Fluorescence As A Tool To Measure Cell Activity Flashcards
What region of wavelengths does the visual spectrum of light occupy ?
380nm (violet) to 760nm (red)
What does our ability to see depend upon ?
Electromagnetic radiation exciting visual pigment molecules in the retina
What do infrared wavelengths range between ?
1mm-760nm
What do ultraviolet wavelengths range between ?
10nm-400nm
How was fluorescence first described ?
It was described by the halide mineral fluorite emitting visible light when it was irradiated by UV energy
How do certain atoms fluoresce ?
They contain a fluorochrome which contains electrons that can absorb quanta of energy such as in the form of photons
What happens to the electrons in a fluorochrome when they absorb the quanta of energy ?
They reach an excited state - any state with a higher energy level than the ground state of that molecule
It only remains excited for about 1 microsecond
It relaxes back to ground state and liberates absorbed energy as photons at a longer wavelength with lower energy
Describe the excitation wavelength
It has a short wavelength which means it has high energy
Describe the emission wavelength
Longer wavelength so it has less energy
What is stokes shift ?
Difference in the maxima between excitation wavelength and emission wavelength
Greater the shift the easier it is to separate the excited and emitted light from one another
What is fluorescent microscopy used for ?
To irradiate both living and fixed specimens with a desired wavelength of light and separate the strong excited light from the weaker emitted light using filters and mirrors
What does the excitation filter do ?
Allows passage of the desired excitation wavelength - it separates this wavelength from the other wavelengths produced by the light source
What is epifluorescent illumination ?
It is when the objective is used to focus the excited wavelength of light onto the specimen to irradiate it
What is the key component of epifluorescent illumination and how does it work ?
Dichroic mirror
Reflects the excited wavelength and transmits the emission wavelength to the detector
What is the transition value of the dichroic mirror and what does it mean?
It’s the wavelength at which 50% of light is transmitted
Mirror reflects wavelengths less than he transition value and transmits wavelengths greater than the transition value
Not perfect though so about 10% of excited wavelength passes through the mirror
What is the function of the emission filter?
It removes any excited light that is transmitted by the mirror ensuring only emission wavelengths of light reach the detector
What is a limitation of traditional wide field fluorescence microscopy ?
All parts of the specimen are excited - you detect all parts of the specimen, above and below the focal point of the specimen - big problem for thick tissues and even single layer
What happens in laser scanning confocal microscopy ?
Laser beam pass through an aperture and is focused by the objective lens into a small focal volume within a fluorescent specimen
In laser confocal microscopy what does the dichroic mirror do ?
It separates the emitted fluorescent light and reflected laser light so only the fluorescent light passes onto he detector
In laser confocal microscopy what happens to the fluorescent light after passing through the dichroic mirror ?
It passes through the “pinhole” and is detected by a sensitive device called a photomultiplier tube which transduces light energy into an electrical signal which can be used by a computer to produce an image
What is the purpose of the pinhole in laser confocal microscopy ?
Obstructs out of focus light
What are the 2 focal planes in laser confocal microscopy ?
An in focus plane and an out of focus plane
They compare the light coming from different plane and prevent the light hitting the out of focus plane being able to passes through the pinhole
What molecules are fluorescent ?
Many such as naturally occurring proteins (NADH) and many amino acids
Can be a problem when using fluorescence in tissue
Give examples of fluorescent molecules
DAPI - blue
FITC- green
TRITC- orange
Cy5- pink
What is lateral resolution and what is the equation ?
It is the ability to separate objects in x and y
Rxy= 0.61wavelength/numerical aperture
What has super resolution microscopy allowed ?
The separation of a structure of less than 50nm apart
What is axial resolution and what is the equation used ?
Ability to resolve 2 structures in 3D or in the z-axis
Rz= (2wavelengthrefractive index)/numerical aperture squared
What are most calcium imaging methods based on ?
Use of specific fluorescence indicators that change their fluorescence intensity as a function of the free calcium concentration in the solution
What are the 2 most widely used modes of fluorescent calcium imaging ?
Single wavelength intensity measurements
Ratiometric measurements
What is single wavelength intensity imaging ?
