * Dimmer

How are Phase Contrast and DIC Microscopy similiar?

Both use the different refractive indexes in the cell to highlight key structures

# Describe: Phase Contrast Microscopy, how it highlights key structures

Uses varying wavelengths: creates well defined edges where the refractive index contrasts a lot

# Describe: DIC Microscopy, how it highlights key structures

Uses polarized light: Softens the edges but resolutes the 3D structure more for better detail

# Define: Emission Light Microscopy

An external source of light is used to excite fluorescent molecules present in the specimen, causing emissino of light from those molecules

# List: Types of Emission Light Microscopy

1. Epifluorescence 2. Confocal

# Describe: Epifluorescence Emission Light Microscopy

Fluorescent molecules throughout the entire specimen are illuminated, and they all emit light

# Describe: Epifluorescence Emission Light Microscopy samples

Sample may look blurry; poor resolution of fluorescent molecules

# Describe: Confocal Emission Light Microscopy samples

Samples will have good resolution of fluorescent molecules

What is the illuminated plane in confocal emission light microscopy known as?

**Optical slice** or **Z stacks**

# List: Staining and fluorescent dye techniques (3)

1. Direct staining 2. Indirect staining (immunofluorescence) 3. GFP tagging

# Describe: The physics/optics of a compound microscope

1. Image 1 is created from the object through the objective lens 2. Image 2 (the one we see) is created from Image 1 through the eyepiece lens

# Define: Direct staining

Dyes known as flurochromes bind directly to molecule/region of interest

What does direct staining work with? (4)

* Membranes * Nucleus (DNA) * Mitochondria * Cytoskeleton

# Describe: Fluorochromes | (How they work)

Have electrons that when excited then relaxed, release energy at a wavelength that is visible

How are fluorochromes excited?

By using UV light which is a shorter wavelength

What is the released energy at a visible wavelength in direct staining known as?

Emission

# Describe: The basic process of direct staining

1. Light is reflected to the specimen 2. The light excites the fluorochromes on the specimen 3. The emission is shown through the lens

# Define: Indirect staining

A primary antibody binds to the molecule of interest; a secondary antibody, with attached fluorescent marker, binds the primary antibody

What is indirect staining also known as?

Immunofluorescence

# For excitation and emission, describe: 1. Wavelength 2. Energy

Excitation * Shorter wavelength * Higher energy Emission * Longer wavelength * Less energy

# Define: GFP tagging

A gene encoding green fluorescence protein is attached to the gene of interest

What is GFP tagging used for?

Used to observe proteins inside a cell

# True or False: In GFP tagging, the GFP gene is transcribed along with the gene of interest

True

Is the GFP gene transcribed? Is the transcription also translated?

1. Yes, the GFP gene is transcribed 2. Yes, the GFP gene is translated, too

# Define: FRET

Fluorescence Resonance Energy Transfer (FRET) * A technique used to look at protein-protein interactions

# In FRET: Explain the setup of FRET

1. One protein is cloned (like GFP) so that it contains a CFP 2. The other protein which is suspected to interaction with the first protein is labelled with YFP

# In FRET: Define and explain CFP

Cyan fluorescent protein * Emits blue visible light * Can also stimulate fluorescence of other molecules

# In FRET: Define and explain YFP

Yellow fluorescent protein * Requires excitation energy to emit yellow light

# In FRET: Explain the results and its purpose

* When the two proteins interact and come together, yellow fluorescence is observed (CFP transfers energy to YFP) * If no yellow is seen, one can assume the proteins do not interact

# Explain: Electron microscopy

Uses electrons to illuminate specimens; can be used to view smaller structures than it is possible with a light microscope

# List: The types of Electron Microscopy

1. Transmission electron microscopy (TEM) 2. Scanning electron microscopy (SEM)

# Describe: Transmission electron microscopy (TEM)

Specimen is cut into extremely thin sections and stained with a heavy metal to increase electron density * An electron beam passes through the section (similar to transmission light microscopy)

# Describe: Scanning electron microscopy (SEM)

An electron beam is scanned across the surface of a specimen coated with a very thin layer of a heavy metal * The metal emits electrons, and a 3D image of the surface can be generated

# True or False: Electron micrographs can only be shown in black-and-white

False, they are often shown in black-and-white but may be coloured during processing

What is the resolution of TEM?

* Resolution of about 0.005-0.1 nm (2000 times more powerful than light microscopy)

# True or False: Resolution of TEM does not depend on the refractive index of the medium | (Explain why)

True, as it uses electrons and it passes through without change

# State: The formula for resolution of TEM

d = 0.61λ / α

# Overview: Types of Microscopy

Light Microscopy * Transmission (Bright/Dark field, Phase contrast, DIC) * Emission (Epifluorescence, confocal) Electron Microscopy * TEM * SEM

# For Light Microscopy, state: 1. Resolution limit 2. Advantages 3. Disadvantages

1. 200 nm 2. Can image live cells, can use coloured stains, can image whole tissues 3. Low resolution

# In TEM, state: 1. Resolution limit 2. Advantages 3. Disadvantages

1. 0.2 nm 2. High resolution and detail 3. Sample prep kills specimen, difficult sample prep, colourless, no 3D information

# In SEM, state: 1. Resolution limit 2. Advantages 3. Disadvantages

1. 0.2 nm 2. High resolution and detail, can view 3D surface 3. Sample prep kills cells, colourless, cannot see inside of structures

# State the relative size of cellular structure (approximate): Cell

10-100 μm

# State the relative size of cellular structure (approximate): Nucleus

3-10 μm

# State the relative size of cellular structure (approximate): Mitochondrion

0.5-3.0 μm

# State the relative size of cellular structure (approximate): Chloroplast

5 μm

# State the relative size of cellular structure (approximate): Endoplasmic reticulum

10-20 μm

# State the relative size of cellular structure (approximate): Golgi apparatus

20-25 μm

# State the relative size of cellular structure (approximate): Lysosome

0.2-1 μm

# State the relative size of cellular structure (approximate): Peroxisome

0.2-1 μm