DAT Bootcamp - Microscopy and Lab Techniques
_____ adheres cells to microscope slides in their most lifelike state, and it makes it easier for those cells to be _____
describe the process of heat fixation:
living cells are placed on a slide --> the slide is passed over a flame to kill/"glue" the cells to the slide --> stain is applied
_____ is the process of adding color to cells, which allows them to be viewed more easily under microscope
fixation and staining are associated with cells that have been _____
most optical microscopy techniques can be used to view _____ samples of cells
_____ microscopy allows for higher magnification than _____ microscopy
can electron microscopy be used to look at living specimens?
no, due to fixation and staining
most viruses are so small that they must be viewed using _____ microscopy.
_____ are light microscopes that focus visible light to produce a 2D image of a sample's surface
stereo-microscopes (dissection microscopes)
what is an advantage of stereo-microscopes (dissection microscopes)?
they are light microscopes that can be used to view living samples
what is a disadvantage of stereo-microscopes (dissection microscopes)?
they are light microscopes that have a low resolution
_____ are light microscopes that focus visible light to produce a 2D image of thin samples (single cell layers)
compound light microscopes usually have have different _____, which gives them the ability to make more resolute images than a stereo-microscope (dissection light microscope)
what is an advantage of compound light microscopes?
they can be used to view 2D images of living samples (1 cell thick)
what are some disadvantages of compound light microscopes?
they only view samples that are 1 cell thick and they have a poor contrast, which means some samples may need to be fixed & stained (killed)
_____ are optical microscopes that use light phase changes and contrast to produce 2D image of thin samples
what are some advantages of phase-contrast optical microscopes?
good resolution and contrast; can be used to observe thin samples of living cells - including their internal structures
what are some disadvantages of phase-contrast optical microscopes?
ineffective on thick samples; halo effect around sample edges
what are some strategies to reduce the halo effect of phase-contrast optical microscopy?
using phase plates to reduce the phase shift; use thinner samples
_____ are fluorescent chemicals that will re-emit light upon being excited by another light source
_____ is the emission of photons (light) from a particle that has absorbed light
_____ and _____ are optical microscopy techniques that use laser light to produce 2D images of samples that have been tagged with fluorophores
fluorescence; confocal laser scanning
what are some advantages of fluorescence optical microscopy?
colorful, 2D images of thin samples of living cells; increased brightness
what are some disadvantages of fluorescence optical microscopy?
fluorescence sometimes creates distortions (artifacts) that reduce the resolution
what are some advantages of confocal laser scanning optical microscopy?
colorful, 2D images of thin samples of living cells; view chromosomes during mitosis; overcomes fluorescence artifacts (higher resolution)
what are some disadvantages of confocal laser scanning optical microscopy?
reduced light intensity and longer illumination times than fluorescence optical microscopy
in _____, only scattered light from the sample is transmitted to produce 2D images of unstained, living cells
dark field optical microscopy
what is an advantage of dark field optical microscopy?
excellent contrast on living samples of unstained cells (black background)
what is a disadvantage of dark field optical microscopy?
low light intensity
in electron microscopy, electrons are shot through a _____ at a sample which has been fixed and
metal coated (cells are dead)
(the vacuum prevents electrons from deviating in path)
_____ captures electrons that are scattered by atoms found on the surface of dehydrated samples
scanning electron microscopy (SEM)
what is an advantage of SEM?
high resolution, 3D images of sample surfaces
what are some disadvantages of SEM?
it is costly, and the fixation/staining/dehydration kills the sample
_____ is like SEM, but the sample is frozen instead of dehydrated
cryo-scanning electron microscopy (cryo-SEM)
what are some advantages of cryo-SEM?
high resolution, 3D images of sample surfaces, which are presented in a more natural form than SEM (due to freezing)
what are some disadvantages of cryo-SEM?
it is costly, and the fixation/staining/freezing kills the sample
_____ captures electrons that are transmitted through a thin slice of a sample
transmission electron microscopy (TEM)
what are some advantages of TEM?
high resolution 2D images of internal sample structures
what are some disadvantages of TEM?
