Some cells reach confluency – a single layer of cells, stopped growing due to social signals of the cells that there will not be enough resources if the cells keep dividing
Primary culture – initial cells placed on slide
Secondary culture – ‘immortal’ cells produced from primary cultures after confluency. (some cells die after confluency)
Oncogenic transformation – cells not as dependant on substrate and divide on top of one another
Terms used in microscopy
Magnification – lets you see how large you can view an object while keeping the components as distinct objects. How big can the object be while maintaining objects as distinct?
R= (0.61*lamba)/(n * sin(angular aperture))
a factor that measures how close two points can be in a sample and still be distinct.
R=194 nm. This means that two objects that are 200nm apart can be seen as distinct objects. If they are closer than this, they will not be seen as distinct structures.
Different types of light microscopy
Cell often appears transparent when there isn’t manipulation
Contrast for organelles – must be dead
Stains used but can be alive
Tagged proteins for induced expression
DIC – differential interference contrast imaging
Insertion of nomarski optic - A prism that splits light into two separate waves through specimen. Changes wavelength of light and the interference leads to increased contrast, largest difference is the edges of cells and organelles.
Differentiation of organelles and differences between them w/out fixing
Good way to see edges of cell
Vesicle transport in cells
Takes advantage of physics of light and the characteristics of it as a waveform. When two waves are in phase, they are at the same frequ and combine to create a better resolution. If out of phase, reduced intensity of light. When in phase, the unstained cell will disrupt one of the light waves creating a slowed speed for the wave form.
Thicker samples and image optical layers for 3d images.
TEM vs SEM
2d vs 3d
Lots of dyes to look at parts of cells at the same times.
Only limited by number of filters one can place in microscope.
Source of light is ultraviolet passed through an excitation filter, which removes wavelengths of light not useful in expressing the fluorescence.
Dyes are chemically derived that bind carbohydrates, lipids, DNA.
One doesn’t need to use antibodies if the dyes bind directly.
Proteins most often identified by fluorescently linked antibodies because few dyes directly link to proteins.
Primarily, the fluorescent microscope does not have a condenser like in a light microscope. Must be thin samples.
has allowed us to use laser light to image specific layers/regions within a thick sample.
Images individual parts and put together to rebuild the picture.
∆Gc = RTln[in/out] + zFVm
F = 23,062
Vm = membrane potential in volts
R = 1.987
Types of ATPases
P type (for phsphorylation)
Used to pump H+ across membrane for pH maintenance