PSC2002/L16 Fluorescence Microscopy Flashcards
Why is fluorescence used? (3)
For high specificity or sensitivity
Multiplexing (simultaneous imaging of multiple targets)
Increased contrast and resolution
Real-time imaging and dynamic studies
Quantitative analysis
What is Brightfield imaging?
Simple light source illuminates specimen, transmitted light passes through sample and into objective lens
Contrast mechanism generated by refractive index and absorption properties of sample
Works well with naturally pigmented samples
Give 2 disadvantages to Brightfield imaging.
Limited ability to make out intracellular organelles
Impossible to identify individual proteins/processes
Give 3 types of (photo)illuminescence.
Luminescence
Phosphorescence
Fluorescence
Define phosphorescence.
Slow emission of light that has been previously absorbed by a substance
Slow (ms to hours)
Define fluorescence.
Emission of light by a substance that has absorbed light
Fast (0.5-20ns)
Define autofluorescence.
Fluorescence from naturally occurring molecules in a sample
Describe the Jablonski energy diagram. (3)
Energy input
Excitation of electrons to higher energy levels
Fluorescence emitted as electrons lose energy and return to ground state
Fluorescence can be seen using which apparatus? (2)
Widefield fluorescence microscope
Dichroic filter block
How does the dichroic filter block work?
Reflects below a specific wavelength
Transmits above it
What is the equation for lateral resolving power?
d = wavelength of light/2x numerical aperture of lens
Define super resolution microscopy.
Imaging beyond the diffraction limits of normal microscopy
What 3 major concepts are used to overcome resolution limit?
Structured illumination (SIM)
Stimulated emission depletion (STED)
Localisation (STORM & PALM)
Describe STED imaging. (3)
Up to 60nm X-Y resolution
Up to 130nm Z resolution
Fixed samples
Give 3 problems with labelling samples.
Getting probe to target
Only label target
Overcome sample autofluorescence
Phototoxicity
Photobleaching
How can dye be delivered to the target?
Dye chemistry
Antibodies - immunofluorescence
How does GFP work?
Genetically manipulates target protein to express fluorescent tag
Describe dye chemistry. (3)
Live-cell imaging applications
Get through cell membrane
Only become fluorescent in certain environments
Accumulate in certain organelles
Very easy to use and visualise
Give 3 problems with organellar-specific probes.
Limited retention time in cell/organelle
Limited targets
Specificity
Toxicity
Give 3 materials and their uses in immunofluorescence.
Formaldehyde - fixation
Mild detergent - permeabilisation
Excess of ‘non-specific’ protein - blocking
+/- direct label - primary antibody
Fluorescent label - secondary antibody
Give 2 disadvantages to small-molecule labelled probes.
Many cannot be fixed
Few specifically target proteins
Give 2 disadvantages to immunofluorescence techniques.
Difficult to use with live cells - only cell surface proteins visible
Getting label to target requires permeabilisation
Describe how GFP works. (3)
Target gene DNA manipulated to contain code for GFP
Host cell ‘transiently’ expresses GFP-tagged gene or tagged-gene gets incorporated into genome (transgenic cells/animals)
DNA inserted into cell
What part of the spectrum is covered by fluorescent proteins?
Most of visible spectrum
eBFP 380-440 - mPlum 590/648
Give 3 problems with fluorescent proteins.
Fusion constructs not native proteins
Strong promoters can ‘enhance’ signal
Transient transfections - higher expression
May perturb protein function
Give 2 requirements for live organelle/protein tracking.
Chemical or genetic tag to label organelles/proteins
Many images per second
Give 3 problems with dynamic imaging (live organelle/protein tracking).
Requires short exposure times
Bleaching issues
Toxicity/phototoxicity
What can be identified using interaction/association ‘colocalisation’?
Cells/organelles that co-express certain proteins
Location of proteins (co-labelling organelles with known markers)
What feature of a microscope limits colocalisation?
Resolution
Describe FRET imaging. (3)
Measures interaction and location of interaction of two proteins or structures
Typically one structure is labelled with donor fluorophore, other with acceptor fluorophore
Emission spectra of donor must be matched with acceptor
When 2 structures become associated (<10nm), energy transfer takes place from donor to acceptor
Give the 2 typical FRET pairs.
CFP (donor) & YFP (acceptor)
Fluorescein (donor) & rhodamine (acceptor)
Give a potential use of FRET and a problem with this.
Visualisation of cAMP signalling
No fluorescent cAMP reporter molecules exist
Describe Fluo-3 dye for ion imaging.
Single excitation - single emission for fast imaging
Only fluoresces when bound to Ca2+
Large increase in fluorescence when bound to Ca2+
Give 2 problems with Fluo-3 dye.
Photobleaching
Difficult to accurately measure Ca2+ concentration
Describe Fura-2 dye for ion imaging.
Dual excitation - single emission
Only fluoresces when bound to Ca2+
Large increase in fluorescence when bound to Ca2+
Ratiometric - easy to correct for photobleaching
Can be used to accurately measure Ca2+
Give 2 problems with Fura-2 dye.
UV exposure
Dual excitation is slow and requires specialised imaging equipment
Describe GCaMP (genetic Ca2+ indicator).
Based on GFP, Calmodulin (Ca2+ binding protein) and M13 (peptide sequence from myosin light chain kinase)
Single excitatio - single emission
Only fluoresces when bound to Ca2+
Large increase in fluorescence when bound to Ca2+
Give an example of using ion imaging.
Imaging intercellular Ca2+ wave
Skin cell monolayer loaded with Fluo-3 Ca2+ indicator
Monolayer ‘wounded’ intercellular Ca2+ signalling
