DAT bio Chapter 8 Microscopes Flashcards
What is fixation
getting cells to stick to the slide and preserving them in their most life like state
2 types of fixation
heat and chemical
heat fixation
put cell on slide
run the bottom of the slide over a bunsen burner.
This heats the cell, preserving and sticking them to the slide
What does staining do
adds color to cells helping us see their structures easier (down side) it kills the cell
Optical microscopy
Cells viewed directly
light shines on sample
used to observe living cells
Electron microscopy:
Cells viewed indirectly using a computer after it is bombarded with electrons that passes through a magnetic field in a vacuum. Used to see smaller objects.
Requirement to see cells on electron microscope
cells must be fixed, stained, and killed
Stereo microscopes (
(dissection microscopes):
use low magnification to view the surface of an object
Compound microscopes
multiple lenses to view sample. cells are one cell thick, and alive.
Cons
without fixing and staining, they have poor contrast. which sucks because fixing and staining would mean the cells would die
example of compound microscope
bright field microscopes
Bright field microscopes:
compound microscopes with a bright light
Phase contrast microscopes:
view: thin samples with live cells.
Due to crazy good contrast, cells are not fixed or stained
How does phase contrast microscopes achieve its high contrast?
Light is refracted
through an annular ring creating a phase shift,
leading to high contrast. Large phase shifts can
lead to a halo effect.
How can you reduce halo effects in phase contrast microscopes?
can be reduced with
phase plates or thinner samples
Fluorescence microscopy
: fluorophores
( fluorescent chemical that will re-emit light upon being excited by another light source) are attached to parts of a specimen. Using different types of fluorophores allows researchers to view different parts of the cell.) A dichroic filter is
used which allows certain wavelengths of light
to be reflected and others to pass through.
Distortions in the image (artifacts) decrease the resolution.
Confocal laser scanning microscopy:
visualizes fluorescent objects in living cells. Can be used
without fluorescence tagging. Artifacts (distortion in the image) are
reduced by focusing a beam of UV light onto the
sample. This reduces intensity so samples must
be illuminated longer
Confocal laser scanning microscopy was made to overcome _____
artifacts or distortions of fluorescence microscopy.
Dark field microscopy
: increases contrast
between the sample and the field around it to
allow visualization of unstained live cells. Only
scattered light is viewed - allows the sample to
be viewed against a black background.
Scanning electron microscopy (SEM)
high
resolution 3D images of the surface of a
dehydrated sample
Cryo-scanning electron microscopy
type of SEM where the sample is
frozen in liquid nitrogen instead of dehydrated.
Costly and produces artifacts (distortions in the image).
Transmission electron microscopy (TEM)
high resolution 2D images of the sample’s
internal structures.
Electron tomography
not a type of
microscopy. Sandwiches TEM images to create
a 3D image of the sample’s internal structure.
3 techniques to count cells
Hemocytometers (counting chambers):
Colony Forming Units (CFUs):
Automated cell counting
Hemocytometers
(counting chambers):
gridded slide under microscope. Cells can be
counted in a known area, and that number can
be extrapolated to find the full volume of the
sample
Colony Forming Units (CFUs):
estimates
number of cells plated on growth medium
assuming that one cell gives rise to one colony
Automated cell counting
includes electrical
resistance (counting cells by observing flow of
electricity) and flow cytometry (cells pass
through a narrow tube and are detected by
laser)
Electrical resistance does what
stops electrical conduction, when this happens, we can count the cells that are stopping the electrical conduction
Bacterial growth curve
describe the growth pattern of a given culture of cells
Bacterial growth curve has 4 stages
- Lag phase: Adaptation prior to cell division
- Exponential (log) phase: Rapid doubling
- Stationary phase: Growth rate = death rate
- Death phase: Decline due to lack of
food/other variable
Cell fractionation
separates cell
contents by centrifugation
what is centrifuge
spins contents to separate them by mass, density, and/or shape. More dense particles collect at the bottom (pellet) and less dense particles remain as supernatant liquid on top.
Differential centrifugation
cells must first be split open so that the components can be separated. (homogenization).
Multiple cycles where supernatant is removed
and spun again allow for fractionation
(isolation) of each organelle.
Process called where cells must first be split open so that the components can be separated
homogenization
What is supernatant
clear liquid that lies above the solid residue after centrifugation,
Density centrifugation:
one cycle where
organelles are separated by density into layers.
○ From most dense to least dense: nuclei >
mitochondria/chloroplast > ER fragments >
ribosomes
Karyotyping:
observing chromosomes under
light microscope during metaphase. Can be
used to diagnose conditions involving
chromosomal aberrations, breakages, or
aneuploidies (e.g. Down’s syndrome or trisomy
21)
DNA sequencing
sequencing nucleotides from cut parts (fragments) of the DNA.. Can sequence complete genomes
piece by piece. In humans single nucleotide
polymorphisms (SNPs) serve as markers for
disease causing genes
2 methods for DNA sequencing
1) dideoxy chain termination (Sanger sequencing) older and more established method)
2) next generation sequencing. (newer)
When is recombinant DNA produced?
when DNA fragments from different sources are joined together. These fragments are produced by restriction enzymes, which tend to cut DNA at palindromic (sequence that reads the same backward as forward… AAT TAA), sequences to produce sticky (unpaired nucleotides) or blunt ends (paired nucleotides).
Restriction fragment length
polymorphisms (RFLPs) function
unique
lengths of DNA from restriction enzymes; allow for comparison between
individuals by analyzing non-coding DNA
(coding DNA is highly conserved). Not different? so wont do us any good in identifying individuals?
