Fluorescence 1
fluorescence microscopy
• what is fluorescence?
• fluorescence excitation and emission
• Stokes' shift and emission intensity
• light source
• epi-illumination
• filter cubes and filter types
• filter sets
• fluorescence in confocal systems
• 2-photon fluorescence
• into the futureDave Johnston
Biomedical Imagng Unit
Fluorescence 2
relaxed singlet excited state (S1)
ground state (S0)
excited electronic singlet states (S1')
1–10 nanoseconds
fFundamentals of fluorescence (1)fluorescence
energy is absorbed to boost electron to higher shell (S0 to S1')
some energy is lost when electron drops to the relaxed singlet excitedstate of higher shell (S1' to S1)
remaining energy is released as fluorescence when electron drops back toground state (S1 to S0)
Fluorescence 3
fundamentals of fluorescence (2) Stokes' shift
peak excitation – peak emission = Stokes' shift
loss of photon energy ( = longer wavelength)
energy loss when electron drops to the relaxed singlet excited stateof higher shell means fluorescence emission is lower energy thanexcitation
lower energy = longer wavelength
Fluorescence 4
fundamentals of fluorescence (3) Stokes' shift
absorption and emission spectra are specific to each fluorescent molecule
Fluorescence 5
Excitation of a fluorophore at different wavelengths does not changethe emission profile
but it does produce variations in fluorescence emissionintensity that correspond to the relative amplitude of the excitationspectrum at the wavelength concerned
fundamentals of fluorescence (4)
excitation and emission
Fluorescence 6
why DAPI and Hoechst are poor on the SP5 Confocal
405 nm laser excitation at far RHS of excitation spectrum means very inefficientexcitation (3 - 7%) and consequently very faint emission
broad fluorescence spectrum means you rarely monitor the whole emission (25%)
monitored emission may only be 1% as bright as staining seen down eyepiece
Fluorescence 7
HBO100 mercury bulb spectrum
http://zeiss-campus.magnet.fsu.edu/articles/lightsources/mercuryarc.html
visible range 380-750nm
Fluorescence 8
epifluorescence
the desired excitation wavelength (λ2) isselected from the spectral output of thelamp by an excitation filter (EX)
the emission filter (EM) selectivelytransmits a portion of the sample'sfluorescence emission (λ4) for detectionand blocks other emission components(λ5).
and directed to the sample via a dichroicbeamsplitter (DB)
the beamsplitter separates emittedfluorescence (---) from back scatteredexcitation light (—)
Fluorescence 9
fluorescence filter block
block is specific tomicroscope and toflurochrome
Fluorescence 10
filter types
Fluorescence 11
Fluorescence 12
Fluorescence 13
Fluorescence 14
Fluorescence 15
Fluorescence 16
Fluorescence 17
Fluorescence 18
Fluorescence 19
Fluorescence 20
Fluorescence 21
Microscope
BIU Olympus IX81BIU Leica SP2BIU Leica SP5
SW Zeiss AxioskopPL Leica DMRBE
BIU Olympus IX81BIU Olympus IX81
BIU Leica SP2BIU Leica SP5
SW Zeiss AxioskopPL Leica DMRBE
BIU Olympus IX81BIU Leica SP2BIU Leica SP5
SW Zeiss AxioskopPL Leica DMRBE
Filter Set
U-MNU2-A02A
U-MWBV2*U-MWB2
I3I310I3
U-MNG2N2.1N2.115
N2.1
Type
"UV"
"Blue"
"Green"
Excitation
360-370-
340-380G365
340-380
400-440450-480450-490450-490450-490450-490
530-550515-560515-560546/12515-560
Dichroic
400-
400395400
455500510510510510
570580580580580
Emission
lp420-
470/40lp420
470/40
lp474lp515lp515lp515
bp515-565lp515
lp590lp590lp590lp590lp590
filter sets on BIU and HRU microscopes
Fluorescence 22
Leica I3 - FITC
Zeiss #10 - FITCZeiss #3 has abandpass emision filterto minimise bleedthrough of rhodaminesignal excited by FITCexcitation wavelengths
filter sets on BIU and HRU microscopes
Fluorescence 23
multilocationFITC/TRITC/UV/Phase
timelapsemicroscopy
Olympus IX81
Fluorescence 24
Leica confocal AOBS
AOBS tuneable crystals inLeica confocal microscopesgives a much cleaner signalcut off than standard optical
filters
Fluorescence 25
Leica confocal detector
fluorescence is focussed intoa parallel "rainbow" lightpath
a slit edged with mirrors canbe moved across the lightpath and opened or closed tospecify which wavelengthsreach the detector behind theslit
other wavelengths aredeflected by the mirrorstowards other detector/mirror / slit units
our SP5 has 4 detectors
Fluorescence 26
Leica DM1600automated, inverted
stand
heated chamber
5% CO2 in air
resonant scanner
Leica SP5
Fluorescence 27
multilocationmultichannelsequential multifocustimelapseconfocal
microscopy
Leica SP5
Fluorescence 28
multiphoton fluorescence microscopy
University of Wisconsin
............
pulsed laser light
2 photons must arrivesimultaneously at the focalpoint to excite fluorophore
summed energies result inemission of a shorter
wavelength
long wavelength
low energy excitation is suited tolive cell imaging
less scatter
good depth penetration
superior resolution
Fluorescence 29
Leica SP5 X white continuum laser
for confocal systems providingmultiple and any wavelengths withnm resolution from a single laser
Leica FLUO LED illumination
bright and long lasting excitationspectra from tuned LED units with
touchscreen brightness control
new advances
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