Fluorescnce Porteins And Their Applicatiosn Flashcards
Why do we use fluorescence?
• High contrast
• High resolution
• High specificity
• Quantitative
• Live cell imaging
Fluorescence
The result of a molecule absorbing light at a specific wavelength and emitting light at another wavelength
Energy diagram (Jablonski)
• Time spent in excited state is an important property ‘excited lifetime’
• Energy released by the photon as fluorescence (Stage 3) is less than the energy absorbed (Stage 1)
• The wavelength of light emitted is longer than that of the light of excitation
• Process is cyclical
Photobleaching
• Irreversible destruction of the fluorophore
• Cannot be promoted to the excited state
• Ability to fluoresce is lost
Fluorescence Detection:
(i) An excitation source
(ii) Afluorophore(insample)
(iii) wavelength filters (to isolate emission photons from excitation photons)
(iv) A detection instrument to record emission photons
Fluorescent Microscope
i) An excitation source
Illumination via Xenon or Mercury lamp, LED or laser
(ii) A fluorophore – Ab or flu protein in the sample
(iii) wavelength filters (to select appropriate excitation
wavelength and detect only excitation photons)
(iv) A detection instrument to record emission photons
Eye or camera
(v) Magnifying lens (objective) (vi) Dichromatic (dichroic) mirror
Applications of fluorescence techniques
• Imaging in fixed cells
• Immunolabelling; Protein localisation and co-localisation, cell structure
• Live cell imaging
• Protein/cellular dynamics – protein-protein interactions by FRET, FRAP, FLIM • Ionic concentrations and signalling e.g. calcium signalling
• Intrinsic (auto) fluorescence and non-invasive diagnostics
Immunofluorescence (IF) in fixed cells/tissues
• Analogous to immunohistochemistry (IHC)
• Cells or tissues are ‘fixed’
• Uses antibodies (Ab) to label a specific biological target (antigen)
• 1° or 2°Ab is coupled to a fluorophore
• Visualise by microscopy techniques
• Specificity of the fluorescent label comes from the specificity of the antibody for its antigen
• Direct IF or indirect IF
DIRECT if
Pros:
Shorter staining time and a simpler workflow
• No cross-reactivity between secondary antibodies
Cons:
• Reduced signal as relies on the finite number of
fluorophores attached to the antibody
• Less colour options
• More expensive
Multi-labelling
Labelling multiple antibodies with different fluorophores allows visualization of multiple targets within a single image.
Cellular and sub-cellular structures
Organelle labelling
Antibodies recognising organelle- specific proteins* coupled to a fluorophore
Genetically encoded flu proteins: fusion proteins
• Induce cells to produce their own flu-labelled proteins
• Fuse a Fluorescent Protein gene to your target protein gene
GFP gene
Gene of protein of interest e.g. protein X
• The host cell will then produce your target protein with the fluorescent marker permanently attached…..SIMPLE! (?)
Cell dynamics: Molecular interactions
FRET
• Fluorescence (Förster) Resonance Energy Transfer
• An excited fluorophore of a higher energy (shorter wavelength) can transfer some of its energy to an acceptor fluorophore of a longer wavelength range
• Applications:
• Protein-protein interactions
• Ligand –receptor interactions
• Protein dimerization
• Protein conformational changes
Requirements for FRET
• Two proteins tagged with different fluorophores
• The emission λ of protein 1 must overlap with the excitation λ of
protein 2
• The interacting molecules must be <10nm apart
So what exactly do we measure when we quantify FRET?
1)
2)
Decreased fluorescence brightness (quantum yield) of the donor
Increased fluorescence brightness of the acceptor upon excitation of the donor
So what exactly do we measure when we quantify FRET?
3)
Decreased donor fluorescence lifetime; FLIM
FRET issues
Autofluorescence
• Photobleaching
• Bleed through* – unwanted emission of the fluorophore into another channel
• Cross excitation* – unwanted excitation of another fluorophore – e.g. exciting CFP may also excite YFP due to spectral overlap.
*Both bleed through and cross excitation need to be subtracted
• Through detecting FRET, theoretically we are ‘Inferring’ P-P interactions
FRAP: Fluorescence Recovery After Photobleaching
• Examine membrane fluidity or mobility of a membrane protein • Dynamics of cytoskeletal proteins or intracellular transport
FRAP Uses
- Membrane fluidity
- Mobility of a membrane protein
Cell dynamics: Calcium signalling
• Calcium imaging: Fluorescent Calcium indicators e.g. Fluo-4
Fluo-4 applications
• Fluorescence changes as function of [Ca2+]i
• Greater the concentration of Ca in the
cytoplasm, the greater the fluorescence • Applications:
• GPCR activation – release of Ca from intracellular stores by agonists
• Contraction assays – change in calcium during muscle contraction
Other uses of fluorescence in BMS
• Reporter genes – gene expression detection
• Automated DNA sequencing – each nucleotide has a flu tag
• DNA detection – visualising DNA on agarose gels with ethidium bromide/SYBR green
• Quantitative PCR – both dye-based and probe-
based qPCR detection methods use a fluorescent signal to measure the amount of DNA in a sample
• Protein quantification – flu Ab in western blotting and proteomics techniques
Diagnostic applications
• Fluorescence assisted cell sorting (FACS)
• labelling antibodies with flu probes enables cells within a biological specimen to be sorted according to their expression of a particular protein
‘Immunophenotyping’
FACS applications
• Oncology
• Clinical diagnosis and classification of blood
cancers
• Efficacy of cancer treatment – minimal residual disease
• Early detection of cancer in liquid biopsies
• Immunology
• Organ transplant and donor matching
• Blood transfusion
• Efficacy of HIV treatment (CD4 cell counting)