it is costly, and the extensive sample preparation kills all living cells
_____ integrates multiple TEM 2D images into a 3D model
(not a form of microscopy)
what are some advantages of electron tomography?
can look at objects and their relative positions in 3D
what are some disadvantages of electron tomography?
it is costly, and the extensive sample preparation kills all living cells
(because it is based on TEM)
is SEM or TEM used to look at surfaces?
what are hemocytometers?
cell counting chambers
_____ are used to estimate the number of cells plated on a growth medium
colony forming units (CFUs)
colony forming units (CFUs) are based on the assumption that each viable cell initially plated gave rise to a _____
what are 2 methods for automated cell counting?
electrical resistance and flow cytometry
as cells show electrical resistance and impede conductance, the _____ in a solution can be estimated by observing the flow of electricity
number of cells
in _____, cells pass through a very narrow tube and can be counted via detection by a laser beam.
_____ is the process where cell contents are separated into their fractions (one part of a whole) by centrifugation
a _____ is a laboratory apparatus that spins in a circular path at very high speeds.
centrifugation separates cell components through _____
mass, density, and/or shape
in centrifugation, the densest and most compact particles will _____ to the bottom of the tube first, becoming pressed together as a _____ (precipitate)
(top liquid is the supernatant)
centrifugation can be used to separate _____ based on solubility
(insoluble proteins pellet out, while the soluble proteins remain in the supernatant)
in _____, cells are split open with a blender and the resulting homogenate is separated based on mass, density, and/or shape
(the homogenate is centrifuged/fractionated)
_____ centrifugation separates cell contents in just 1 spin step, creating multiple layers separated by density
arrange the following organelles from most to least dense: endoplasmic reticulum (ER), ribosomes, mitochondria, nuclei, chloroplasts
nuclei > mitochondria/chloroplast > ER fragments >
_____ is the observation of chromosomes under a light microscope using staining
a karyotype shows both the _____ of chromosomes and their _____
number; physical appearance
karyotyping is preformed during _____
_____ is a condition that results in a third copy of chromosome 21, and _____ allows for substantiation of its diagnosis
Down syndrome (or trisomy 21); karyotyping
for the most part, the human genome is the same, with slight differences in the sequence every ~ 1000 nucleotides (called _____), which serve as markers for genes that cause disease
single nucleotide polymorphisms (SNPs)
what are the 2 most common methods for DNA sequencing?
dideoxy chain termination (Sanger sequencing) and next generation sequencing
_____ is an older and more established method of DNA sequencing, while _____ is used more often now because it is quicker and cheaper
dideoxy chain termination (Sanger sequencing); next generation sequencing
_____ is produced when DNA fragments from different sources are joined together
a _____ occurs when there is a block of nucleotides that are inverted mirrors of each other
the DNA fragments that get incorporated into recombinant DNA are produced by _____, which tend to cut DNA at palindromic sequences to produce _____ ends
restriction enzymes; sticky or blunt
sticky ends have _____, which makes it easy for complementary sticky ends to hybridize
(complimentary sticky ends are made by the same restriction enzyme)
_____ are less common than sticky ends, and they do not have unpaired nucleotides
(blunt ends are harder to hybridize because of the paired nucleotides)
_____ are unique lengths of DNA that result from restriction enzymes, allowing for the comparison between individuals.
restriction fragment length polymorphisms (RFLPs)
a _____ is a group of nucleotides that repeats again and again in a stretch of DNA
short tandem repeats (STRs)
RFLPs and STRs are _____ amongst all individuals, except _____
RFLPs and STRs are used in _____, which is a technique that may be used in paternity and forensic cases
the _____ is an automated biotechnology process that can quickly create millions of
copies of DNA, and it requires no cells
polymerase chain reaction (PCR)
PCR can be carried out in a single container - list the components that container needs to contain in order for PCR to take place:
DNA to be cloned; nucleotides; DNA primers; heat-resistant DNA polymerase (Taq polymerase)
what are the 3 cyclical steps of PCR?
