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1. Microscopy
Cytochemistry/histochemistry
Sectioning and Fixation of samples for microscopy.
Sectioning
Tissues should be cut (using a microtome) in slices thin enough to avoid complete
absorbance of the incident light or electrons.
For example, the sections for electron microscopy must have only 50-100 nm,
which is about 0.2% of the thickness of a single cell.
Fixation
Fixation aims to denature and cross-links groups on adjacent molecules of proteins
and nucleic acids, rendering them insoluble and stable for subsequent procedures
and observation.
Disruption of cell activity and possibly cell death
Groups of fixatives: Aldehydes: (formaldehyde, glutaraldehyde), Alcohols
(methanol, ethanol) or Oxidizing agents (potassium permanganate, potassium
dichromate and osmium tetroxide).
Light microscopy
Most cellular constituents (e.g. nuclei, mitochondria) are not colored and absorb
about the same degree of visible light, so that they are hard to distinguish under a
light microscope and staining is needed.
Staining
Many chemical stains bind to molecules that have specific features.
1. Microscopy 1
, eosin binds to basic amino acids (lysine and arginine) and hematoxylin binds to
acidic molecules (such as DNA, and aspartate and glutamate side chains).
Modifying a substrate into a colored product or into a precipitate by an endogenous
enzyme.
In situ hybridization
Allows localization and detecting of specific mRNA (also DNA) sequences in tissues
by the hybridization of the complementary strand of a nucleotide probe
Immunocytochemistry/immunohistochemistry
Dyes
Dyes (colorimetric, electron-dense or fluorescent) have a low and nonspecific
affinity for biological molecules.
but they can be chemically coupled to antibodies specific for almost any desired
protein - immunocytochemistry
Examples
Localization of catalase by immunocytochemistry and TEM: the antibody is
allowed to interact with a specific antigen (catalase-protein that we want localize)
and then incubated with gold nanoparticles.
Localization of actin by immunocytochemistry and fluorescence microscopy: a
fibroblast was stained with a fluorescent (fluorophore) anti-actin antibody.
By staining a specimen with two or three dyes that fluoresce at different wavelengths,
multiple proteins can be localized within a cell.
Fluorescence microscopy
Main advantage: live cells! But low resolution compared with electron microscopy.
Probes for fluorescence super-resolution imaging:
1. Microscopy 2
, Genetic Fusion - Labelling by fusion with reporter Fluorescent proteins
(genetically encoded probes)
gene for the protein (e.g., GFP) is transferred to a cell and expressed
continuously with a protein of interest
Photoactivatable-FPs: convert from a dark state to a bright fluorescent state
Photoshiftable-FPs: change fluorescence wavelength on irradiation
Organic small-molecule fluorophores (non-genetically encoded probes)
Synthetic fluorophores can be advantageous over fluorescent proteins for super-
resolution imaging due to their high intrinsic brightness, excellent photostability,
good contrast, and greater fatigue resistance.
couple with antibodies
Optogenetics
Involves the use of light to control neurons that have been genetically modified to
express light-sensitive ion channels.
Mechanism:
Light sensitive protein (photoactivatable protein) which responds to a certain
colored light opening certain channels (by causing change in the membrane
voltage).
The DNA that encodes this protein is added to neurons using gene therapy.
Neurons express the DNA and manufacturing copies of that protein installing them
into their membrane.
Now it’s possible activate that neurons using lights (and without affecting
neighboring neurons).
Not only for study neurons, also for tissue muscle.
FRET (Fluorescence resonance energy transfer)
Mechanism describing energy transfer between two chromophores (part that absorb
particular wavelengths of visible light, confering colour)
1. Microscopy 3
, to study molecular interactions inside living cells
protein-protein interactions
ligand binding to a receptor
Mechanism:
is needed 2 proteins tagged with different flurophore
this flurophores bind to the two proteins we want analyse
one of the flurophores acts as excitation wavelenght for the other (show spectral
overlap)
if the proteins interact FRET will occur and can be seen by yellow fluorescence light
if they don’t interact, FRET don’t occur and a cyan light is visible
1. Microscopy 4
1. Microscopy
Cytochemistry/histochemistry
Sectioning and Fixation of samples for microscopy.
