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Leica TCS SP8Looking Forward to Your Discoveries

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Contents

Leica TCS SP8 – Looking Forward to Your Discoveries 4

From Whole Organisms to Highest Level of Detail 6

Innovations at a Glance 8

Sensitivity by Design 10

Faster for New Biology 12

Focus on Your Research – Automation & Reliability Redefined with Leica TCS SP8 14

Leica Microsystems Innovation Synergies 16

Ready to Grow – Future-proof with the Leica TCS SP8 18

Leica HyD™ – All-purpose Super-Sensitivity 20

Leica Confocal Objectives – The Best Objectives for Superior Images 22

LAS AF 3 – Intuitive Software that Makes Your Life Easier 24

Leica TCS SP8 X – Freely Tunable Excitation 26

Leica TCS SP8 STED – Opening the Gate to Super-Resolution 28

Leica TCS SP8 MP – Look Deeper into Tissues with Multiphoton Microscopy 30

Leica TCS SP8 CARS – Imaging Native Specimens without Dyes 32

Leica HCS A – Amplify the Power of Screening 34

Leica TCS SP8 SMD – Learn More from Single Molecules 36

Find the Right Leica TCS SP8 for Your Research 38

Captures and Acknowledgements 39

The Leica TCS SP8 Scan Head 40

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5LEICA TCS SP8 – LOOKING FORWARD TO YOUR DISCOVERIES

Looking Forward to Your DiscoveriesFrom routine imaging to live cell research, from super-sensitivity to super-resolution,

from multiphoton imaging to CARS – whatever your research, Leica Microsystems

has the confocal for your application.

SENSITIVITY BY DESIGN

The design of a confocal microscope

requires excellent separation of

excitation and emission light, high scan

speed, and flexibility. Each photon emitted

by the sample is precious and needs to

be preserved. Almost all components in

the optical path have an impact on the

preservation of photons emitted from

the sample. The best photon trans-

mission is achieved by matching each

optical component with the entire

system. This central concept is integral

to the design of confocal instruments by

Leica Microsystems. Read more on page 11.

FASTER FOR NEW BIOLOGY

Biological research is advancing at an

ever increasing rate as is the range of

questions to be answered. Fast scanning

provides a twofold advantage: it allows

new insights into biological phenomena,

and it helps meet tight deadlines. Leica

Micro systems now takes speed another

step further by introducing a new Tandem

Scanner, along with many other

innovations, to give you the results you

need – faster. Read more on page 13.

READY TO GROW

As new scientific approaches unfold, so

too do the instrumentation requirements

to support them. With the Leica TCS SP8

you will be prepared for the new

directions your research will take in the

future. Leica Microsystems always

ensures the path is open for the future,

to help your confocal grow with changes

in your experiments.

Backed by an all new user interface, for

intuitive and tight control of every

imaging parameter, the Leica TCS SP8 is

versatile and adaptable to suit both

small laboratories and large shared

facilities alike. Read more on page 18.

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Large field – whole organism. By using an X2Y scan mirror architecture, Leica confocal systems support the largest field of view in point scanning.

You can take images of whole animals or plants, often without stitching. See your specimens in widescreen.

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From Whole Organismsto Highest Level of Detail

7LEICA TCS SP8 – SUBCHAPTER

Mosaicking (stitching) is available for samples larger than the field of view. Save time on stitching by covering the same area with a smaller number of images.

Cover more ground, while preserving even the finest detail.

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Leica TCS SP8 – Innovations at a Glance

FOV (Field Of View) scanner

› See the full specimen in one shot (save time on stitching)

› Increased time resolution

› Higher throughput in screening experiments

White Light Laser

› LightGate technology for higher image contrast

› Freely tunable excitation from 470 – 670 nm (1 nm steps)

› Up to 8 fully tunable laser lines simultaneously from over

3 trillion unique combinations

› Comprehensive spectroscopy imaging with 2D

excitation-emission scans

› Pulsed excitation source for FLIM and gSTED

› Develop new imaging strategies with full spectral freedom

Tandem Scanner

(FOV and resonant scanner combined)

› High-speed FRAP experiments with soluble proteins

› Improved cell viability

› Rapid 3D particle tracking

Galvo flow (3D stacking without inertia)

› Vibration-free, continuous movement

› Real-time XZ-slices

› Faster 4D imaging

› Ideally complements rapid scanning with Tandem Scanner

Leica HyD™ (Hybrid photodetector)

› Reduced light dosage improves cell viability

› Ideal for high-speed imaging

› Descanned or non-descanned detection

› Quantitative through single photon counting (selectable)