DNA fingerprinting
identifies individuals
through unique aspects of DNA such as RFLPs
and short tandem repeats (STR’s). Used in
paternity and forensic cases
CRISPR
used to edit specific genomic regions
of interest by adding or deleting specific
targeted sequences of DNA. Used in gene
therapy.
Polymerase Chain Reaction (PCR):
automated
process creating millions of copies of DNA
3 steps for PCR
I. Denaturation (~95 °C): heating separates
DNA into single strands.
II. Primer annealing (~65 °C): DNA primers
hybridize with single strands.
III. Elongation (~70 °C): nucleotides are added
to the 3’ end of DNA using Taq
polymerase.
Bacterial cloning
cloning eukaryotic gene
products in prokaryotic cells. Used to produce
medicine
Steps (protocol for bacterial cloning) PART 1
Processed mRNA for eukaryotic
gene is isolated then treated with reverse
transcriptase to make cDNA
Steps (protocol for bacterial cloning) PART 2
cDNA incorporated into plasmid (transfer vector) using restriction enzymes and DNA ligase → vector taken up by competent bacterial cells (can undergo transformation; made competent using electroporation or heat shock)
Steps (protocol for bacterial cloning) PART 3
undergo
transformation → gene of interest is
found using antibiotic resistance
(antibiotic resistant gene attached to target
gene) or color change (vectors containing
genes making cells blue) methods
Gel electrophoresis
: separates DNA
fragments by charge and size. An electric field is
applied to agarose gel (top = negative cathode,
bottom = positive anode). Smaller fragments
travel further from top of gel
Southern blotting:
identifies fragments of
known DNA sequence in a large population of
DNA. Electrophoresed DNA is separated into
single strands and identified via complementary
DNA probes
Northern blotting
identifying fragments of
known RNA using an RNA probe.
Western blotting:
quantifies amount of target
protein in a sample using sodium dodecyl
sulfate polyacrylamide gel electrophoresis or
SDS PAGE (proteins denatured and given
negative charge proportional to their mass).
Treated with primary antibody (binds to
target protein) and secondary antibody
(attached to indicator and binds to primary
antibody).
What is a DNA probe?
NA probes are single stranded DNA, so they only hybridize with complementary DNA sequences.
- Enzyme-Linked Immunosorbent Assay
(ELISA):
: determines if a person has a specific
antigen. Important to diagnose diseases (e.g.
HIV). Antibodies are placed on a microtiter
plate with a sample and change color if
antigens are present.
Pulse chase experiments:
useful for researchers that want to know more about how proteins move through a cell. This is beneficial because it gives researchers information about gene expression for any given cell type. Also, it illustrates the fate of those same gene products (proteins).
During the pulse phase amino acids are
radioactively labeled and then incorporated
into proteins. The chase phase prevents
radioactively labelled protein production.
Using simple staining, the radioactive proteins
can be tracked
Genomics
study of all genes present in an
organism’s genome and how they interact.
genomic library
stores the DNA of an
organism’s genome
DNA microarrays
contain thousands of DNA
probes that bind to complementary DNA
fragments, allowing researchers to see which
genes are expressed.
Protocol for DNA microarrays
isolate a cell and remove mRNA (because it represents the active transcription of that cell type)→ synthesize cDNA from mRNA using reverse transcriptase → hybridize cDNA with DNA probes → examine microarray for fluorescence → compare microarray with the sequenced genome
Transgenic animals
models used to
identify the function of a gene. A gene is taken
from one organism and inserted into another.
Can be used for mass medication production
(e.g. clotting factors for hemophiliacs). This
process is labor intensive.
Reproductive cloning:
producing a genetic
copy of an organism from a somatic cell (any cell of a living organism other than the reproductive cells) . A
multipotent cell must be converted to a
totipotent cell. E.g. Dolly the sheep
Reproductive cloning
- totipotent cells
Single cell with the ability to divide and produce an entire organism. . E.g. zygote → morula.
Pluripotent cells:
Stem cell that can differentiate into any of the three germ layers: endoderm, mesoderm, or ectoderm. Cannot develop an entire organism because they cant develop extraembryonic tissue, like the placenta.
Multipotent cells
can give rise to some of
the three germ layers - not all. These cells are most differentiated. cannot develop entire organisms
Chromatography:
separating components of
a heterogeneous sample using differential
solubility. The sample is dissolved in the solvent
(mobile phase) and placed in an apparatus
containing the stationary phase. The mobile
phase climbs up the stationary phase and the
different components ascend to different
heights
Fluorescence Recovery After
Photobleaching (FRAP):
: quantitative measure
of how and where biomolecules move in a live
cell.
Fluorescence Recovery After
Photobleaching (FRAP): protocol
baseline fluorescence is measured → area of the sample is photobleached (Photobleaching causes pigmented molecules to irreversibly lose their fluorescence.) → photobleached molecules are replaced by unbleached molecules overtime due to cell dynamics → area gradually recovers fluorescence.
2 types of live cell visualization that utilize fluorescence.
FRAP and FLIM
Fluorescence Lifetime Imaging Microscopy
(FLIM):
provides a quantitative measure of the
concentration of various ions, molecules, and gases
in a cell. Cell is irradiated with light and fluorescence
lifetime (amount of time it takes for an exited molecule to release all its fluorescence) is measured
Knockout mice:
selected gene is ‘knocked out’
and changes between knockout and wild type
are observed
