2 - Imaging Cells Flashcards
What was important in the release of Hooke’s essays in 1665?
He gave a detailed description of a section of cork as being a honeycomb of chambers or cells.
What was significant about Leeuwenhoek’s work in 1675?
His microscopes could resolve to about 1.5 microns. He reported ‘a host of little animals’ in a drop of rain water, which was protozoa.
What important events happened in the 19th century for microscopes?
The maximum theoretical resolution of the light microscope was attained (0.25 microns)
What important event happened in the 1930s for microscopes?
The electron microscope was developed = cell organelles could be seen.
Ultrastructure
The level of detail obtainable with the electron microscope.
What are the 3 important parameters of microscopy?
magnification, resolution, contrast
Magnification
The ratio of an object’s image size to its real size.
Resolution
The measure of the clarity of the image or the minimum distance of two distinguishable points.
Contrast
Visible differences in brightness or colour between parts of the sample.
Light microscope
The light passes through the specimen, which must be at least partially transparent.
Stained to enhance contrast and must be fixed.
Advantages of light microscopes
The ability to image living cells.
Disadvantages of light microscopes
Limited resolution (0.2 microns)
What is the only way to improve resolution of a microscope?
Use a shorter wavelength of radiation.
Fluorescence microscopy
Shows the location of specific molecules in the cell by absorbing short wavelength, ultraviolet radiation and emit longer wavelength, visible light. Usually by tagging molecules.
phase contrast light microscopy
Enhances contrast in unstained cells by amplifying variations in density within specimen.
Useful for living unpigmented cells.
Differential-interference contrast light microscopy (Nomarski)
Uses optical modifications to exaggerate differences in density.
Confocal Light Microscopy
Uses lasers and special optics for ‘optical sectioning’.
Narrow depth are imaged.
Above and below plane of view = black rather than blurry.
Usually fluorescently stained.
Sample preparation for light microscopes
- Whole mounts
- Tissue sections
- Fixation
- Dehydration and clearing
- Embedding
- Sectioning
- Staining
Whole mounts in LM
Small relatively transparent specimens can be mounted directly onto slides.
Tissue sections in LM
Most tissues need to be sectioned before they can be examined.
Fixation in LM
using chemical fixatives to prevent cell autolysis and to preserve the structure of the tissue.
Dehydration and clearing in LM
Removes water from tissue in preparation for wax impregnation.
Embedding in LM
Specimen is infiltrated with molten wax after which it’s transferred to a mould.
Sectioning in LM
Thin sections approx 5 microns thick are cut on a microtome and collected onto a glass slide.
Staining in LM
Wax removed and tissue stained with coloured dye
Eosin
Coloured dye used for the cytoplasm
Haematoxylin
A coloured dye used for nuclei.
Confocal scanning microscopy 3D
Generated 3D images of living cells.
Removes out of focus images by optical sectioning and can look inside thick specimens e.g. embryos.
Deconvolution microscopy
Algorithms remove out of focus light and sharpens the image to improve resolution
How is the resolution limit broken?
Super resolution gathers light from individual fluorescent molecules and records their position, combining these breaks the limit.
Who developed electron microscopes?
Ruska and Knoll
What is the theoretical limit of electron microscopes?
~0.002nm
Why is the electron microscope kept under vacuum?
Electrons have poor penetrating power.
What can electrons be focused by in the electron microscope?
Magnetic fields
Electron gun
Heated tungsten filament which produces electrons by thermionic emission
How do transmission electron microscopes work?
Electrons from electron gun > passes through specimen > focused and magnified.
How is the image from a TEM focused and magnified?
By magnetic objective and projector lenses.
What happens to the electron image after it is focused and magnified?
Converted into a visible image by a fluorescent screen, which is viewed through a glass window.
Photos taken with digital camera.
Sample preparation for TEM
- Whole mounts
- Fixation
- Dehydration
- Embedding
- Sectioning
- Staining
How can whole mounts be used for a TEM?
Bacteria and viruses can be examined directly as well as tissue sections.
Fixation in TEM
Glutaraldehyde for protein crosslinking and then osmium tetroxide for lipid crosslinking.
Dehydration in TEM
In an ethanol series
Embedding in TEM
Embedded in plastic resins such as epoxy resins.
Sectioning in TEM
50nm thick sections cut using ultramicrotome.
Staining in TEM
Biological tissue has little contrast under the electron beam, heavy metal stains such as lead are used to improve contrast.
X-ray cystallography
X-rays scatter as they pass through a crystallized protein, resulting in waves that interfere with each other, creating a diffraction pattern which shows the position of atoms.
Cryo-electron microscopy
A beat of electron is fired at a frozen protein solution, the emerging scattered electrons pass through a lens to create a magnified image on the detector, allows structure to be found.
Scanning electron microscope
Electron beam scanned across specimen, used for looking at the surface of specimens.
How does the SEM work?
Electrons are reflected from the surface of the specimen, collected by a electron detector and converted into an electronic signal, which is displayed on a screen.
Sample preparation for SEM
- Fixation
- Dehydration
- Critical point drying
- Coating
Fixation in SEM
The same as TEM
Dehydration in SEM
Water is replaced with ethanol.
Critical Point Drying
Allows all of the ethanol to be removed from the sample in a way that minimises shrinkage.
Coating
Specimens coated with a thin layer of gold to protect them from electron beam damage.
Cell fractionation
Allows major organelles to be individually separated out so they can be studied in isolation.
What must happen to the cells before the organelles can be released?
Must be homogenised.
Differential centrifugation
Isolates cell components on the basis of size and density by using increasing durations and g forces.
