Cell
Discovery of Cells
Birth of Modern Cell Biology
Schleden and Schwann
Introduced first 2 rules of the Cell Theory
- all life is made from 1+ cells
- cell is basic unit of structure for all organisms
Virchow
Modified Schleden and Shwann’s Cell Theory
- all cells arise from pre-existing cells
Craig Venter
participated in human genome project
created first artificially created cell
Development of Cell Biology
Micrometer (um)
One-millionth of a meter, 1/25000 of an inch.
- unit of choice for describing cells and organelles
Nanometer
Unit of choice for molecules and subcellular structures; 1/billionth of a meter.
Microtome
Instrument developed for the rapid and efficient preparation of thin tissue slices.
Limit of Resolution & Resolving Power
LR: A quality refering to how far apart adjacent objects must be to appear as seperate entities.
RP: A microscope quality refering to the ability to see fine details of structure
Brightfield Microscopy
White light is passed directly through a stained or unstained specimen while the field (background) is illuminated.
-limitation: specimen must be unalive and specifcally prepared to highlight transparent features
Types of Microscopy
Phase Contrast Microscopy
Enhances contrast in unstained cells by amplifying variations in refractive index within specimen; especially useful for examining living, unpigmented cells
- breaks waves which are in phase as a sample of light hits a specimen, then brings them back together at the eyepiece
Fluorescence Microscopy
Shows locations of specific molecules within cell. Fluorescent substances absorb ultraviolet radiation (short wavelength) and emit visible light (long wavelength)
- utilizes DICHROMAIC MIRROR; reflects short wavelength and transmits longer wavelengths
- fluorescing molecules may occur naturally but more often are made by tagging the molecules of interest with fluorescent dyes or antibodies.
- ideal wavelength for filter must be inbetween absorbance and reflective cutoffs (ie. abs. at 470, ref. at 550; dichoric mirror ideal at 470)
- resolution is not increased, you can just see more because it is illuminated
Differential Interference Contrast/ Nomarsky Microscopy
uses optical modifications to exaggerate differences in refractive index; good for living cells
Confocal Microscopy and Z-stacks
Specimen image is scanned on multiple focal plates and images are stacked upon eachother (z-stacks)to create a 3D image
Antibody
Protien molecule produced by the immune system which binds to one target molecule/antigen
- allows microscopers to visualize and identify soecific molecules within cells
Angstrom (Å)
0.1 nm, size of a hydrogen atom
Sizes of Cells & their Components
Typical eukaryotic cell: 10-100um
Typical prokaryotic cell: 1-10um
Light microscope limit of res: 0.2nm
Electron microscope limit of res: 0.2 nm/ 2 Å
Resolution Equation
resolution = 0.61 λ /NA
λ = wavelength (nm)
NA = numerical aperture = nsinθ
Importance of Wavelength
We are limited by the range of violet light (390nm), which is too large to hit small objects under a microscope; electromagnetic waves (0.004nm), allows for greater visibility.
Digital Video Microscopy
attatches light-sensitive video camera to a light microscope to collect digital images for storage.
-allows cells to be observed for extended period of time
Immunofluorescence
A fluorescent molecule is attached to an antibody, which in turn recognizes and binds to one specific complementary target molecule/ antigen
- for fluorescence or confocal microscope