2.1.1: Cells and Microscopy Flashcards
Describe the four ways in which a sample can be prepared (light microscope examination)
- Dry mount
- Wet mount
- Squash slides
- Smear slides
How light microscope works:
1) Light passes from bulb
2) Through condenser lens which focuses rays
3) Through specimen
4) Then focused through one of the 4 objective lenses to view specimen at different magnification
5) Light passes into eyepiece –> specimen can be viewed
Why must the cover slip by placed on a wet mount at an angle?
• To avoid trapping air bubbles
Why should the refractive index of the medium for a wet mount be close to that of glass?
- To avoid excess refraction of light
* Thereby keep image clear
Why is staining necessary?
- Increase the contrast between different components of cells
- Thereby make them easier to identify
What is a simple stain?
- A process that involves using one solution of a single dye.
- Can make it possible to discern the shapes of different organisms like bacteria.
What is differential staining?
- A process that involves using more than one chemical stain.
- Can help distinguish between different microorganisms or provide contrast between different organelles in an organism.
What is resolution?
The ability to see individual objects as separate entities. Allows more detail to be seen.
Points closer than…
1/2 the wavelength of light cannot be seen as separate entities.
What is magnification?
The degree to which the size of the image is larger than the size of the object itself.
How does Laser Scanning Confocal Microscopy work?
1) Laser excitation source emits rays of light of certain wavelength
2) Light passes through illumination pinhole
3) Light reflected by dichromatic mirror
4) Reflected light refracted through the objective lens
5) Light focused on single focal plane of the specimen (illumination point)
6) Light causes fluorescence from components stained with a fluorescent antibody tag
7) Light emitted back through objective lens, passed through dichromatic mirror
8) Light enters detector through confocal pinhole
Why does the fluorescent light emitted pass through the dichromatic mirror?
Because this light has a different wavelength to the light emitted by the laser excitation source.
What does the confocal pinhole do?
Prevents out of focus light from a different focal plane being detected.
What does confocal mean?
Both the light waves from the laser and radiated when the sample fluoresces have the same focal plane and both follow the same paths.
What is the beamsplitter?
The dichromatic mirror, which reflects light of the wavelength emitted by the laser but lets light of other wavelengths pass through.
How can a 3D image be produced from LSCM?
- 2D image slices layered on top of each other.
* Reconstructed into 3D model by computer.
How can antibodies be used for fluorescence microscopy?
- Antibodies specific to a particular molecule are dyed.
* They then attach to this feature in the cell.
DNA in eukaryotic cells is…
- linear
- contained in nucleus
- wrapped around histomes
DNA in prokaryotic cells is:
- circular
- ”naked” = not wrapped around histomes
- exists in nucleoid region or in plasmids
Organelles in prokaryotic cells are:
Not membrane bound
Organelles in eukaryotic cells are…
Membrane bound
Size of eukaryotic cell:
10-40 µm (micrometers)
Size of prokaryotic cells:
0.5-5 µm (micrometers)
Cell walls in a prokaryotic cell:
- always present
* made of peptidoglycan
Cell walls in a eukaryotic cell:
- sometimes present
* made of cellulose or chitin
Prokaryotic cells reproduce by:
Binary fission (asexual reproduction)
Eukaryotic cells reproduce by:
Sexual or asexual reproduction
Flagella in eukaryotic cells:
• larger and more flexible that prokaryotic cell flagella
Flagella in prokaryotic cells:
- thinner and more rigid that flagella in eukaryotic cells
* rotate to move cell
Arrangement of microtubules in cilia:
9 + 2 arrangement
Functions of the cytoskeleton:
- provides structural support
- aids movement of cells
- helps transport of substances
What are microtubules made of?
Protein
What are intermediate fibres (filaments) made of?
Protein
What are microfilaments made of?
Protein
Size of microtubules
~ 25 nm (diameter)
Size of intermediate fibres:
~ 10 nm (diameter)
Size of microfilaments
~ 7nm (diameter)
Function of intermediate fibres
• provide structural support to the cell -> help resist mechanical stress and enable cell to retain shape
Example of microtubules function:
In trachea, cilia waft mucus
Example of intermediate fibres function
Anchors nucleus
Example of microfilaments function
Pull membrane in during cell division
Composition of cilia
Plasma membrane
Microtubules
Prokaryotic flagella function
- enable cell mobility
* as a sensory organelle detecting changes in an environment
Function of cilia
Cause fluids/objects adjacent to the cell to move
Bacterial flagella have a 9+2 arrangement?
No
Prokaryotic flagella lack a 9+2 arrangement
Prokaryotic cells:
Have no nucleus, e.g. bacteria.
Eukaryotic cells:
Have a nucleus, e.g. plant and animal cells.
What is the ultrastructure of a cell?
Sections of the internal structure of the cell that can only be seen with an electron microscope.
