LS
FLUORESCENCE
BASIC NOTES ON FLUORESCENCE
A COLORFUL EXPRESSION
Fluorescence is a molecular phenomenon of spontaneous light emission from a substance after being excited. The emitted light
is always of lower energy (longer wavelength) than the excitation energy.
The excitation/emission process is extremely sensible, making fluorescence the most complex contrast method in widefield
microscopy. An incorrect setup may cause more than a bad image: there might be no visible signal at all.
A SIMPLIFIED APPROACH TO FLUORESCENCE
• Specific dyes have been developed to selectively stain
parts of a specimen. These dyes are called fluorochromes.
• A selected wavelength is applied to the sample (excitation);
this wavelength refers to the absorption maximum of the
fluorochrome.
• Energy is absorbed by the fluorochrome, shifting it to a
higher energy level.
• The excited state of the fluorochrome lasts only a split
second; falling back to the original energy level is accom-
panied by an emission of light.
• As the emission comes along with a partial energy transfor-
mation, all emission is less energetic than its corresponding
excitation.
• “Stokes shift” describes this effect: The emission is always
shifted to a longer wavelength (color).
UV excitation
BLUE excitation
GREEN excitation
BLUE emission
GREEN emission
RED emission
As the energy relation of excitation to emission is 10.000:1,
a maximum efficient hardware setup has to be targeted.
The Mercury bulb as fluorescence light source ensures a
broad energy spectrum for excitation from 300nm to 600nm,
including several intensity peaks.
To optimize the performance, specific objectives with
maximum Numerical Apertures and maximum transmission
(especially in UV) are recommended. A good choice is
Motic’s selection of PLAN FLUAR PL FL objectives.
Long working distance (LWD) objectives and Phase contrast
lenses may be necessary because of sample characteristics,
but will not allow the brightest possible signal.
Principle of excitation/emission Stokes Shift Mercury Arc Lamp Spectral Distribution
E
Intensity
Absorption
Stokes Shift
Emission
Wavelength
PLAN FLUAR OBJECTIVES
0,10
0,08
0,06
0,04
0,02
Spectral Intensity [W/sr/nm/1000cd]
300 400 500
Objectives
Plan Fluar PL FL 4X
Plan Fluar PL FL 10X
Plan Fluar PL FL 20X
Plan Fluar PL FL 40X
Plan Fluar PL FL 50X
Plan Fluar PL FL 100X
Wavelength
N.A.
0.13
0.30
0.50
0.75
1.0 Oil
1.3 Oil
600 700 800
W.D. (mm)
20.5
10.5
1.9
0.58
0.17
0.20
Motic’s fluorescence filter cubes can be used on both upright and inverted microscopes.
Following the microscope type, the fluorescence ray path requests a different filter cube orientation.
UPRIGHT MICROSCOPE - BA410E
INVERTED MICROSCOPE - AE31E
FILTER CUBE ORIENTATION
Barrier filter
(emission)
Dichroic Mirror
in a filter cube
Microscope
Objective
FILTER CUBE ORIENTATION
Microscope
Objective
Dichroic Mirror
in a filter cube
Excitation
Filter
Glass slide
Glass slide
Excitation
filter
Barrier filter
(emission)
A standard Fluorescence filter cube consists of a
combination of 3 independent yet inter-coordinated filters:
- Excitation filter
- Dichroic Mirror
- Barrier filter (emission)
The excitation filter has a band pass characteristics, opening a
defined sector within the spectrum of the Mercury light source
to match the absorption peak of the fluorochrome.
In a given filter specification, the excitation peak of the filter is
specified by the first numeric value, while the width of the ex-
citation sector at 50% of the maximum intensity is displayed by
the second number (peak width at half height).
A value of 480/30 (nm) for FITC describes a curve of excitation
from 465 to 495nm, with a peak around 480nm.The broader
the sector, the more energy will excite the sample. A broad
sector will result in a strong but unspecific excitation. A narrow
sector effectuates a more specific excitation. Filters with narrow
excitations are especially desired in case of multiple staining,
where excitation maxima of diverse fluorochromes (e.g. GFP
mutants) may be close to each other.