Sectioning
Tissues should be cut (using a microtome) in slices thin enough to avoid complete
absorbance of the incident light or electrons.
For example, the sections for electron microscopy must have only 50-100 nm,
which is about 0.2% of the thickness of a single cell.
Fixation
Fixation aims to denature and cross-links groups on adjacent molecules of proteins
and nucleic acids, rendering them insoluble and stable for subsequent procedures
and observation.
Disruption of cell activity and possibly cell death
Groups of fixatives: Aldehydes: (formaldehyde, glutaraldehyde), Alcohols
(methanol, ethanol) or Oxidizing agents (potassium permanganate, potassium
dichromate and osmium tetroxide).
Light microscopy
Most cellular constituents (e.g. nuclei, mitochondria) are not colored and absorb
about the same degree of visible light, so that they are hard to distinguish under a
light microscope and staining is needed.
Staining
Many chemical stains bind to molecules that have specific features.
1. Microscopy 1
, eosin binds to basic amino acids (lysine and arginine) and hematoxylin binds to
acidic molecules (such as DNA, and aspartate and glutamate side chains).
Modifying a substrate into a colored product or into a precipitate by an endogenous
enzyme.
In situ hybridization
Allows localization and detecting of specific mRNA (also DNA) sequences in tissues
by the hybridization of the complementary strand of a nucleotide probe
Immunocytochemistry/immunohistochemistry
Dyes
Dyes (colorimetric, electron-dense or fluorescent) have a low and nonspecific
affinity for biological molecules.
but they can be chemically coupled to antibodies specific for almost any desired
protein - immunocytochemistry
Examples
Localization of catalase by immunocytochemistry and TEM: the antibody is
allowed to interact with a specific antigen (catalase-protein that we want localize)
and then incubated with gold nanoparticles.
Localization of actin by immunocytochemistry and fluorescence microscopy: a
fibroblast was stained with a fluorescent (fluorophore) anti-actin antibody.
By staining a specimen with two or three dyes that fluoresce at different wavelengths,
multiple proteins can be localized within a cell.
Fluorescence microscopy
Main advantage: live cells! But low resolution compared with electron microscopy.
Probes for fluorescence super-resolution imaging:
1. Microscopy 2
, Genetic Fusion - Labelling by fusion with reporter Fluorescent proteins
(genetically encoded probes)
gene for the protein (e.g., GFP) is transferred to a cell and expressed
continuously with a protein of interest
Photoactivatable-FPs: convert from a dark state to a bright fluorescent state
Photoshiftable-FPs: change fluorescence wavelength on irradiation
Organic small-molecule fluorophores (non-genetically encoded probes)
Synthetic fluorophores can be advantageous over fluorescent proteins for super-
resolution imaging due to their high intrinsic brightness, excellent photostability,
good contrast, and greater fatigue resistance.
couple with antibodies
Optogenetics
Involves the use of light to control neurons that have been genetically modified to
express light-sensitive ion channels.
Mechanism:
Light sensitive protein (photoactivatable protein) which responds to a certain
colored light opening certain channels (by causing change in the membrane
voltage).
The DNA that encodes this protein is added to neurons using gene therapy.
Neurons express the DNA and manufacturing copies of that protein installing them
into their membrane.
Now it’s possible activate that neurons using lights (and without affecting
neighboring neurons).
Not only for study neurons, also for tissue muscle.
FRET (Fluorescence resonance energy transfer)
Mechanism describing energy transfer between two chromophores (part that absorb
particular wavelengths of visible light, confering colour)
1. Microscopy 3
, to study molecular interactions inside living cells
protein-protein interactions
ligand binding to a receptor
Mechanism:
is needed 2 proteins tagged with different flurophore
this flurophores bind to the two proteins we want analyse
one of the flurophores acts as excitation wavelenght for the other (show spectral
overlap)
if the proteins interact FRET will occur and can be seen by yellow fluorescence light
if they don’t interact, FRET don’t occur and a cyan light is visible
1. Microscopy 4
