q
Olympus offers a range of other high numerical aperture
objectives whose reduced autofluorescence and specially selected
glass contribute to improved fluorescence S/N ratios.
Especially the PLAPON60XO has outstanding N.A., which is 1.42.
N.A.
W.D. (mm)
UPLSAPO 10X2
0.40
3.1 mm
UPLSAPO 20X
0.75
0.6 mm
UPLSAPO 40X2
0.95
0.18 mm
UPLSAPO 60XO
1.35
0.15 mm
UPLSAPO 100XO
1.40
0.13 mm
PLAPON60 XO
1.42
0.15 mm
UPLFLN40XO
1.30
0.2 mm
LUCPLFLN 20X
0.45
6.6 — 7.8 mm
LUCPLFLN 40X
0.60
2.7 — 4 mm
LUCPLFLN 60X
0.70
1.5 — 2.2 mm
q
8
7
Improved S/N ratio enables efficient detection of
even weak fluorescence.
Better S/N ratio delivers brighter, higher-contrast
images in fluorescence observation.
The ideal microscope allows bright, high contrast fluorescence
observation from the minimum amount of excitation light in order to
minimize cell damage or fluorescence fading. To detect a weak
fluorescence signal (S) efficiently, all other light noise (N) must be
reduced. Therefore, it is very important for fluorescence observation
to maximize the signal (S) and to minimize the noise (N).
Measures to enhance the signal (S)
q
Fluorescence objectives with high N.A.
w
Filters matched to the wavelength
characteristics of individual
fluorochromes
Measures to reduce noise (N)
q
Objectives without autofluorescence
w
No crossover between excitation &
emission light with new introduced
filters.
e
Optical system that prevents entry of
stray light
r
Ring slit illumination to reduce
autofluorescence
Fluorescence Observation Units
w
The S/N ratio of certain interference type fluorescence mirror
units is now improved, thanks to the application of new coating
technology to narrow the gap between excitation (Ex) and emission
(Em). The line-up has been extended for wide variety of choice.
U-MNIBA3
U-MGFPHQ
400
450
500
550
600
650
0
10
20
30
40
50
60
70
80
90
100
T
ransmittance (%)
Wavelength (nm)
BP470-495
DM505
BA510-550
T
ransmittance (%)
400
450
500
550
600
650
0
10
20
30
40
50
60
70
80
90
100
Wavelength (nm)
DM485
BP460-480
Ex
BA495-540
Em
High S/N ratio objective with reduced autofluorescence
High-performance fluorescence mirror units for fluorescent proteins
Improved performance of interference type fluorescence mirror unit
High performance mirror units
FL
S
N
S
N
S
N
S
N
FL
w
w
e
r
e
The slight transmission of stray light when excitation light is
reflected in the dichromatic mirror causes a rise in the level of noise.
Olympus mirror units absorb more than 99% of this stray light
through their light absorber.
Light
source
Stray light
Excitation filter
Emission filter
Stray light
reducing
function
Fluorescence light
for observation
Dichromatic
mirror
Objectives
Specimen
Excitation light:
Illumination light
●
A multi-band dichromatic mirror is normally used to obtain multi-
color images of multiple stained fluorescent samples by using filter
wheels on the excitation and emission sides. However, this kind of
mirror encounters the problem that each fluorescence image gets
darker as the number of color dyes increase, because the
transmission spectrum becomes narrower and the transmittance
falls to lower than 90% at best. Olympus has therefore developed
the world’s first glass reflector that is not wavelength-dependent,
offering a high transmittance of 94% across a wide wavelength
range from 430 nm to 700 nm. Used in combination with the filter
wheels on the excitation and emission sides, a wider variety of color
dyes can be used and
fluorescence images are captured
more efficiently.
*Special order basis product
■
Glass reflector specifications
26 X 38 mm (t=1 mm) glass substrate
Transmittance 94% (at 430-700 nm)
26 X 38 mm
* Observations through eyepieces may have
some restrictions
Sapphire-pm.
CFP-CaM
YFP-mt
DsRed-nu
Simultaneous imaging of Sapphire, CFP, YFP, and RFP. HeLa cells were imaged for
Sapphire-pm, CFP-CaM, YFP-mt, and DsRed-nu. The images were obtained using the glass
reflector in a normal cube.
Optical components used for a 4-fluorophore imaging
Dye
ND Filter*
1
Excitation Light Path
Reflector
Emission Light Path
Sapphire-pm
400DF15
535DF2
CFP-CaM
—
440DF20 Glass
480DF30
YFP-mt 490DF20
535DF25
DsRed-nu 546DF10
595RDF60
*
1
ND filters in the holder of the illuminator.
Glass reflector captures fluorescence of multiple color dyes
Stray light reduction function equipped on all mirror units
Ring slit illumination unit to reduce noise / IX2-RFRS
■
Usage examples of the glass reflector
r
The ring slit illumination IX2-RFRS makes the ring shape
illumination on the objective to allow excitation light to pass through
the objectives outer portion
to not to excite the objective
auto-fluorescence generated
at the center of an objective
.
Annular illumination
Auto-
fluorescence
Specimen
Objective
Ring slit
Excitation
filter
Excitation
light
Hg lamp
Xe lamp
General observation
S
N
N
Glass reflector transmission properties
*Solid line: excitation, Dotted line: emission
Excitation filter
Emission filter
Sapphire
CFP
YFP
DsRed
400
500
Wavelength (nm)
600
700
100
80
60
40
20
0
T
ransmittance (%)
Normal illumination
Ring slit illumination
➔
Illumination
Normal
Annular
SIGNAL
408
479
NOISE
36 18
S/N 11.3
26.6
New FL system
The sharp performance of the dichromatic mirror in the new mirror unit minimizes crossover with the excitation filter and reduces
excitation light leakage to less than a tenth of our conventional models. Combined with the light absorbing mechanism (which absorbs
more than 99% of stray light), a high S/N ratio is achieved without the need for any special mechanism to prevent excitation light leakage.
IX2-RFRS and IX2-RS40/60/100
w
Olympus has developed outstanding filter coating technology,
which gives the high efficient transmission and the reflection as well
as sharp cut off characteristics. This newly developed coating
results in optimized mirror units for the various fluorochromes
included ECFP/EGFP/EYFP/DsRed.
