MCDB 428 Exam 1 ACTUAL EXAM/ MOSTLY TESTED
QUESTIONS AND CORRECT DETAILED ANSWERS| 100%
CORRECT SOLUTIONS/GRADE A+
Normal tissue culture cell size Normally 20-30 µm in diameter
Size of most eukaryotic cells 5 -100 µm
Transmission
Kinds of light microscopy
Fluorescence
White light that has passed through the sample is
Transmission microscopy
collected at the eyepiece
Specimen is illuminated with the light of a specific
wavelength. This light is reabsorbed and only re-
Epifluorescence microscopy
emitted fluorescence of a specific wavelength is
collected at the eyepiece.
, Major challenge in transmission microscopic
techniques
Contrast
Because individual cells are predominantly
transparent at high levels of magnification
Bright field Light transmitted straight through the specimen
Phase alterations of light transmitted through the
specimen are translated into brightness changes
Phase contrast
Can see details
Highlights edges where there is a steep change of
refractive index
DIC
Different wavelengths
Specimen lit from side and only scattered light is
seen
Dark field
Used to look at fluorescence
Because you can't see details by just microscopy
alone
Staining of cell components
Paraffin section of urine-collecting ducts are stained
with H&E
Hematoxylin Basic dye that stains the nucleus
Eoisin Acidic dye that stains the cytosol
, First barrier filers - only allows blue light through from
light source
Beam-splitting mirror - reflects light below 510 nm
Fluorescence microscopy
but transmits light above it
Second barrier filter - passes green light only to
eyepiece
Often antibodies are used to recognize a protein,
located on a particular structure, followed by a
Multi-probe fluorescence fluorescent second antibody
microscopy
Can reveal subcellular compartments and cell
dynamics
Rabbit antibody directed against antigen
Primary antibody
Not usually labeled
Marker-coupled antibody directed against rabbit
Secondary antibody
antibodies
Fluorescent molecules, for immunofluorescence
Kinds of tags that can be
coupled to secondary antibodies
Enzymes for immunoblotting or ELISA
Fluorescing label in cell
Fluorescence Recovery After Shine UV light to spot to bleach
Photobleach - FRAP
See how fast the label can recover, how the label
moves into the bleached area
Also a key challenge in microscopy
Physical limit of resolution for a light microscope is
~0.2 micrometers
Resolution
Visible light is 0.38-0.75 micrometers
Super resolution - 30-50 n
EM - 0.1 nm
, Roughly equivalent to light microscopy
Transmission Electron Uses electrons instead of a light source
Microscopy - TEM
Need a column of vacuums so the electrons can
move straight down
EM has a resolution down to ~0.1 nm
-can see ribosomes, and large
Advantages of EM
protein complexes
-membrane bilayer is ~5 nm wide
EM techniques require fixed samples
-use of electrons requires sample
Disadvantages of EM observed to be in a vacuum
-optimal imaging requires use of heavy
metal stains to provide contrast
Collects reflected electrons off surface of samples
Scanning Electron Microscopy
Spray heavy metal, electrons shoot on surface and
reflect back
Used in electron microscopy
Secondary antibody has gold particle
Cro-EM and immunogold
Gold particle is electron dense and can be seen as a
labeling
black dot in the microscope
Different antibodies can have diff sized gold particles
to recognize diff proteins
Inject mouse with antigen
Monoclonal antibody production Antigen causes immune response in mouse
Makes antibodies
Because the mouse can't be kept alive forever, can't
keep making antibody
Polyclonal antibodies
Fuse with tumor cell that divides forever, makes
hybridoma cells that divide and make the antibody
QUESTIONS AND CORRECT DETAILED ANSWERS| 100%
CORRECT SOLUTIONS/GRADE A+
Normal tissue culture cell size Normally 20-30 µm in diameter
Size of most eukaryotic cells 5 -100 µm
Transmission
Kinds of light microscopy
Fluorescence
White light that has passed through the sample is
Transmission microscopy
collected at the eyepiece
Specimen is illuminated with the light of a specific
wavelength. This light is reabsorbed and only re-
Epifluorescence microscopy
emitted fluorescence of a specific wavelength is
collected at the eyepiece.
, Major challenge in transmission microscopic
techniques
Contrast
Because individual cells are predominantly
transparent at high levels of magnification
Bright field Light transmitted straight through the specimen
Phase alterations of light transmitted through the
specimen are translated into brightness changes
Phase contrast
Can see details
Highlights edges where there is a steep change of
refractive index
DIC
Different wavelengths
Specimen lit from side and only scattered light is
seen
Dark field
Used to look at fluorescence
Because you can't see details by just microscopy
alone
Staining of cell components
Paraffin section of urine-collecting ducts are stained
with H&E
Hematoxylin Basic dye that stains the nucleus
Eoisin Acidic dye that stains the cytosol
, First barrier filers - only allows blue light through from
light source
Beam-splitting mirror - reflects light below 510 nm
Fluorescence microscopy
but transmits light above it
Second barrier filter - passes green light only to
eyepiece
Often antibodies are used to recognize a protein,
located on a particular structure, followed by a
Multi-probe fluorescence fluorescent second antibody
microscopy
Can reveal subcellular compartments and cell
dynamics
Rabbit antibody directed against antigen
Primary antibody
Not usually labeled
Marker-coupled antibody directed against rabbit
Secondary antibody
antibodies
Fluorescent molecules, for immunofluorescence
Kinds of tags that can be
coupled to secondary antibodies
Enzymes for immunoblotting or ELISA
Fluorescing label in cell
Fluorescence Recovery After Shine UV light to spot to bleach
Photobleach - FRAP
See how fast the label can recover, how the label
moves into the bleached area
Also a key challenge in microscopy
Physical limit of resolution for a light microscope is
~0.2 micrometers
Resolution
Visible light is 0.38-0.75 micrometers
Super resolution - 30-50 n
EM - 0.1 nm
, Roughly equivalent to light microscopy
Transmission Electron Uses electrons instead of a light source
Microscopy - TEM
Need a column of vacuums so the electrons can
move straight down
EM has a resolution down to ~0.1 nm
-can see ribosomes, and large
Advantages of EM
protein complexes
-membrane bilayer is ~5 nm wide
EM techniques require fixed samples
-use of electrons requires sample
Disadvantages of EM observed to be in a vacuum
-optimal imaging requires use of heavy
metal stains to provide contrast
Collects reflected electrons off surface of samples
Scanning Electron Microscopy
Spray heavy metal, electrons shoot on surface and
reflect back
Used in electron microscopy
Secondary antibody has gold particle
Cro-EM and immunogold
Gold particle is electron dense and can be seen as a
labeling
black dot in the microscope
Different antibodies can have diff sized gold particles
to recognize diff proteins
Inject mouse with antigen
Monoclonal antibody production Antigen causes immune response in mouse
Makes antibodies
Because the mouse can't be kept alive forever, can't
keep making antibody
Polyclonal antibodies
Fuse with tumor cell that divides forever, makes
hybridoma cells that divide and make the antibody