FLUORESCENCE EMISSION
Barrier filter
(emission)
Dichroic Mirror
Excitation filter
INCOMING LIGHTWAVES
The dichroic mirror (from the Greek dikhroos, meaning two-colored)
is a long pass filter which starts transmission from the specified
wavelength on (e.g.505nm in FITC). For shorter wavelengths
the dichroic is impervious and thus acts as a mirror. So the
separation of excitation light and emission signal is achieved.
The emission (also called barrier) filter may be designed with
long pass or band pass characteristics. The long pass displays
all emissions from the stated wavelength on (515nm in FITC),
resulting in a mixed color image, while the band pass construction
cuts out a pure color. Multiple staining always requests band
pass emission filters, as the single signals have to be separated.
To select the correct filter cube, the applied dyes and their
excitation/emission maxima have to be carefully taken into
consideration. A standard equipment may include DAPI, FITC
and TRITC filter cubes, which will cover common dyes in clearly
separate sectors of the Mercury bulb spectrum.
MOTIC’S STANDARD FILTER COMBINATIONS
DAPI / Hoechst FITC / RSGFP / Fluo3 FITC Long pass
Exciter D350/50X Dichroic 400DCLP Barrier D460/50m
100
90
80
70
60
T (%) T (%) T (%)
50
40
30
20
10
0
300 350 400 450 500 550
Wavelength (nm) Wavelength (nm)Wavelength (nm)
Exciter D480/30X Dichroic 505DCLP Barrier D535/40m
100
90
80
70
60
50
40
30
20
10
0
400 450 500 550 600
TRITC (Rhodamine) / CY3 Texas Red / Cy3.5 Cy5 / Fluor 633 / Alexa Fluor 647
Exciter D540/25X Dichroic D565DCLP Barrier D605/55m Exciter HQ620/60X Dichroic Q660LP Barrier HQ700/75mExciter D560/40X Dichroic 595DCLP Barrier D630/60m
100
90
80
70
60
T (%) T (%) T (%)
50
40
30
20
10
0
500 550 600 650 700
Wavelength (nm) Wavelength (nm)Wavelength (nm)
100
90
80
70
60
50
40
30
20
10
0
500 550 600 650 700
Cyan GFP Endow GFP Yellow GFP
Exciter D436/20X Dichroic 455DCLP Barrier D480/40m Exciter HQ500/20X Dichroic Q515LP Barrier HQ535/30mExciter HQ470/40X Dichroic Q495LP Barrier HQ525/50m
100
90
80
70
60
T (%) T (%) T (%)
50
40
30
20
10
0
400 450 500 550 600
Wavelength (nm) Wavelength (nm) Wavelength (nm)
100
90
80
70
60
50
40
30
20
10
0
400 450 500 550 600
Exciter D470/40X Dichroic 505DCLP Barrier E515LPv2
100
90
80
70
60
50
40
30
20
10
0
400 450 500 550 600
100
90
80
70
60
50
40
30
20
10
0
580 630 680 730 780
100
90
80
70
60
50
40
30
20
10
0
450 500 550 600 650
MOTIC’S STANDARD FILTER COMBINATIONS ARE:
Filter combinations
DAPI/Hoechst
FITC/RSGFP/Fluo3
FITC Long Pass
TRITC (Rhodamine) / CY3
Texas Red/Cy3.5
Cy5/Fluor633/Alexa Fluor 647
Cyan GFP
Endow GFP
Yellow GFP
Exciter
D350/50X
D480/30X
D470/40X
D540/25X
D560/40X
HQ620/60X
D436/20X
HQ470/40X
HQ500/20X
Dichroic
400DCLP
505DCLP
505DCLP
D565DCLP
595DCLP
Q660LP
455DCLP
Q495LP
Q515LP
Barrier Filter
D460/50m
D535/40m
E515LPv2
D605/55m
D630/60m
HQ700/75m
D480/40m
HQ525/50m
HQ535/30m
100.0
50.0
400.0 550.0 700.0
0.0
T (%)
Wavelength (nm)
400.0 550.0 700.0
Wavelength (nm)
FLUORESCENCE
LIGHTING UP THE INVISIBLE
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Official Distributor:
*CCIS® is a trademark of Motic Incorporation Ltd.
Motic Incorporation Limited Copyright © 2002-2015. All Rights Reserved.
Design Change: The manufacturer reserves the right to make changes in instrument design in
accordance with scientific and mechanical progress, without notice and without obligation.
Designed in Barcelona (Spain)
October 2015
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