denaturation; primer annealing; elongation
bacterial cloning is an important technique to produce medicines because _____ are cloned in _____
eukaryotic gene products; prokaryotic cells
(note that the insulin gene is obtained as cDNA from processed human mRNA)
_____ corresponds to a eukaryotic gene with all introns removed (used in microarrays and bacterial cloning)
_____ is DNA made from RNA, and it is used in microarrays and bacterial cloning
complementary DNA (cDNA)
_____ produces complementary DNA (cDNA) from mRNA, and it is relied upon for microarrays and bacterial cloning
_____ catalyzes phosphodiester bonds between the ends of DNA restriction fragments (used heavily in genomic libraries and bacterial cloning)
_____ are circular pieces of extrachromosomal DNA in bacteria (used in bacterial cloning and genomic libraries)
a _____ is a piece of DNA (such as a plasmid) that can be taken up by competent cells
(used heavily in genomic libraries and bacterial cloning)
_____ is a process that occurs when a cell's genome is changed by the addition of DNA that was once floating freely in the environment
competent bacterial cells can undergo _____, and they can be made competent through _____
_____ is a process where electricity is applied to cells, creating temporary holes in the plasma membrane
what are 2 key methods for selecting bacterial cells that have undergone transformation in bacterial cloning?
antibiotic resistance and color change
gel electrophoresis separates macromolecular fragments on their _____ and _____
gel electrophoresis has a (positive/negative anode/cathode) at the top and a (positive/negative anode/cathode) at the bottom
negative cathode at the top; positive anode at the bottom
in gel electrophoresis, the (smallest/largest) fragments travel the furthest
a _____ is a fluorescent or radioactively labeled tool that allows scientists to identify a specific sequence within a large sample
_____ is an electrophoresis technique for separating DNA fragments, and it uses _____ probes
southern blotting; DNA
_____ is an electrophoresis technique for separating RNA fragments, and it uses _____ probes
northern blotting; RNA
_____ is an electrophoresis technique for separating proteins, and it uses _____ as the probes
western blotting; primary and secondary antibodies
southern and northern blotting tend to use _____ gel, whereas western blotting tends to use _____
_____ is a technology to determine if a specific antigen exists in a person, aiding in the diagnoses/exposure to certain diseases
enzyme-linked immunosorbent assay (ELISA)
ELISA is based on the idea that a person will have _____ for a given disease's _____ if they have the disease, or have been exposed to it
_____ experiments allow for the visualization/tracking of molecules of interest throughout a cell
describe the general idea of a pulse chase experiment for proteins:
add radioactive amino acids during the pulse --> radioactive protein synthesis --> wash away radioactive amino acids --> add regular amino acids during the chase
_____ studies all the genes of a genome by looking at their structure and function, as well as their interactions with each other
a _____ is a way to store all the DNA of an organism's genome
a _____ is a chip containing thousands of probes that are complementary to a certain sequence
_____ have genes from different species in order to study gene effects and/or mass produce medications
_____ is the process of reverting a multipotent somatic cell to totipotency, such that it can develop into another identical animal
_____ cells have the ability to produce extraembryonic membranes, and ultimately, an entire organism
a _____ cell can give rise to any of the 3 germ layers, but they cannot develop an entire organism because they can't develop extraembryonic tissue
what are the 3 germ layers, from superficial to most deep?
ectoderm; mesoderm; endoderm
_____ cells can give rise to some of the 3 germ layers but not all; so, they are the most differentiated and cannot develop an entire organism
what are the 3 components of a chromatography apparatus?
sample; mobile phase; stationary phase
chromatography is based on _____ of sample components in the _____ phase
differential solubility; mobile
_____ gives quantitative insight into cell dynamics because bimolecular movements in live cells can be traced
fluorescence recovery after photobleaching (FRAP)
_____ provides a quantitative measure of the concentration of various ions, molecules, and gases in a cell
fluorescence lifetime imaging microscopy (FLIM)
_____ refers to the amount of time it takes for an excited molecule to release all its fluorescence
knockout mice have a selected gene of interest 'knocked out' by _____
recombinant DNA technology