AOBS –The Most Versatile
Beam Splitter®

Acousto-Optical Beam Splitter for the TCS SP8 Confocal• High transparency and narrow excitation bands increase cell
viability and reduce photobleaching.
• The freely programmable AOBS perfectly adapts to challenging
multicolor experiments, new laser lines, and the tunable
White Light Laser of the TCS SP8 X.
• Provides fast line sequential imaging of living cells having
spectrally close ﬂuorophores.

2

Discover the AOBS® –Changing Confocal Imaging
Fluorescence imaging is performed in an incident light configuration: the excitation lightenters the specimen on the same side from which the emission is collected. This setup
requires a device that separates excitation and emission light: a beam splitter. The AOBS
is a uniquely flexible, efficient, and fast beam splitting device.
FLEXIBILITY AND VERSATILITY SIMPLIFYGAIN MORE LIGHT WITH MAXIMUM

FAST, RELIABLE SWITCHING

MULTICOLOR IMAGINGSENSITIVITY

The position of the band for excitation

The reflection bands for excitation

Reprogramming the AOBS is a matter ofis tunable. Consequently, a single AOBS

light through the AOBS are very narrow,a few microseconds. Even complicatedis programmable for any visible laser

leaving broad bands for the collection ofillumination routines can be switchedcolor – even for lines that are installed

the precious fluorescence photons. The

between scan lines without delay or pixel

during laser upgrades or modifications

result: maximum detection sensitivity.

shift.

of the laser configuration.

The efficient separation between excitationand emission is shown in the transmission Also, complex region-of-interest scans

While the use of dichroic mirrors forcurves (Fig. 1).

more than three simultaneous excitations
is complicated and inefficient, the AOBS

can be defined. The AOBS minimizes
crosstalk at high acquisition speeds

The fraction of injection is tunable. Thiseasily operates up to eight tunable colors allows the AOBS to be programmed to a
by design, even with narrow distances

50/50 device, which is optimal for reflected

between excitation lines.

light imaging. Reflected and flourescenceimaging can be performed simultaneously,with the light from each mode detectedby distinct channels.

by switching excitation lines in linesequential multichannel scans.

3Transmission Curves for Different Beam Splitting Devices
561 nm488 nm561 nm633 nm
458 nm 514 nm
594 nm633 nm

100

100

80

80

Efﬁciency [%]Efﬁciency [%]488 nm

60
40
20

60
40
20

Dichroic

AOBS

0

0
450

500

550

600

650

700

450

Emission [nm]

500

550

600

650

700

Emission [nm]Fig. 1a: Dichroic. Non-flexible reflections on bands.

Fig. 1b: AOBS. All wavelength are fully flexible, adjusting to your experiment.

Comparing the transmission curves of a typical triple dichroic mirror (blue) and AOBS (red) for excitation with 488, 561,
and 633 nm shows two major advantages of the AOBS:• The AOBS reﬂection bands have steep edges and narrow bandwidth – allowing the collection of more emission light.
• The AOBS is more ﬂexible and can accomodate up to eight reﬂection bands – facilitating the simultaneous imaging
of multiple ﬂuorophores.
488 nm561 nmNow we add the emission curves of633 nmAlexa 488 and Alexa 568 to an overlay
100of the transmission curves of a dichroic
mirror and AOBS tuned to 488, 561,
and 633 nm. The advantage of the AOBS

80
Efﬁciency multichroic

becomes clear. Notice the increased

Efﬁciency [%]detection efficiency for simultaneous
60
Efﬁciency AOBS

excitation of Alexa 488 and Alexa 568(shown in solid orange).

Alexa
488

40

Alexa
568

Leica advantage
Signal gain by AOBS

20

For sequential excitation with the twolaser lines the spectral detection window
can be increased so that the advantageof the AOBS becomes even larger

0

500

550

600

650

700

Emission [nm]Fig. 1c: Overlay of emisson curves of Alexa 488 and Alexa 568 with transmission sof dichroic and AOBS.

(hatched orange area).

4

A Comparison of Beam Splitting Methods
Classically, beam splitting is done by dichroic mirrors that reflect certain color bands andtransmit between them. The reflecting part is used for excitation; the emission is collected
through the transmitting band. An AOBS also separates excitation and emission light,
but works in a completely different way as compared to a dichroic mirror. It is an active,
programmable device that is uniquely flexible, efficient, and fast.