Visible light lasers

Gas lasers

› Most flexible choice of laser lines

› Atomic energy transitions for monochromatic light

› Monochromatic light source for ideal spectral separation

› Ideal for multicolor imaging with > 4 colors

Solid state lasers

› Supply unit with small footprint

› Flexible combinations of excitation lasers

› Ideal for FRET with 448/514 nm and 488/552 nm pairs

Leica Confocal Scanner

9LEICA TCS SP8 – INNOVATIONS AT A GLANCE

Intermediate optics

IRAPO objectives (dedicated apochromats

for MP and OPO imaging)

› High transmission in visible and infrared for

brighter images from multiphoton and CARS

› Exceptional axial and lateral color correction in the infrared

for multiphoton excitation of multiple fluorophores

LIAchroics:

Low incident angle dichroic beam splitters

› High suppression of excitation light for high contrast

› Custom-designed low incident angle dichroics

› Cost-efficient beam splitting

AOBS: Acousto-optical beam splitter

› Programmable beam splitter provides the highest flexibility

› Support of White Light Laser (WLL)

› Completely transparent for unhindered spectroscopy

› Steep cut-off for maximum photon efficiency

› Longer sample viability with low power of excitation laser

› Switching in microseconds for fast multicolor kinetics

› Be quantitative with constant laser power using Setlight

SP Detector (Spectral detector)

› Gapless emission bands for faithful rendition

of fluorescence spectra

› Multi-band spectrophotometer

› Prism-based dispersion for highest efficiency

› Free from photon waste

Square pinhole

› Ideal pinhole geometry for high resolution

› Optimal separation of adjacent objects

Scan optics dedicated to specific application ranges

› Optimal transmission for each application

› Dedicated intermediate optics for maximum photon

efficiency

› HIVIS from 400 – 800 nm (reduced reflection by 60%)

› VISIR from 400 – 1300 nm

› UVIS from 350 – 800 nm

Objectives and scan optics

König-Rotator: Optical scan field rotation

› Perfect image geometry independent of rotation angle

› High transmission for minimal losses

› Interactivity due to direct user access by control panel

CS2 objectives

› Improved color correction throughout the whole field of view

› Perfect VIS – 405 overlay for best colocalization results

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Color-coded maximum intensity projection of Zebrafish muscle. Acquired using second harmonic generation (SHG) signal excited by IR laser.

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11LEICA TCS SP8 – SENSITIVITY BY DESIGN

A STRONG TEAM

Efficient routing of fluorescence light

between excitation and detection is

achieved by harmonizing each optical

component within the entire system.

Dedicated scan optics (such as HIVIS or

VISIR), optical scan field rotation, a

square pinhole, and the patented SP

Detector design using a Pellin-Broca

prism, provide recycling-loop free

dispersion. The SP Detector resembles a

powerful multiband spectrophotometer,

offering adaptive dynamic range because

of the individual gain on each detector.

The result is gapless emission bands for

faithful rendition of fluorescence spectra.

Consequently, the Leica TCS SP8 benefits

from an overall system design philosophy

that ensures all components team up to

give the best results.

COUNT ON IT

Leica‘s digitally sampled photomultiplier

tubes (PMT) pioneered the transition

from analog capacitor detection to digital

read-out, making pixel intensities

statistically robust and optimizing

detector duty cycles. In addition, the

hybrid photon detectors (HyD™) by Leica

Microsystems integrate next generation

detection – fully multispectral and with

individual gain per detector. Photon

counting avoids many inherent problems

of older detection principles, such as

pixel convolution, noise, and voltage-

related scaling issues. Backed by

lightning-fast counting electronics, even

very bright signals become accessible for

photon counting, making quantitative

measurements a routine part of your

daily work.

PRE-PROGRAMMED FLEXIBILITY

For coupling a wide range of excitation

lasers into the confocal scanner, Leica

Microsystems offers two options: the

acousto-optical beam splitter (AOBS) and

newly developed low incident angle

dichroics (LIAchroics). The AOBS is an

active optical crystal that is completely

transparent and offers the highest photon

efficiency of any beam splitting device.

The AOBS maximizes the advantages of

the WLL – fast tuning, using multiple

laserlines simultaneously or rapid kinetic

and spectroscopical analysis. Unlike filter

wheels, it switches within microseconds

by simply changing the radiofrequency of

the acoustic wave coupled into the

crystal. LIAchroics are Leica's high-effi-

ciency beam splitters for customized

performance to produce improved image

contrast when compared to standard

dichroics.

Sensitivity by DesignDesigning a high precision optical instrument such as a confocal microscope is the

ultimate balancing act of excellent beam splitting, high scan speed, and flexibility.

At the same time, every photon emitted by the sample needs to be preserved so it can

contribute to your brilliant imagery.

› Flexible and future-proof

› Publication ready images

› Photon efficient for live cell imaging

› Quantitative through photon counting4

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No Galvo Flow

Galvo Flow

time

GALVO FLOW – RAPID Z-STACKING

By continuously propelling the insert rather

than in discrete steps, the system minimizes

inertia. This avoids an otherwise rate limiting

repeated acceleration and deceleration and

improves the throughput and overall speed

with large 4D stacks.

