Topic 1

a cell is “the vehicle for the hereditary information that defines the species… the cell includes the machinery to gather raw materials from the environment, and to construct out of them a new cell in its own image, complete with a new copy of the hereditary information

1590 Z. Janssen + H Jansse produced first compound microscope by putting eyeglass in a tube (maybe)

1665 Robert Hooke described chambers in cork, the dead remnants of cells, coined term “cell” in Micrographia

1673 Anton van Leeuwenhoek made his own microscope with 300x. First to observe and describe living cells “animacules” in pond water, bacteria in tooth scraping Leeuwenhook had better resolution than Hooke with his microscope, not entirely known how he made his lenses

• in 1839 Schleiden and Schwann proposed the first two parts of cell theory 1. all life is made from 1 or more cells 2. cell is the basic unit of structure for all organisms

• in 1855, Virchow added the third rule 3. all cells arise from pre-existing cells he added this rule after spontaneous generation had been disproven

What about viruses and artificial cells? Viruses aren’t considered living, nor cells Artificial cells were made by Craig Venter by taking DNA from one bacteria, and replacing it with DNA from another bacteria in the cell. not in nature, doesn’t count

• All cells store hereditary information in the same linear chemical code (DNA) • All cells replicate their DNA by templated polymerization • All cells transcribe portions of their DNA into the same intermediary form (RNA) • All cells use proteins as catalysts • All cells translate RNA into protein the same way • The fragment of genetic information corresponding to one protein is one gene* *sorta, see bio230 • Life requires free energy • All cells function as biochemical factories dealing with the same basic molecular building blocks • All cells are enclosed in a plasma membrane across which nutrients and waste materials must pass • There are living cells with fewer than 500 genes, but most require more

Prokaryotic cells are between 1-10 um in size

Eukaryotic cells are 10-100 um

unaided a human eye can see 100-200 um a light microscope can see ~200 nm an electron microscope goes to ~200 pm

Resolution is the minimum distance at which we can determine two separate objects are distinct “better resolution” = “higher resolution” = a smaller number

Using violet light in a light microscope we can get a limit of resolution of just under 200 nm.

Resolution = 0.61 λ/NA λ = wavelength NA = numerical aperture = n sin ø n = the refractive index, a value representing the material light travels through. air = 1.00, oil = 1.5

the closer the lens is to the specimen, the higher ø is, the higher n sin ø is. So the lens being closer to the specimen increases resolution The lower the light’s wavelength, the better resolution Violet = 390 nm, is the limit since lower is not visible to use

• brightfield • fluorescence • TEM • SEM

• compound microscope, essentially same as Hooke’s • Light passes through sample, then 2 lenses (objective and ocular), to the eye • Staining can be used to highlight specific cells, structure, etc • Stains can be used on specific features, or generally, multiple stains can be used at once, sometimes on living samples

How phase shift works • light moving through a samples gets out of phase with other light ex 1/4 out of phase • It then passes through a phase plate to bring it further out of phase. ex 1/2 out of phase • Then the waves pass through a lens and are diffracted together Takes advantage of the phase shift to compare direct and diffracted light to improve contrast.

• two waves in phase lead to bright light • two waves out of phase lead to dim light

Light Polarization • When polarized light travels in a sheet (Nomarsky) utilizes changes in phase and polarization (light rotation) that are induced by travelling through sample. Looks 3D but is an illusion.

Fluorescent microscopy • based on fluorescence, whereby a fluorochrome absorbs light at a short wavelength and emits light at a higher wavelength • A dichroic mirror reflects light below a certain wavelength, and transmit it above a certain wavelength Laser excitation source emits light of a shorter wavelength it reflects off a dichroic mirror and hits a fluorochrome die in the sample slide. It then emits light of a longer wavelength which passes through the dichroic mirror to the eyepiece. Fluorescence does not increase resolution

Confocal microscopy and Z stacks: • The image is scanned using fluorescence on multiple focal planes and the images are then stacked upon each other to create a 3D image • A specific plane is excited causing emissions at that specific level • Can be used to increase resolution by spacing planes by less than 200 nm

For both an electron gum emits an electron beam with a very small wavelength For TEM the beam passes through the sample, then hits a detector below it For SEM the electrons bounce/reflect off the samples and are detected by a scattered electron detector. For TEM the samples must be cut very thin and stained with heavy metals. Resolution = 1 nm or with very specialized equipment and techniques and expertise can be reduced to 0.1 nm SEM usually have a resolution of 10 nm. Sample must be covered in thin layer of gold. SEM have a large depth of field samples are frozen or dried. Gives a photo that is not 3D. The photo is Black and White but colour is often added later.

The surface area / volume ration decreases as a cell increases in size and thus the surface area becomes limiting for exchanging materials with the environment at adequate rates. Some other possibilities. • It takes time for substances to diffuse from one side to another, may be too long for bigger cells. Largely disproven. • There is a limiting rate at which a cell can produce its required RNA