SPLITTING MIRRORS HAVE FIXEDAOBS: A SINGLE, FLEXIBLE, AND RELIABLE

SPECIFICATIONSOPTICAL ELEMENT

For different applications, the dichroic

Because of its fast electronic programming,

mirror needs to be mechanicallythe AOBS does not rely on the previous

exchanged to fit spectral requirements.

selection of spectral specifications.

Changing mechanical parts takes time,

It is suitable for any set of laser lines

is prone to misalignment (pixel shift),and combinations of those in the visible

and requires a large set of mirror types

range, and features maximum transmis-in the repository.

sion between the excitation intervals.

Dichroic

AOBS

Acousto-Optical Beam SplitterThis device replaces all conceivabledichroic and multichroic mirrors andwheel- or slider-based arrangements or
combinations of these. The AOBS is asingle programmable optical element forvisible range laser scanning microscopy.

5

Acousto-Optical Beam Splitting
Technology in Detail
Beam splitting by the AOBS is based

The grid constant is variable and depends

on acousto-optical diffraction in a TeO2

on the frequency and amplitude of thecrystal. These crystals are fully transparent applied wave, which makes it easily
from below 400 nm to more than 4 μm.

tunable.

Applying a mechanical wave at radio-

If excitation light of the desired color

frequency results in periodic density

enters the crystal at an appropriate

variations within the crystal. The crystal

angle, the light will be acousto-opticallydensity affects the refractive index.

refracted and will merge with theThus, the acoustic wave creates a

principal beam, which coincides withrefractive index grid.

the optical axis of the microscope.

Understanding Physics: The Principle of Acousto-Optics
The incident angle, wavelength, and grid constant must fulfill

Excitation

the Bragg-condition. As the grid constant is tunable, any color
can be merged with the optical axis.

λ1st order

SpecimenαFurthermore, the refractive index grid of the AOBS canδaccommodate several wavelengths simultaneously for
multicolor excitation. The design of the AOBS ensures thatthe first order injection of all visible wavelengths is coaxial.
The emitted light is stokes-shifted, i.e. of different color,and so passes the crystal without being affected by
the refractive index grid. The emission is then fed into
the multichannel spectral detector of the TCS SP8.

Detector

0th order
Principal beam

Bragg condition:
νλ = 2δ sin α

458
594

561

514

488

Excitation

6468-4822496-514523-558579-597618-670
Emission

plasma membrane

nucleusmitochondria

early endosomes

tubulin3

7

A Versatile Beam Splitter for
all Confocal Imaging Applications
The AOBS is ideal for both single parameter fluorescence and sophisticated multichannel
imaging – without changing dichroic mirrors. Upon selecting a set of colors for excitation,
the AOBS will automatically be programmed to direct these lines onto the specimen and transmit
the emission between them. No need to think about which mirror to use. The TCS SP8 with
AOBS is perfect for core imaging facilities, where so many different fluorochromes need to
be imaged each day. It largely reduces the training time needed for novice confocal users.

514 nmThe spectral detector of the TCS SP8comprises an array of up to five sensors

543 nm100

that collect emissions of truly tunable80

In combination with the AOBS, it allows
simultaneous or fast line sequential
imaging of many channels without theneed to move optical parts or remove
crosstalk after recording.

Efﬁciency [%]bands with individual gain and offset.

Leica advantage
Signal gain by AOBS (30%)

60

Fig 4: Gain of efficiency:
double excitation at 514

Efﬁciency AOBS

EYFP

40

and 543 nm.
Green: Emission of EYFP,

Efﬁciency multichroic

20

Red: transmission of AOBS
(2 nm width), blue: typicaltransmission of dichroic

The AOBS is the ideal solution foradvanced multichannel confocal

0(20 nm width).

510

540

570
Emission [nm]600

Orange area: 30 % efficiency increase by AOBS.

microscopy, e.g. with many different
fluorescent proteins, chemical dyes or
combinations of those. The steep
band-edge allows wider emission bands
as compared with plan-optical mirrors

Fig. 2: NIH3T3 cells transduced with five individual fluorescent protein (FP) vectors.

and filters, therefore the AOBS offers

FPs: Cerulean, EGFP, Venus, tdTomato and mCherry. Each FP was visible only in the cells transduced with the corresponding

maximum transmission efficiency.

