Wavelength Characteristics of Fluorescence Filters
Images courtesy of : 1: Michael W. Davidson, National High Magnetic Field Laboratory, USA 2: Dylan Burnette, Paul Forscher Laboratory, Yale University, USA
Qdot, and the Q logo are trademarks of Quantum Dot Corporation.
Trademarks of other companies that are referred to in this brochure are the following: Texas Red® (Molecular Probes, Inc.), Cy®(Amersham Biosciences, Ltd.)
Company and product names included in this brochure are the registered trademarks of the respective companies. Monitor images are simulated.
Specifications and equipment are subject to change without any notice or obligation on the part of the manufacturer. May 2005. ©2005 NIKON CORPORATION
WARNING
TO ENSURE CORRECT USAGE, READ THE CORRESPONDING MANUALS CAREFULLY BEFORE USING THE EQUIPMENT.
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Code No. 2CE-MRJH-1
ISO 14001
Nikon Technology Maximizes
the Potential of Fluorescence Images
Nikon offers a wide range of filter blocks, from general epi-fluorescence use to those dedicated for
a specific fluorescent reagent, for supporting today's variety of fluorochromes. With a standard filter
diameter of 25 mm, commercially available fluorescence filters can be exchanged by users according
to the desired use.
Now you can capture fluorescence images with higher contrast than ever. In addition to the superb
optical performance of its filters, as well as its high signal-to-noise ratio optical system, Nikon employs
a proprietary Noise Terminator in its fluorescence systems. This enables clear images, even with
weak fluorescence.
Example with an inverted microscope
Noise Terminator
Fluorescence filter blocks
General fluorescence filters corresponding to various excitation colors such as B-2A and G2A. Since many of the barrier filters are the long-pass type, numerous reagents can be
supported by a single filter.
Deciphering filter names
Example:
B-2A
Excitation color
B is blue, G is green,
and so on
Excitation light that barely passes through without being totally
reflected by the dichroic mirror can be a source of noise. The noise
terminator is installed to appropriately process excitation light (stray
light) that passed through the dichroic mirror, thereby preventing
the light from reflecting in the filter block and leaking into the
observation side. This makes it possible to obtain fluorescence
images with extremely low background noise and a high S/N ratio.
The Noise Terminator comes standard with Nikon fluorescence
microscopes.
To objective lens
Excitation light
Filter type
Excitation filter
bandwidth
Absorbing
material
Stray light
A
m bs
at or
er bi
ia ng
l
Product Lineup
Noise terminator
A and B are long-pass types
(B has a longer cutoff wavelength)
E is a band-pass type
(Example with an inverted microscope)
1 is narrowband (up to 20 nm)
2 is wideband (30 to 50 nm)
3 is super wideband (60 nm and up)
High-performance filter cassette holder
Filter blocks for fluorescent reagents
and fluorescent proteins
1)
Filters corresponding to specific fluorescent reagents such as DAPI and FITC. Since the bandpass type is common on the barrier filter side, autofluorescence of plants, for example, is
suppressed, enabling clear images with low background noise.
The epifluorescence filter cassette holder for the TE2000E/U/S
delivers superb performance, especially when combined with TIRF
systems. It is the optimum choice for TIRF illumination in all types
of excitation methods because it minimizes the deviation of the
focal point of the light source on the objective pupil plane due
to filter cassette switching.
Freedom of switching filters
The optimal combination for the purpose of your observation can
be created by easily removing the excitation filter, barrier filter,
and/or the dichroic mirror.
Excitation filter
High-quality filter blocks for fluorescent protein
The Wavelength cut-on and cut-off rise to peak is very steep, much more than for ordinary
filter blocks for fluorescent proteins, thereby enhancing transmittance. Employing this filter
makes it possible to obtain extremely clear, bright and non-overlapping fluorescent images.
Dichroic mirror
Multiband filter blocks
Filters that enable the simultaneous observation of double or triple staining techniques such
as DAPI-FITC-Texas Red. Since there is no need to switch filter blocks in multi-stained
fluorescence observation, there will be no position deviation of fluorescence filters, and no
need to use the merge function of capture software when shooting with a color camera.
