Topic 1: Studying Cells, Techniques Flashcards
What is the Schleiden and Schwann Cell Theory?
all organisms are composed of cells
the basic unit of life is a cell
all cells arise from pre-existing cells
What are prokaryotic cells?
bacteria and archaea
does not include any membrane-bound organelle
no true nucleus
most are unicellular
What are eukaryotic cells?
contains membrane-bound organelles
contains one true nucleus
can either be unicellular or multicellular
What does unicellular mean?
one cell = one organism
eg. yeasts and protists
What does multicellular mean?
often consist of differentiated cell types (specialized cell types)
eg. plants, fungi, animals
How small are cells?
plant cells (20-30 um)
animal cells (20-30 um)
bacterium (1-2 um)
ribosome (25 nm)
microfilament (7 nm)
nm scale requires electron microscopy to resolve
How do wavelengths impact microscopy?
light waves travel through objects and can change their path
when paths intersect of different waves of light they can reinforce or interfere with each other
two wavelengths of light can interact
two waves in the same place = reinforcement = appear brighter
if two waves are out of phase = interference = dimmer areas of a specimen
What is resolution?
the minimum distance that two objects have to be in order to be distinguished as seperate
What is light microscopy?
as light passes through the specimen, the phase and frequency of light can change
limit of resolution of any light microscope is 200 nm because of the visible spectrum of light cannot be used to prove structural details much smaller than it’s own wavelength
What is the formula for resolution?
R = 0.61(wavelength)/NA
Low R = good microscope = better resolution
What is interference microscopy?
increase contrast by manipulating the light source
phase contrast microscopy
differential interference contrast (DIC) microscopy
What is phase contrast microscopy?
generates an image where contrast depends on refractive index of specimen
What is differential interference contrast (DIC) microscopy?
use a prism to change phase of light passing through specimen
contrast is generated by differences in the index of refraction of the object and it’s surrounding medium
What is fluorescence microscopy?
a type of light microscopy
very powerful technique for visualizing subcellular structures and even individuals proteins
fluorescent molecules are added to cell
see only the signal from the fluor
What is fluorescence?
a molecule gets excited by one wavelength of light and emits another wavelength within visible spectrum
What is immunofluorescence?
using fluorescently labelled antibodies
antibodies are immunoproteins made to detect a specific antigen
they can be used to detect specific proteins in cells
advantage: very specific and good resolution
disadvantage: requires fixed (dead specimens)
What is antibody staining?
use 1o antibody = protein produced by immune system that is specific to antigen
first step: 1o binds antigen
second step: 1o antibody is detected by a 2o antibody, 2o antibody has been covalently linked to a fluor
What is a green fluorescent protein (GFP)?
naturally occurring fluorescence protein
gene was cloned and added to DNA of other proteins
fusion protein retains normal protein function and glows
advantage: real-time localization, time-lapse images, many different GFP variants available in all sorts of colors, localization of known proteins
disadvantage: requires complicated genetic engineering and DNA reintroduction techniques, some proteins don’t like it
What is confocal microscopy?
type of fluorescence microscopy
laser excites the specimen in a single plane of focus
get crisp images of only one plane of focus
can use it for live species, but time consuming
advantage: high resolution images with distinct focal planes
disadvantage: takes time to scan an entire specimen plane by plane
What is electron microscopy (EM)?
much higher resolution because it is a beam of electrons, not wavelengths of light
requires fixed and sectioned cells (dead cells)
two types: scanning EM and transmission EM
approx. 40,000x better than light microscopy for theoretical limit of resolution
What is transmission EM?
use e- beam instead of light
a very thin section of a fixed specimen encased in wax in a vacuum (this can distort specimen)
e- bounce off electron-dense structures
often use a gold-conjugate antibody to increase e- density of specific proteins
What is scanning EM?
same concept as TEM but now it measures amount of electron scatter off the surface of a heavy-metal coated object
only visualize surfaces
can freeze fracture to visualize internal surfaces
How do you study subcellular components?
many studies on cell structure and function require isolated organelles and cellular structure
a cell homogenate is first prepared by: sonication, chemical lysis, pressurized homogenates among others
need to disrupt the tissues/cells in a controlled way = called homogenate
What is centifugation?
differential centrifugation allows separation of a filtered homogenate by sinning the suspension at increasingly higher speeds
separation based on density
What is electrophoresis?
separation of proteins based on size
1: take sample of prep proteins
2: mercaptoethanol breaks S-S, denature proteins using SDS = detergent that denatures proteins and adds a net negative charge to unfolded protein
3: add proteins to gel
larger proteins stay near top
shorter proteins run further down gel
proteins migrate through the gel at a rate that reflects their size and number of amino acids
What is western analysis?
method to find a specific protein from a mixture on a gel
“blot”: transfer proteins out of gel onto a filter paper where pattern from gel is maintained
use 1o and 2o antibodies to detect protein of interest
Why use?: detect presence of proteins, size, amount (thicker the band the more protein)