Image courtesy of Daniela Malide, NIH Bethesda, MD USA

Excitation power can be reduced toFig. 3: PAE cell line stably expressing five fluorescent subcellular markers (FPs) with ex and em: EBFP2-Nuc (nucleus)

decrease photobleaching and increase

405/430-450, mTFP1-FYVE (early endosomes) 458/485-510, mCitrine-Tub (tubulin) 514/525-545, mCherry (mitochondria)

specimen viability.

Image courtesy of Philipp Berger, Paul Scherrer Inst. Villingen, CH

vector. AOBS fast line sequential scan with five excitations and five emission bands. No unmixing.

543/585-620, mPlum-PLC-PH (plasma membrane) 633/640-800.

8

5
t=0 s

6

t=10 st=20 st=30 st=40 s9Live Cell Imaging with Spectrally Close
Fluorophores
With dichroic beam splitters, fluorophores with a large spectral separation between
excitation peaks must be selected for multiple staining of biological specimens. Otherwise,
there is insufficient space for emission collection. Additionally, multichroic mirrors do
not allow for closely adjacent excitation lines. This limitation is removed with the AOBS.
CHOOSE SIMULTANEOUS OR

FAST SWITCHING FOR LIVE CELLFAST SPECTRAL SEPARATION OF GFPSEQUENTIAL MODE

IMAGINGAND YFPThe AOBS offers excitation with laser

The AOBS offers sufficiently short

The efficiency of the AOBS plus the freelines that are spectrally close and inswitching times (< 10 μs) even for living

tunability of the detection bands in thevarious imaging modes.

specimens, whereas sequential scanning

SP detector open the door to imaging dye

only makes sense in line mode; otherwise combinations that were previously not› In simultaneous mode, with simul-

the spatial correlation is lost.

separable without post processing.

channels.

› Fast switching of the illumination

GFP and YFP, for example, require› In simultaneous mode, with mathe-routine for line sequential scanningsequential scanning with different

matical separation by unmixing.

in living specimens.

excitation lines that are comparably

taneous emission collection of all› In sequential mode, switching theclose together. No existing dichroic

excitation fast between lines or in a

mirror can accomplish this task, themore relaxed way between frames.

AOBS is the only solution.
With the AOBS one can separately
excite with 488 nm and 515 nm, and theillumination can be changed betweenlines.
› The AOBS allows spectral separationof two close excitation lines.

Fig. 5: Separation of GFP and YFP in living cells. HeLa cells expressing three different fluorescent proteins: GFP-tubulin(green) Ex 476 nm, Em 485-509 nm, YFP-GPI-filipodia (yellow) Ex 514 nm, Em 517-556 nm, mCherry-H2B-nucleus (red)Ex 561 nm, Em 571-671 nm. Three channels, simultaneously recorded.
Fig. 6: 4D live cell imaging with GFP, YFP, and mCherry. Per time point, a stack of images is recorded and visualized as a
maximum projection. Movement of the filipodia becomes visible in the YFP channel, here shown in yellow. GFP is simultaneously recorded and mCherry (nucleus) as reference. Sample courtesy of J. Bulkescher, EMBL Heidelberg, Germany.
Simultaneous recording. No unmixing.

10

Detect Fluorescence and Reflection
Simultaneously
The AOBS can easily convert to a 50/50 splitter for a desired wavelength for optimalreflected light contrast. Simultaneously, other laser lines can still be used for fluorescence
excitation and corresponding emission.
FREELY COMBINE FLUORESCENCEFAST SWITCHING BETWEEN REFLECTIONSTUDY OF CANCER CELL INVASION

WITH REFLECTION

AND FLUORESCENCE

BY REFLECTION AND FLUORESCENCE*

The AOBS is not just a programmable

Any excitation color can be employed for

An interesting application example forchromatic splitter. It can be used as areflected light. And you may even wish

combining reflection and fluorescence

50/50 beam splitter as well. You can

to collect different colors in reflection

imaging is the study of cell movement

choose which wavelength you want tomode, for example in a sequential scan,through tissue. It is accompanied by lysis

use for the reflected imaging mode.

where fluorescence and reflection are

and removal of collagen fibers – visual-collected from the very same illumination ized by reflection imaging – to make way
In reflection mode, the AOBS willcolor.

for the invading cells.

perform as a 50/50 splitting device(this ratio is the most efficient for* Multi-step pericellular proteolysis controls thereflected light imaging). Simultaneously,transition from individual to collective cancer cellup to seven additional lines can be

Overall C, Stack MS, Friedl P, Nature Cell Biology

used for fluorescence excitation.