Barrier filter
2)
2
Nikon, Chroma Technology:
The direction of the arrow on the excitation and barrier filter is the dichroic mirror side
Omega Optical, Semrock:
The direction of the arrow on the excitation/barrier filter is the direction of light
3
Fluorescence Filter Blocks from Nikon
Spectral Characteristics Table for Filters
UV excitation
V excitation
UV-2A
Filter Color
B excitation
V-2A
Filter Color
G excitation
B-3A
Filter Color
G-2A
80
80
80
80
60
40
20
0
60
40
60
40
20
20
300
350
400
450
500
550
600
650
0
700
0
350
400
450
DM400
500
550
600
650
700
BA420
EX380-420
UV-1A
DM430
350
60
40
400
450
500
550
600
650
0
700
EX420-490
DM505
BA520
Filter Color
80
Transmittance (%)
80
Transmittance (%)
80
Transmittance (%)
100
60
40
20
20
1)
350
400
450
500
550
600
650
0
700
DM400
350
400
450
EX450-490
BV excitation
UV-2B
Filter Color
BV-2A
Filter Color
500
550
600
650
0
700
DM505
B-1A
Filter Color
450
500
550
600
650
0
700
350
400
450
Wavelength (nm)
EX330-380
DM400
60
40
450
500
550
600
650
0
700
350
400
450
EX400-440
DM455
EX470-490
BA470
BV-1A
Filter Color
80
Transmittance (%)
80
Transmittance (%)
100
60
40
20
0
500
550
600
650
600
650
700
DM575
BA590
60
40
700
0
350
400
450
DM505
500
550
600
650
700
Wavelength (nm)
BA520
EX510-560
B-1E
100
550
Filter Color
Wavelength (nm)
Wavelength (nm)
BA435
500
20
20
20
400
Transmittance (%)
Transmittance (%)
Transmittance (%)
80
Transmittance (%)
80
20
BA590
G-2B
80
350
400
EX546/10
80
0
350
BA520
100
40
DM575
Wavelength (nm)
100
40
700
40
100
60
650
60
100
60
600
Filter Color
Wavelength (nm)
BA400
550
20
Wavelength (nm)
EX365/10
500
G-1B
100
0
450
EX510-560
B-2A
40
400
Wavelength (nm)
100
60
350
Wavelength (nm)
BA450
Filter Color
Filter Color
20
Wavelength (nm)
Wavelength (nm)
EX330-380
Transmittance (%)
100
Transmittance (%)
100
Transmittance (%)
100
Transmittance (%)
100
DM575
BA610
Filter Color
60
40
20
350
400
450
500
550
600
650
700
0
350
400
450
Wavelength (nm)
EX435/10
DM455
500
550
600
650
700
Wavelength (nm)
BA470
EX470-490
DM505
BA520-560
EX: Excitation filter DM: Dichroic mirror BA: Absorption filter
4
5
Fluorescence Filter Blocks from Nikon
Special filter blocks for fluorescent reagents and fluorescent proteins
DAPI
Filter Color
Texas Red
Filter Color
Cy7
(Chroma Technology equivalent: 41009)
Filter Color
BFP
80
80
80
80
60
40
20
0
60
40
60
40
20
20
335
400
450
500
550
0
600
525
550
600
Wavelength (nm)
EX340-380
DM400
Transmittance (%)
100
Transmittance (%)
100
Transmittance (%)
100
Transmittance (%)
100
650
0
700
FITC
EX540-580
Filter Color
Cy3
60
40
500
550
600
650
700
750
800
850
0
900
DM595
BA600-660
EX710/75
(Chroma Technology equivalent: 41007a)
Filter Color
DM750
BA810/90
GFP LP (Long-pass type)
Filter Color
CFP
80
60
40
500
550
0
600
350
400
450
500
Wavelength (nm)
EX465-495
DM505
60
40
20
20
450
Transmittance (%)
80
Transmittance (%)
80
Transmittance (%)
80
Transmittance (%)
100
0
550
TRITC
EX535/50
600
Filter Color
Cy5
DM565
650
700
0
750
350
400
450
500
EX480/40
BA610/75
Filter Color
550
600
650
700
0
750
DM505
GFP BP (Band-pass type)
Filter Color
YFP
20
500
550
600
