Cell Theory and Microscopy Flashcards
What are the 3 main facts about Cells (Cell Theory)
- All organisms are composed of one or more cells
- Cells are the smallest units of life, forming the basis of organization in all living things
- Cells arise only by division of a pre-existing cell
How does cell structure relate to function
Cell structure and biology varies according to its function, as a result of evolution, hence can predict/understand different types of cells existing
What is the main way of studying cells in biology
Microscopy
What is Magnification
producing a larger image using a microscope
What is resolution
ability to discriminate parts of image
How does light microscopy work
Broad beam of illuminating light is focused on a specimen by condenser lens
Light will pass through specimen in different absorbencies, leading to darker and lighter regions
What are the different types of light microscopy you need to know about
Bright Field
Phase-contrast
Differential interference contrast (DIC)
Fluorescence microscopy
Laser Scanning Confocal
Describe what Bright-Field microscopy is and its issues
Classic’ light microscopy.
Image illuminated with white light.
But poor contrast (area between dark and light) so requires staining, often involves cell death
Describe Phase-Contrast Microscopy
Generates contrast through differences in refractive index of sample.
Good for intracellular components of living cells.
Glass lenses detecting difference in refractive index
Refractive index (dimensionless number that gives the indication of the light bending ability of that medium)
Describe Differential interference contrast (DIC) microscopy
Rates of change in refractive index produce apparent 3D images
Describe Fluorescence microscopy and where can the fluorescent come from
specimen stained with fluorescent dye or protein.
Ultraviolet light used to excite specimen, producing bright high contrast image against dark background.
Green fluorescent protein (GFP) from jellyfish very useful.
Now get blue, yellow derivatives and red fluorescent protein from sea anemone
How would one prepare a section for microscopy
- Fixation: to stop tissue from falling apart, you can immobilise cells using fixatives like formaldehyde/alcohol
- Embed: Provide mechanical support using wax/resin
- Section: cut thick section (0.5/10um) using microtome with metal/glass/diamond blade
- Staining: Improves visibility of section
Describe Lazer Scanning Confocal light microscopy
Allows imaging of a thin plane within a thicker (non-sectioned) specimen.
Focused laser beam excites fluorescent molecules in cells and tissues, scanning at successive single points in planes.
Gives sharp images.
Optical sections stored on computer can be pooled to reconstruct a three- dimensional model of object.
Very useful for living cells in real time
How does Electron Microscopy work
Uses a beam of electrons rather than light. Resolving power of 2 - 20 nm
What are the two types of Electron Microscopy
Transmission Electron Microscopy
Scanning Electron Microscopy
How does Transmission electron microscopy work
Electrons pass through a very thin specimen (⩽ 0.1 µm).
Allows magnification up to 1,000,000 x or 106.
Specimens generally must be fixed, dehydrated, sectioned, and stained with electron-dense heavy metals - bind to various parts of cell contents, helping give better contrast
What is an Alternative Method for doing Transmission electron microscopy
Alternatively, samples may be rapidly frozen (cryofixation) and used in ‘freeze-fracture’ to reveal cell internal structure. Involves cleaving cell then coating split surfaces with covering of heavy metal (e.g. platinum) to form replica for viewing
Describe Scanning Electron Microscopy
An electron beam is scanned over the surface of a specimen, and electrons bounced off (back-scattered) strike a detector. Provides information about surface details, which appear three-dimensional. Magnification from 15 - 150,000x standard instruments (not as high). Specimens are usually* fixed, dehydrated and coated with a thin layer of metal (often gold).
Describe the Recent advancement with Cryo-Electron Microscopy
Less harsh method. Use deep frozen molecules in solution and gentler electron beams to determine structure of biomolecules.It stops the damage from heavy metals to the cell
Describe the Recent advancement with super-resolution light microscopy
Fluorescence-based microscopy techniques overcomeing traditional light resolution limit by various complex interference/tunnelling methods. Allows resolution to ca. 100 nm.
Describe the Recent advancement with Atomic Force Microscopy
Method to visualise surfaces at molecular scale. Uses fine pointed tip linked to cantilever arm, can move up and down as moves across surface. Detect fine movement by reflected laser beam - helpful in looking at 3D strucuture
