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