650
0
DM565
60
40
350
400
450
500
550
600
650
700
750
0
350
400
450
Wavelength (nm)
BA605/55
EX620/60
DM660
400
450
500
550
600
650
700
750
DM455
BA480/40
(Chroma Technology equivalent: 41028)
Filter Color
60
40
500
550
600
650
700
750
0
300
350
400
EX480/40
DM505
450
500
550
600
650
700
Wavelength (nm)
Wavelength (nm)
BA700/75
Filter Color
20
20
Wavelength (nm)
EX540/25
Transmittance (%)
Transmittance (%)
80
Transmittance (%)
80
Transmittance (%)
80
0
350
EX436/20
80
20
750
BA460/50
(Chroma Technology equivalent: 31044v2)
BA510
100
40
700
Wavelength (nm)
100
40
650
40
100
60
600
DM420
Wavelength (nm)
(Chroma Technology equivalent: 41008)
550
60
100
60
500
20
Wavelength (nm)
BA515-555
450
EX380/30
100
20
400
Wavelength (nm)
100
40
350
Wavelength (nm)
100
60
Filter Color
20
Wavelength (nm)
BA435-485
(Chroma Technology equivalent: 31021)
BA535/50
EX500/20
DM515
BA535/30
1)
6
7
Fluorescence Filter Blocks from Nikon
Reagent Compatibility Table/
Specific Energy Distribution
Multiband filter blocks
DAPI/FITC
Reagent Compatibility Table
(Chroma Technology equivalent: 51000)
Filter Color
DAPI/FITC/TRITC
80
80
Transmittance (%)
100
Transmittance (%)
100
60
40
20
0
40
20
350
400
450
FITC/TRITC
500
550
600
650
700
0
750
350
400
450
(Chroma Technology equivalent: 51004)
Filter Color
DAPI/FITC/Texas Red
80
Transmittance (%)
80
Transmittance (%)
100
60
40
20
550
600
650
700
750
(Chroma Technology equivalent: 61002)
Filter Color
60
40
20
300
350
400
450
500
550
600
650
700
0
350
400
Wavelength (nm)
FITC/Texas Red
(Chroma Techno logy equivalent: 51006)
Filter Color
80
60
40
20
350
400
450
500
550
Wavelength (nm)
450
500
550
Wavelength (nm)
100
Transmittance (%)
500
Wavelength (nm)
100
0
Filter Color
60
Wavelength (nm)
0
(Chroma Technology equivalent: 61000)
600
650
700
750
1)
600
650
700
750
Typical reagents
ACMA
Acridine Orange (DNA+RNA)
Alexa Fluor 350
Alexa Fluor 488
Alexa Fluor 568
Alexa Fluor 647
Allophycocyanin (APC)
BCECF (high ph)
BFP (Blue Fluorescent Protein)
Calcein
Calcium Green-1
Cascade Blue
CFDA (Carboxyfluorescein)
CFP (Cyan Fluorescent Protein)
Cy2
Cy3
Cy5
DAPI
DiOC6
Dil
DsRed (Red Fluorescent Protein)
Ethidium bromide
FITC
Fluo3
FluoroGold
FM1-43
Fura2
Fura Red
Hoechst 33342 & 33258
Indo1
JC-1
Lissamine rhodamine B
Lusifer Yellow
Lyso Tracker Green
MitoTracker Green
MitoTracker Orange
Monochlorobimane
NBD (amine)
Nile Red
Pacific Blue
R-phycoerythrin
POPO-3
Propidium lodide (PI)
Pyronine Y
RH795
Rhodamine123
SYTOX
Texas Red
TMR (Tetramethylrhodamine)
TO-PRO-3
TOTO-3
XRITC (X-rhodamine-5)
YFP (Yellow Fluorescent Protein)
YOYO-1
EX
430
440-480
347
495
579
653
650
503
381
494
506
376
495
458
489
550
649
358
480
549
558
545
494
506
368
502
335
472
352
330
514
570
428
505
490
551
380
460
549
405
480/546/565
534
536
555
530
507
504
577
555
642
642
580
513
491
EM
474
520-560
442
519
604
669
660
528
445
517
531
425
520
480
506
570
670
461
501
565
583
605
518
526
565
625
505
646
461
401
529
590
536
511
516
576
461
534
628
455
578
570
617
580
712
529
523
620
580
661
660
605
527
509
Nikon-compatible filter blocks
BV-2B
B-2A
UV-2A
