Lecture 2 Flashcards
Is structure related to function in cell biology?
Structure is always related to function in cell biology.
What happened in 1665?
Robert Hooke published a collection of assays under ‘Micrographica’.
Described seeing a honeycomb of chambers or cells.
What happened in 1675?
Van Leeuwenhoek improved the art of polishing lenses, able to resolve to 1.5µm. Could see protozoa. Then went on later to discover bacteria.
What happened in the 19th century regarding light microscopes?
Maximum theoretical resolution was obtained (0.25µm).
What was developed in the 1930s? And what term was given to the new level of detail observable?
Electron microscope.
Ultrastructure.
Best resolution of LM?
0.2µm.
6 types of light microscopy?
Brightfield (unstained), Brightfield (stain), Fluorescence, Phase-contrast, Differential-interference-contrast, Confocal.
Seven steps in light microscope sample preparation?
Whole mounts - small, translucent specimens can be mounted directly.
Tissue sections - most tissues need sectioning first.
Fixation - prevents cell autolysis, preserves structure.
Dehydration and clearing - Removes water from tissue to prepare for wax impregnation.
Embedding - specimen infiltrated with molten wax.
Sectioning - sections 5 microns thick cut on microtome, collected onto glass slides.
Staining - wax removed, tissue stained with coloured dye.
What does Eosin stain?
Cytoplasm.
What does haematoxylin stain?
Nuclei.
What light microscopy method can be used to generate 3D images of living cells? What other benefits does this method confer?
Confocal.
Can also remove out of focus images, and look inside thick specimens.
2 light microscope techniques that break the resolution limit? How do they work?
Deconvolution microscopy - algorithms remove out of focus light. Sharpens image and improves resolution.
Super resolution microscopy - gathers light from individual fluorescent molecules and records their position. Combining information from these individual molecules breaks limit.
Who were electron microscopes developed by?
Ruska and Knoll.
What is the theoretical resolution limit for EMs? And why?
0.002nm. Electrons have very short wavelength.
What is the currently achieved maximum resolution of the EM?
0.1nm.
As electrons have poor penetrating power, what state is the EM kept under?
Vacuum.
How are electrons focussed?
Magnetic fields.
Two types of EM?
Transmission electron microscope.
Scanning electron microscope.
How does a TEM work?
Electron gun, usually heated tungsten, produces electrons by thermionic emission.
Electron beam passes through specimen, having been focussed and magnified by magnetic objective and projector lenses.
Electron image then converted into visible image by fluorescent screen, viewed through a glass window.
How is a TEM sample prepared?
Whole mounts - bacteria and viruses can be examined directly.
Fixation - Usually glutaraldehyde (protein cross linking), followed by second fixation in Osmium Tetroxide (lipid cross linking).
Dehydration - in ethanol series.
Embedding - specimens embedded in plastic resins, e.g. epoxy.
Sectioning - 50nm thick sections cut using ultramicrotome.
Staining - Heavy metal stains e.g. lead used to improve contrast under electron beam.
Why is an SEM called an SEM?
Because electron beam scanned across surface of the specimen.
How does an SEM work?
Electron beam scanned across surface of the specimen, reflected from its surface, before being collected by an electron detector and converted into an electronic signal displayed on TV screen.
What gives SEM images a 3D appearance?
Depth of focus of SEM.
How are SEM samples prepared?
Fixation - Glutaraldehyde (protein cross linking), then Osmium Tetroxide (lipid cross linking).
Dehydration - water replaced with ethanol.
Critical point drying - enables all ethanol to be removed in a way that minimises shrinking.
Coating - Coated in a thin layer of gold, to protect from electron beam damage.
