advanced imaging laboratory


epi-fluorescence with the microscope

Epi-fluorescence microscopy is a technique which is widely used by biologists. In some cases it involves auto or primary fluorescence which occurs with some substances such as chlorophyll. More often it involves staining with a special type of histological stain called a fluorochrome which is only taken up by specific material or hybridization of fluorescently labeled complementary DNA sequences. High energy, short wavelength (eg. UV or blue) light is directed onto the preparation and lower energy, longer wavelength (eg. green or red) light is emitted, according to Stokes' Law. The Jablonski Diagram, seen below, shows a number of possible routes by which an excited molecule can return to its ground or room temperature state via unstable triplet states. A rapid return results in fluorescence and a delayed return results in phosphorescence.

Jablonski diagram

For example, excitation using invisible ultra-violet light results in the emission of visible blue fluorescence light from a label such as DAPI. Another common example involves blue excitation resulting in a brighter yellow-green emission from FITC.

epi-fluor light path

Traditional fluorescence microscopy used transmitted light, at first with carbon arc sources and then later with 200 watt super-pressure mercury lamps. It is now usual to use much more efficient epi-illuminators with small 50 or 100 watt super pressure mercury or xenon lamps. Using a matched optical set that consists of an exciter filter, a dichroic beamsplitter and a barrier filter, the epi-illuminator allows only light of the correct short, exciting wavelength to strike the preparation and only light of the longer, emitted wavelength to reach the eyepiece, detector or the camera.

To use the Zeiss Universal with epi-fluorescence turn on the left-hand transformer for the mercury lamp and the right-hand one for the transmitted light tungsten lamp. You will first need to search your slide using brightfield or phase contrast. Because fluorescence is quite faint it is usual to work in dim light with the door closed. On finding the correct material, cut off the visible light by putting a lens cap in the light path of the microscope and open the blocking slide in the epi-illuminator. Position I on the epi-fluorescence condenser contains the #02 filter set with maximum transmission for the mercury line at 365/366 nm. and emission beyond 420 nm. Position II contains the #09 filter set used for FITC (fluorescein isothiocyanate), transmitting from 450 to 490 nm and allowing emission beyond 520 nm. Position III contains the #15 filter set used for Rhodamine, transmitting at 546 nm and allowing emission beyond 580 nm. Do not use position IV because it is blocked off.

The next figure shows the spectra of the HBO light bulb used for epi-fluorescence. It show a spectra with very strong lines at particular wavelength, such as the one at 365 nm or the one at 436 nm. Some regions of the spectrum are very weak, such as the region between 450 nm and 530nm and excitation at those wavelengths is problematic.

DAPI excites at 359 nm and emits at 461 nm. Rhodamine excites at 570 nm and emits at 590 nm. The figure below shows the FITC excitation at 494 nm and fluorescence emission at 518 nm

The figures below show the transmission curves for the exciter filter, the dichroic beam splitter and the barrier filter for the Zeiss 02, 09 and 15 filter sets. There are many other fluorochromes in use and this fluorochrome list shows some of the large number of labels and conjugates available today.


Zeiss filter set 02 excites at 365 nm. and allows emission at 420 nm.

Filter set 02 is used with the following Fluorochromes:

1,5 IAEDANS
1,8-ANS
4-Methylumbelliferone
7-Amino-4-methylcoumarin
7-Hydroxy-4-methylcoumarin
ABQ
Alexa Fluor 350™
AMC, AMCA-S
AMCA (Aminomethylcoumarin)
AMCA-X
Aminocoumarin
Aminomethylcoumarin (AMCA)
Anthrocyl stearate
Bisbenzamide
Bisbenzimide (Hoechst)
Calcein Blue
Cascade Blue™
Dansyl
Dansyl Amine
Dansyl Cadaverine
Dansyl Chloride
Dansyl DHPE
DAPI
Dapoxyl 2
Dapoxyl 3
DIDS
Dopamine
ELF 97
Fast Blue
FluoroGold (Hydroxystilbamidine)
Granular Blue
Hoechst 33258
Hoechst 33342
HPTS
Hydroxycoumarin
Hydroxystilbamidine (FluoroGold)
Indo-1, low calcium
Intrawite Cf
Laurodan
Leucophor PAF
Leucophor SF
Lyso Tracker Blue
LysoSensor Blue
LysoSensor Yellow/Blue

Marina Blue
Noradrenaline
Nuclear Yellow
PhotoResist
Phorwite AR
Phorwite BKL
Phorwite Rev
Phorwite RPA
PhotoResist
Pyrozal Brilliant Flavin 7GF
Serotonin
sgBFP™ (super glow BFP)
SITS
SITS (Stilbene Isothiosulphonic Acid)
SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium)
Stilbene
Thioflavin TCN
Thiolyte
Tinopol CBS (Calcofluor White)
True Blue
Uvitex SFC
Y55F
Y66H


Zeiss filter set 09 excites between 450-490 nm. and allows emission at 515 nm.

Filter set 09 is used with these Fluorochromes:

5-Carboxyfluorescein (5-FAM)
5-FAM (5-Carboxyfluorescein)
Acridine Orange + RNA
Acridine Orange, both DNA & RNA
Acridine Yellow
Alexa Fluor 488™
ATTO-TAG™ CBQCA
ATTO-TAG™ FQ
Auramine
BCECF (high pH)
BCECF (low pH)
Calcein
CFDA
Chlorophyll
CMFDA
Coriphosphine O
CyQuant
Di-8-ANEPPS (non-ratio)
DiA (4-Di-16-ASP)
DTAF
FDA
FITC
FITC Antibody
Fluo-3
Fluo-4
Fluorescein (FITC)
Fluorescein Diacetate
Fluoro-Emerald
FluorX
FM 1-43™
Genacryl Pink 3G
GFP (S65T)
GFP red shifted (rsGFP)
LIVE/DEAD Kit Animal Cells
Calcein/Ethidium homodimer
Lyso Tracker Blue-White
Mitotracker Green FM
Oregon Green 488-X
Oregon Green™ 488
Oregon Green™ 500
PE-Cy5
PerCP
PerCP-Cy5.5
PE-TexasRed [Red 613]
Phosphine 3R
PKH67
Procion Yellow
Red 613 [PE-TexasRed]
Rhodamine 110
Rhodamine 5 GLD
Sulphorhodamine G Extra
SYTO 16
SYTO 21
SYTO 23
SYTO 24
TruRed

Zeiss filter set 15 excites at 546/12 nm. and allows emission at 590 nm.

Filter set 15 is used with these Fluorochromes:

7-Aminoactinomycin D (7-AAD)
Acid Fuchsin
Alizarin Complexon
Alizarin Red
LDS 751 (DNA)
Rhodamine B
Rhodamine B 200

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September 21st, 2005