What is fluorescence?
Fluorescence = property of some atoms or molecules to absorb light as a wavelength and subsequently emit light of longer wavelengths (stokes shift) after a brief time interval (fluorescence lifetime)
Is based on the ideas of excitation and emission
(Stokes shift basically = excitation has higher greater energy than emission because some energy is lost as heat)
Fundamentals of excitation and emission
Excitation light is of a lower wavelength (higher energy)
Cells/targets/markers absorb photons of excitation light → electrons become excited
Going from excited back to ground state means some energy is lost in the form of emission
Emitted light is always of a ______ wavelength than excitation light
always of a LONGER wavelength than excitation light
ex: might absorb blue or violet and emit green
From lowest to highest energy: ROYGBIV
(The smaller the wavelength, the greater the frequency, the greater the energy)
Fluorescence microscopy allows us to
- examine morphological structure over time
- observe protein expression (i.e. by tagging w/ GFP)
- study cellular and subcellular function without disturbing membrane
- obtain information about the kinetics and spatial distribution of cell
- optically record from individual synapses
What kind(s) of imaging can you do with fluorescence microscopy?
static and dynamic imaging
static: fluorescent antibodies against subcellular structures
dynamic: measure cellular dynamics i.e. by dye loading with patch pipette
Basics of IHC
Primary antibody binds antigen
Secondary antibody binds primary antibody
Secondary antibody is labeled with fluorochrome
GFP
- bimodal absorption
- absorbs blue/violet light (395 and 475 nm) and emits green (509 nm)
Tagging proteins with GFP (how to and why)
- Inject/insert GFP before stop codon for protein
- Can be used for cell lineage/gene expression marking, protein tagging, and protein-protein interaction monitoring
Brainbow
Uses stochastic and combinatory expression of a few spectrally distinct fluorescent proteins
- Expresses fluorescent proteins in different ratios within each cell
- Three separate FPs are arranged sequentially in transgene along with a pair of lox sites (i.e. loxP-loxP, lox2272-lox2272)
Classes of fluorescent indicators
- antibodies (labeled w/ fluorophore)
- GFP
- Ion/environment-sensitive indicator dyes
What is the basic principle of epifluorescence? How many filters are there?
Epifluorescence principle = how light flows through a microscope
Excitation photons flow from light source through excitation filter (short-pass) to dichroic mirror → reflected from dichroic mirror through objective onto specimen → emission photons pass through objective, mirror, and emission filter (long-pass) through detector
Parts of microscope for fluorescence imaging
- Light source
- Filter block
- Objective
- Photon detector
Light source in different types of imaging
Fluorescence: either tungsten halogen lamp or mercury arc lamp (HBO)
Confocal: Laser (gives off monochromatic light of same wavelength that is coherent and linear polarized)
Two-photon: Ti/Sa laser (gives very short pulses in the fs range)
Chromatic aberration?
Light rays passing through lens focus at different points, depending on their wavelength
Colors bend light to different degrees
There are lenses that correct this
Can be axial or lateral
Objective he’ps focus the different wavelengths of light to the same point
What are the 2 types of abberrations/aberration correct?
chromatic aberration and spherical aberration
What is spherical aberration?
= When light rays enter at different points of a spherical lens are not focused to the same point of the optical axis, creating different focal points
(light that hits at the edge of a lens bends more than light that hits at the center)
What is numerical aperature
= how much light you can gather and how much detail you see
= nsine(mu)
Which medium is usually used for its high refractive index?
Oil
-refractive index close to glass, meaning the light bends less and more light can reach the objective
What is a resolution limit?
The minimum distance 2 points can be told apart as separate
Why is the use of filters important?
Excitation light has much greater energy than the secondary fluorescence/emission light, so you need to be able to block the bright excitation light from reaching the detectors
What does the filter block contain?
- Excitation filter (short pass filter)
- Dichroic mirror (reflects low wavelengths and allows longer wavelengths to pass through)
- Emission/barrier filter (long pass filter)
Photon detectors used in each type of microscopy
Conventional: Photomultiplier tube (PMT) and/or CCD camera
Confocal: PMT (colorblind system based on a pseudo-color look up table)
Two-photon: PMT
Basic idea of two-photon microscopy
Two photons enter at same time and same place with doubled wavelength
Uses photons from infrared spectrum (>750 nm)
Have high photon density
Excite w/ two photons of longer λ and less energy
Single photon: E= hv c = λv E ~ 1/λ
two-photon: E* = 1/2E = ~ 1/2λ
Confocal vs two-photon
- Confocal has sufficient excitation at any time
- Two photon only has sufficient excitation in the focal plane w/in a laser pulse
- Focal plane in confocal is established by the pinhole, but don’t need pinhole in two-photon
- Both have same general structure, but with different laser(?) and two photon doesn’t require a pinhole
- Several emission signals can be sent, but in confocal microscopy, only the ones that enter through the pinhole are the ones observed
- Two-photon has excitation only within a narrow range of focal range but has better Z resolution
- Less bleaching in two-photon because excitation can only occur in a super narrow focal plane