A single wavelength measurement of fluorescent intensity is carried out using a dye for which its fluorescent intensity depends on the intracellular calcium concentration - fluo-4
When calcium binds to this dye the fluorescence intensity increases to over 100 fold
What is ratio metric measurement ?
Use dyes which undergo calcium dependent spectral shifts
These shifts are detected by measuring fluorescence at 2 different wavelengths and expressing it as a ratio of the fluorescent intensities - fura-2
Shift is expressed as a ratio of F340/F380
What do the ratio values mean ?
Fura-2 absorbs maximally at 365nm in calcium free solution and absorbs maximally at 335nm in saturated calcium solution
Shift is measured at 340nm and 380nm
Increasing free calcium concentration causes the F340 to increase with a decrease in F380 so higher ratio values means a higher free intracellular concentration of calcium
What happens to a graph of emission intensity vs excitation in calcium imaging technique with fluo-4?
Represents single wavelength intensity
The curve increases upwards as the intensity increases due to to increased levels of calcium
What happens to a graph of emission intensity vs excitation in calcium imaging technique with Fura-2?
Represents ratiometric method
Curve increases in size and is shifted to the left as intracellular calcium levels have increased
Suggest some experiments that would test the source of calcium for each of the agonists applied (bradykinin and capsaicin)
Exclusion of calcium from extracellular media to prevent calcium influx through calcium channels
Pharmacological inhibition of IP3 receptors or SERCA pump to prevent release of calcium from intracellular stores
What are the advantages and disadvantages of ratiometric measurement ?
Advantages
- doesn’t depend on concentration of dye because it reports the spectral shift rather than fluorescence intensity
- allows quantification of inctracellular calcium concentration which is good as dye loading is variable from cell to cell and uptake is a poorly controllable biological process
- can be calibrated against intracellular calcium concentration used to quantify the intracellular calcium concentration
Disadvantages
- requires sophisticated and expensive equipment
What are the advantages and disadvantages of single wavelength intensity measurements?
Advantages
- done with a simple stationary set up
Disadvantages
- only used for semi-quantitative evaluation
What is fluorescence immuno-histochemistry ?
Technique which utilises the properties of antibodies and fluorescent probes to illuminate specific areas
What is the primary antibody ?
It is the antibody which is specific to the protein area of interest
How can labelling to fluoresce a protein occur ?
1- a primary antibody for the specific protein can be conjugated to a fluorophore
2- first the primary antibody binds to the specific protein and then a secondary antibody specific to the constant region of the primary antibody is incubated with the sample so it binds to the primary antibody secondary antibody is conjugated to a fluorophore to allow identification of the primary antibody
Why is using the 2 step process in fluorescent immuno-histochemistry better ?
Allows greater sensitivity and flexibility
- sensitivity is better because multiple secondary antibodies can bind to the primary antibody so increasing the number of fluorophore molecules associated with the primary antibody complex
- flexibility is better because fluoresce toy labels secondary antibodies can bind to any primary antibody as long as it’s raised ina species which it can be recognised
- more flexibility arises from the use of different coloured fluorochromes enabling simulate nous staining of different target proteins
What is fluorescence ?
Absorption and immediate re-radiation of light by organic and inorganic specimens
What are the advantages and disadvantages of confocal microscopy ?
Advantages - good spatial resolution - reduction of out of focus haze Disadvantages - full scan mode take time - cost
What are the advantages and disadvantages of multiphoton microscopy ?
Advantages
- allows imaging of live tissue and whole animals
Disadvantages
- high power lasers often damage the specimen
- cost
What is TIRF microscopy ?
Total internal reflection microscopy
Used to focus exclusively on plasma membrane
It can be used to isolate membrane events
What are the advantages and disadvantages of TIRF ?
Advantages - excellent z axis resolution - 150nm - optical isolation of plasma membrane Disadvantages - only adherent cells can be imaged - impossible to focus inside the cell
What is FRET ?
It’s fluorescence resonance energy transfer
Used to study protein to protein interaction
What is FRET efficiency dependent upon ?
Distance
What are the advantages and disadvantages of immunocytochemistry ?
Advantages
- specific labelling of virtually any protein
- co staining of different proteins in one prep
Disadvantages
- most applications the prep has to be fixed
- many antibodies suffer from low specificity
- in most cases the antibodies are cell impermeable so the period has to be extracellular or cells have to be permeabilised