2007.

Structures that give good contrast in reflected light without staining:• collagen ﬁbers and similar structures in connective tissue• lipid conglomerations• cytoskeleton components

invasion, Wolf K, Wu YI, Liu Y, Geiger J, Tam E,

11Fig. 7: NIH3T3 fibroblasts. Actin Alexa 488,
Ex 488 nm, Em 500-550 nm (green). Reflectionof focal contacts, Ill 476 nm Det 467-492 nm (gray).

7

Paxilin Cy3, Ex 561 nm, Em 570-627 nm (red).
Image courtesy of M. Bastmeyer, KIT Karlsruhe,
Germany.

Fig. 8: Arabidopsis thaliana, root tissue. PINI-GFP(membrane, green), REV-2xYFP (nucleus, red),reflection (gray).
Image courtesy of M. Heisler, EMBL Heidelberg,
Germany.

8

12

9

Background Information:
White Light
With supercontinuum fiber lasers,
a light source is available that covers
a wide spectral range and fulfills therequirements for confocal microscopy.
A truly white source emits photons ina spectral range, e.g. visible light,
without gaps.
Light composed of three laser lines,
e.g. red, yellow, and blue, isperceived by the human eye as“white” as well, but is far fromcovering a spectral range without
gaps. Such devices should not becalled “white sources”.

13

A Freely Tunable Beam Splitter for theWhite Light Laser
The White Light Laser (WLL) used in the TCS SP8 X combines a white light source thatcovers the spectral range from 470 nm to 670 nm and an acousto-optical tunable device
for the selection of single lines. Up to eight lines can be selected from the spectrum,
each line being freely tunable in both color and intensity.
TUNABLE EXCITATION REQUIRES FREELYFAST AND EASY AUTOMATIC SETUP OF

TRUE SPECTRAL DETECTION WITHTUNABLE BEAM SPLITTING

THE BEAM SPLITTERTHE TCS SP8The WLL provides any color combination

The above-mentioned acousto-optical

The continuously tunable illumination

within the visible range. Illumination

color selector (which in essence is anand splitting is efficiently complemented

routines are switched within a few

AOTF) is easily co-controlled with theby the continuously tunable detection of

microseconds.

AOBS, the sister component in such

Leica‘s SP detector.

a setup. All you need to do is select the
The WLL is tunable in 1 nm steps,

colors for excitation from a continuous

It can collect the transmission bands

resulting in 200 different colors, plus

spectrum.

between the excitation by the tunable

the permutations that you can create

spectral photometer bands – at utmost

with one to eight colors simultaneously.

The setup is automatically recognizedtransmission efficiency and in five true

The number of required mirror designs

by the AOBS control, setting thedye emission channels simultaneously.

would dramatically exceed all limits.

appropriate beam splitting characteristics. If any selected colors from the

To separate the channel information,

The AOBS, which is also freely tunablesource are changed, the AOBS instantly

mathematical cleaning is just an option

to up to eight illumination lines, perfectly

follows.

you can add on demand.

matches the beam splitter requirements
for the White Light Laser.

Fig. 9: Primary culture of rat cortical neurons imaged withWhite Light Laser. Excitation and detection settings were
adjusted to optimal image contrast.

14

Application Example:

White Light Laser combined with AOBS
The following example covers most of the applications and benefits achieved by combining
WLL and AOBS. Any of the steps might be applied individually; the sequence shown here
explains the multiple operation possibilities.
1. To identify the best excitation colors

2. The result is a sequence of images

3. These in situ excitation spectra show

for the four visible stains (DAPI isthat shows the maximum fluorescence

the actual excitation properties of theexcited with a separate 405 nm laser),detectable as a function of excitationdyes in the specimen. Most dyes

an excitation spectrum is recorded.

wavelength, i.e. each pixel contains

show shifts in their spectral behavior,

Here, the emission of the WLL isan excitation spectrum. From that

depending on the molecular micro-

automatically incremented, by steps

data, the excitation spectra of theenvironment. Thus, the spectra allow(adjustable) with 1 nm accuracy. The

regions of interest (ROI) are extracted,

the optimal excitation color to be

AOBS will automatically follow thiswhich are associated with the different

found for the fluorochromes. In

incrementation. The detector collects

fluorochromes (Fig. 10), either in

combination with the emission spectra,

all fluorescence emitted on the red

selected frames from the series,

the excitation is also optimized inside of the excitation by automaticallyor from a maximum projection. The

order to minimize crosstalk from theincrementing the blue edge with thespectra from these regions arevery beginning. The TCS SP8 X's

illumination color.

