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