B-2A, B-1A
G-2A
Cy5
Cy5
B-2A
BFP
FITC, B-2A
B-2A
UV-2A
FITC, B-2A
CFP
B-2A, GFP-BP
Cy3, G-2A
Cy5
DAPI, UV-2E/C
B-2A, FITC-HYQ
Cy3, G-2A
TRITC, G-2E/C
G-2A
FITC, B-2E/C
B-2A
UV-2A
B-2A
Fura-2
B-2A
UV-2A
Indo-1
B-2A, YFP
Cy3, G-2A, G-2B
B-3A
B
B-2A, FITC
Cy3, G-2A
UV-2A
FITC, B-2A
G
V-2A
Cy3, G-2A*B-2A
Cy3, G-2A
G-2A
Cy3, G-2A
Cy3, G-2A
B-2A, FITC
B-2A, FITC
Texas Red, Y-2E/C
TRITC, G-2E/C, Cy3
Cy5
Cy5
Texas Red, Cy3, Y-2E/C
YFP
B-2A, FITC, GFP-BP
Specific Energy Distribution
Specific Energy
546.1
404.7/407.8
400
500
Wavelength (nm)
8
577.0/579.0/1
302.3/6
300
100
50
692.4
5
0
296.8
15
10
334.2
312.6
313.2
20
150
275.3
289.4/5/6
289.4
Specific Energy
30
25
Xenon lamp
434.8/435.8
365.02/4
35
366.29/3
Mercury lamp
600
700
0
300
400
500
600
700
800
900
1000
1100
1200
1300
Wavelength (nm)
9
Special Filter for Detecting Qdot® Conjugates
GFP HQ/CFP HQ/YFP
HQ High Quality Filter Blocks
Qdot®conjugates have several special features, including extremely slow color fading, and it is winning
acclaim as a new labeling tool for fluorescence observation. Nikon now offers a dedicated Qdot®
detection filter, which maximizes the performance of the fluorescent probe.
Nikon's newly developed high-quality filter block employs a dichroic mirror with an extremely sharp
rising edge and band-pass filter with a high S/N ratio. The filter minimizes overlap between signals
for high-quality fluorescence images that are bright and clear. Nikon offers filter blocks corresponding
*We also provide DS-Red filters in the USA.
to CFP, GFP, and YFP, respectively*.
Qdot® nanocrystals
The quantum dot conjugate is made from nanometer-scale crystals
of semiconductor material, and the color of light that they emit
differs depending on the particle size. Qdot® conjugates are
nanocrystals for labeling biomolecules such as antibodies and
streptavidin. Unlike conventional organic dyes, Qdot® offers the
following advantages:
(1) Extremely slow color fading and long-term photo stability
(2) Extremely high fluorescence intensity
(3) Extremely sharp detection of wavelength distribution, enabling
the simultaneous detection of different colors without any overlap
(4) Compatible with all manner of optical microscopes, including
confocal models
Absorbance spectra (solid line) and emission spectra
(dotted line) of Qdot® Streptavidin Conjugates
and fluorescence detect filter sets (Chroma Technology)
500,0000
400,0000
300,0000
200,0000
Basic Knowledge on
Selecting Fluorescence Filters and Mirrors
100,0000
0
400
450
500
Qdot® Conjugates
Qdot®525
Qdot®565
Qdot®585
Qdot®605
Qdot®655
Qdot®705
Qdot®800
550
600
650
700
Wavelength (nm)
Color
Green
Chartreuse
Yellow
Orange
Dark red
Near IR
Near IR
750
800
850
900
Filter block no. (Chroma Technology equibalent)
32006 (20wide), 32010 (40wide)
32005 (20wide), 32009 (40wide)
32004 (20wide), 32008 (40wide)
32003 (20wide), 32007 (40wide)
32011 (20wide), 32012 (40wide)
32014 (20wide), 32015 (40wide)
32020 (30wide), 32021 (50wide)
Note: The emission of Qdot® 705 and 800 is not visible to the naked eye and must be detected with
an IR-sensitive detector.