displayed graphically (Fig. 11).

software also offers an automatic
excitation-emission scan: “lambda
square imaging”.
1.0

0.8

Intensity

0.6

0.4

0.2

0
470

11

10

510

550

590
Excitation [nm]

630

670

15

4. When the system is set up to record

5. Then, the SP detector is set to select

6. Once the optimal imaging parameters

images with the optimal excitation

only the emission bands. Here again,

are worked out, they can be stored

colors, the AOBS is automatically

it is possible to control the red andand retrieved for later use on the

programmed to inject the desired

blue edge of each band online in ordersame or similar specimens. Personalcolors into the specimen and transmit

to optimize signal separation while

imaging parameters are collected in

the emission bands between them.

observing the resulting image on thea library, together with the factory-

The positions of excitation can still bescreen. All operations are instant, i.e.

supplied library.

manually fine-tuned, if desired, andwithout delays for mechanical or other

the AOBS will always automaticallysystem adjustments. The outcome isset the correct spectral bands.

immediately revealed on the display.

A practical implementation of a white light source, i.e. a freely tunable excitation color,
is only possible with a freely tunable beam splitting device, the AOBS.

Fig. 10: Definition of regions of interest for analysis of an excitation spectrum.
Fig. 11 (inset): Excitation spectra in the regions of interest, as indicated in the image above.
Fig. 12: Five-channel confocal imaging with insitu optimized excitation and emission. The five signals are shown
individually in the upper row. Overlay of all five channels below.
Quintuply stained CHO cells. Sample courtesy of J. Lippincott-Schwartz, NIH, Bethesda, MD (USA).

Hoechst (nucleus)

70

12

YFP (Golgi, cell

M-tracker 633

membranes)(mitochondria)

GFP (cytoplasmic)

RFP (ER)

www.leica-microsystems.com

What is the AOBS?
Leica's acousto-optical beam splitter (AOBS) replaces all collections of mirror-based
beam splitters (gray splitters, dichroic splitters, and multichroic splitters) for visible
light illumination in confocal microscopy.
• Highly efﬁcient for maximum sensitivity – get more light from the specimen
to the detector.
• Freely tunable for the most versatile multicolor imaging – no more hassle
with ﬁlters or combinations thereof.
• Quick reprogramming for efﬁcient multicolor live cell – adopt to your specimen’s
excitation without the limitations of ridgid reﬂection bands.

When to use the AOBS?
• The AOBS is the perfect choice for all imaging applications as it is the most ﬂexible, easy-to-use and efﬁcient beam splitter.
It is fully future-proof because it easily adapts to any new visible laser line.
• Sites that use different ﬂuorochromes, or will often use new ﬂuorochromes with classically stained samples. Imaging core facilities
and dye development laboratories must have the AOBS.
• Experimental situations that are not supported by mirror-type beam splitters; if the ﬂuorochromes’ emissions are very close,
or if, e.g. reﬂected light imaging is to be combined with ﬂuorescence imaging. In these cases, the AOBS is the solution of choice.
• Using tunable excitation sources (WLL) combined with an AOBS is the only viable solution. This combination, linked witha freely tunable multiband SP detector is ideal – you can have it now!
• Ultimately, if you want to research with the latest and most advanced technology, there is no other choice; the TCS SP8 X “white
confocal” with White Light Laser and AOBS leaves nothing to be desired.

AOBS is a registered trademark of Leica Microsystems CMS GmbH.
AOBS ist protected by the following patents in the US and elsewhere:
US6510001B1, US6654165B4, US6850358B2, US7016101B2, JP4435977B2,

CONNECT
WITH US

DE19906757B4, EP1281997B1
Additional patents may be pending in the US and elsewhere.

www.leica-microsystems.com/
beam-splitters.

Order no.: English 1593003010 ∙ Copyright © by Leica Microsystems CMS GmbH,
Mannheim, Germany, 2014. Subject to modifications. LEICA and the Leica Logo are
registered trademarks of Leica Microsystems IR GmbH.