Free ribosomes - size in prokaryotic cell
70s
Free ribosomes - size in eukaryotic cell
80s
Production and secretion of proteins:
1) Gene, found on chromosomes, codes for the production of a protein
2) An mRNA copy of gene is produced
3) mRNA leaves nucleus through nuclear pore –> attaches to a ribosome –> to be secreted or turn into lysosome, attached to rER
4) Ribosome ‘reads’ the instructions in the mRNA and synthesises a protein which passes into the cisternae
5) Vesicle containing assembled proteins in rER buds off –> Golgi via transport function of cytoskeleton
6) Vesicles fuse with cis face of Golgi
7) Proteins in Golgi modified and packaged
8) Secretory vesicles bud off from trans side
9) Fuse with plasma membrane
10) Released by exocytosis
Suggest two processes inside cells that rely on the cytoskeleton for movement:
- cell division especially in cytokinesis stage
* transportation of vesicles
Function of the Rough Endoplasmic reticulum:
• synthesis of proteins
Both the rER and the smooth ER have
Cisternae
Stage micrometer
Graduated measuring scale placed on the microscope stage.
Formula for magnification
Magnification = Image size / Object size
Magnification and resolution: light microscope
x 1,500
200nm
Magnification and resolution: LSCM
x 1,500
160nm
Magnification and resolution: TEM
x 2,000,000
0.1 nm
Magnification and resolution: SEM
x 200,000
0.1 nm
Appearance of images by Light microscope, LSCM, SEM, TEM
- Light: Coloured, some components stained
- LSCM: Fluorescent
- TEM: Black and white, 2D - denser parts of specimen = darker
- SEM: Black and white, 3D (can be coloured by computer)
Nucleus (structure)
- Contains nucleolus
- Contains chromatin (DNA wrapped around histomes and proteins)
- Surrounding by nuclear envelope
Nucleus (function)
- Houses cell’s genetic material
* Has instructions for making proteins
Nucleolus (structure)
• Dense spherical structure inside nucleus
Nucleolus (function)
• Makes RNA and ribosomes
Nuclear envelope (structure)
- Surrounds nucleus
- 2 membranes with fluid between
- Nuclear pores go through envelope
Nuclear envelope (function)
• Pores allow passage of large molecules e.g. hormones and mRNA
rER (structure)
- Flattened membraneous sacs = cisternae
- Continuous with outer nuclear membrane
- Has ribosomes attached to surface
sER (structure)
- Flattened membraneous sacs = cisternae
* No ribosomes
sER (function)
- Synthesis of lipids and carbohydrates
* Involved in lipid adsorption from the gut
Golgi apparatus (structure)
- Stack of membrane bound flattened sacs
* Vesicles often seen around the edges
Golgi apparatus (function)
• Packages and modifies (folding, adding sugar) proteins from rER
Flagella (structure)
- Extension from cell
- Cylinder contains nine microtubules in circular arrangement
- Long
- Usually present alone or in pairs
Flagella (function)
• Enables movement
Mitochondria (structure)
- 2-5 µm long
- Spherical or sausage shaped
- Double membrane
- Highly folded inner membrane forms cristae
- Fluid interior = matrix
- Contain own DNA; can replicate themselves
Mitochondria (function)
• Site of aerobic respiration and ATP production
Lysosomes (structure)
• Small spherical sac surrounded by single membrane
Lysosome (function)
- Keeps hydrolytic enzymes separate from rest of cell
* Fuse with old organelles/foreign substances to digest them
Chloroplasts (structure)
• Double membrane
• Flattened membraneous sacs = thylakoids
• Stack of thylakoids = granum
• Fluid inside = stroma
⟶ Found in plant cells and some protoctists
Chloroplasts (function)
• Site of photosynthesis
Plasma membrane (structure)
• Continuous outer membrane, phospholipid bilayer with intrinsic and extrinsic proteins
Plasma membrane (function)
- Selectively permeable
* Controls exchange between cell and environment
Cilia (structure)
- Hair-like extensions from cell surrounded by plasma membrane
- Contain microtubules in 9 + 2 arrangement
Cilia (function)
- Move material (e.g. waft mucus)
* Act as antennae in cell signalling
Vacuole (structure)
• Fluid-filled sac surrounded by tonoplast
⟶ Large and permanent in plant cells
⟶ Small and transient in animal cells
Vacuole (function)
• Maintain cell turgor (plants)
Free ribosome (structure)
• Each consists of 2 subunits (think translation)
Free ribosomes (function)
• Site of protein synthesis (for proteins to remain in cell)
Centrioles (structure)
• Microtubules (small tubes of protein fibres)
Centrioles (function)
• Take part in cell division (produce spindle fibres)
Cell wall (structure)
- Cellulose in plants
* Chitin in fungi
Cell wall (function)
- Maintains cell shape
* Permeable - allows substances to pass in and out