Select fluorescence filters and mirrors according to the following procedures.
Checking the wavelength properties of
fluorescent substances
Selecting an barrier filter
Feel free to refer to the characteristic listed in catalogs and other
sources to check the wavelength properties of fluorescent
substances, but since these properties change slightly depending
upon solution conditions, use a fluorometer or similar instrument
to compare excitation and fluorescence wavelength properties
while selecting the optimal optical element.
Selecting an excitation filter
Multi-color (5-color) immunostaining of fixed human epithelial cells
Selecting a dichroic mirror
Select a filter that satisfies the excitation wavelength of the
fluorescent substance. Especially when using a mercury lamp as
a light source, making the most of the emission lines of the lamp
into the excitation wavelength enables highly efficient excitation.
Since applying intense light in a wavelength range other than the
excitation wavelength of the fluorescent substance being observed
causes the background noise to rise and unnecessarily damages
the sample, this situation should be avoided whenever possible.
Relationship between dyes and dichroic mirrors
100
Anti-mouse IgG antibody labeling/
Mitochondria
Qdot® 565
Anti-rat IgG antibody labeling/
Microtubules
Qdot® 605
Anti-rabbit IgG antibody labeling/Ki-67
Transmittance (%)
80
Qdot® 525
Excitation
wavelength
properties
Fluorescence
wavelength properties
60
40
20
0
400
450
500
550
600
650
700
Wavelength (nm)
*All images displayed using pseudo color
Nikon Eclipse E800 upright microscope was used
100W mercury lamp used for excitation
Qdot® 655
Anti-human IgG antibody labeling/
Nuclear antigen
* For Qdot® products, we recommend that the excitation area be changed according to your application.
Exciter 1: For researchers observing living cells (excitation at 402.5 nm to 447.5 nm) => D420/40
Exciter 2: For researchers observing fixed cells or who emphasize brightness and are not concerned about UV toxicity
(excitation at 365 nm to 465 nm) => E460SPUV
Nikon also carries Qdot®-compatible filters for the C1 plus confocal laser microscope.
10
Generally, a long-pass filter that transmits up to long wavelengths
will be selected, but when observing a multi-stained specimen using
discrete wavelength filter blocks, or when using a camera with high
sensitivity in the high red or IR wavelength bands, select a bandpass filter that does not transmit the longer wavelengths.
Qdot® 705
Streptavidin Conjugates labeling/Actin
When comparing the spectral property curve of a dichroic mirror
measured at a 45º angle, select a mirror for which the rising wavelength
in the transmission range is in between the excitation wavelength
and fluorescent wavelength of the fluorescent substance. When the
excitation wavelength and fluorescent wavelength of the fluorescent
substance are close to each other, select a dichroic mirror that rises
closer to a short wavelength in order to transmit the fluorescence
signal as much as possible. With a dichroic mirror, it is necessary
to pay attention not only to the rising wavelength, but also the
inclination of the rise. A dichroic mirror with a gentle rising edge
may end up lowering the transmittance of the fluorescence signal
and produce an unwanted crossover fluorescence emission signal.
Combining an excitation and
an barrier filter
When the wavelength properties of an excitation filter and barrier
filter overlap, ideally, no light at all will pass through. Since
fluorescence is a weak beam, even the slightest bit of light leakage
will cause the background noise to rise, inviting degraded image
quality.
11