TANDEM SCANNER – TIGHT SPEED CONTROL

Leica Tandem Scanners contain switchable

galvanometric mirrors. The high-speed mirror

resonates at either 8 or 12 kHz, which results in

frame rates of more than 420 fps (at 512 x 16

scan format).

This high speed is possible because of intricate

real-time control using an internal frequency

calibration system. In spite of the rapid scanning

the image geometry is faithfully recorded.

13LEICA TCS SP8 – FASTER FOR NEW BIOLOGY

Faster for New BiologyUnveiling the processes of life is a never-ending challenge. Leica Microsystems

combines innovative elements that amplify each others' performance to provide

accelerated confocal image recording. Arrive at new discoveries with greater speed.

A TANDEM FOR LIVE SPECIMENS

The observation of rapid biological

processes requires high-speed imaging

systems. Traditional confocal scanning

microscopy has an inherent limitation of

serial image collection. Camera-based

systems or other non-point scanning

approaches sacrifice image resolution

or the capability of simultaneous

multichannel acquisition.

Leica Microsystems overcomes this with

the Tandem Scanner, which combines the

FOV scanner with a switchable resonant

scanning system with either 8 or 12 kHz.

The combination of photon efficient scan

optics and HyD™, results in low-dosage

effects for better cell viability, high-speed

and perfect confocality in one.

BROADEN YOUR SCOPE

Some moments in life sciences are too

precious to be missed, which is why

sequential recording and stitching is not

always an option. Employing their

X2Y-scanner design, Leica confocal

scanners allow you to see more through

a wider field of view. With large

specimens, you will always see the

bigger picture.

SWEEP THROUGH LIFE

The unique z-stacking strategy of using a

galvanometric stage insert provides

benefits such as real-time xy-scans,

beam parking in xy, and both rapid and

precise 3D stacks. These are all standard

on Leica confocal instruments. This tried

and true approach is now extended

towards high-speed using galvo flow.

This results in lightning fast 4D time

series acquisition, with the tandem

scanner and Z Galvanometer acting in

perfect harmony to give maximum xyz

results.

› High cell viability

› New high-speed experiments

› Large throughput in 4D series

› Time-saving with large specimens

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Focus on Your ResearchBiological knowledge is expanding, but your time is not. Thankfully, technology can

take away some of the pressure by automating the tedious and repetitive parts

of experiments. Leica Microsystems can be your confocal partner, giving you the

confidence to push your experimental limits and trust in your data.

READY, SET, ... LIGHT!

For quantifiable results, you need both a

reliable detection system and a stable

light source. A much overlooked source

of imaging artifacts can be fluctuations

in laser intensity. With Setlight, you

maintain tight control of laser power

using a built-in feedback loop. It

represents an enhanced power

stabilization of either white light or

tunable lasers. In Leica confocal

scanners with AOBS, this real-time

power control system is available to

reduce backtracking by creating

reproducible results. Sit back and let

Setlight take care of your laser power

fluctuation worries.

PEACE OF MIND WITH

LEICA REMOTECARE

› Save time managing your lab equipment

› Safe and auditable

› Pro-active maintenance of your confocal

instrument

› Behaves like a virtual service engineer –

constantly monitors all subsystems

› Minimizes down-time in mission-critical

environments

› Faster diagnosis – faster fixing

› All you need is an internet connection

and Leica RemoteCare

› More output through automation

› Quantifiable results through power control

› Highest reliability through optional 24/7

remote service monitoring

15LEICA TCS SP8 – SUBCHAPTER

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Leica Microsystems Innovation Synergies

PHOTONIC EXPLORATION

The AOBS and second generation White Light Laser

(unique Lightgate, FLIM, pulse picker) complete the

most innovative light path concept in confocal

imaging. Combine this with trillions of excitation

combinations and the unique “lambda squared” scan,

and you are ready to explore completely new research

areas.

SPECTRAL FREEDOM IMAGING

For multi-channel imaging, the Leica TCS SP8

confocal prism-based spectral detection system

optimizes the detection of your valuable sample’s

emissions. Unlike diffraction grating and barrier

filter-based systems, it works at lower excitation

powers for prolonged fluorescence stability.

17LEICA TCS SP8 – LEICA MICROSYSTEMS INNOVATION SYNERGIES

FOV SCANNER

Leica Microsystems offers the largest field of view

(FOV) of any confocal point scanner. This saves you

time by increasing throughput and improving image

quality. This patented design is optimally backed up

by the objective lenses and high pixel scan formats,

as well as accelerated scan speed of a new Tandem

Scanner option. With Leica confocal scanners, you

get uncompromised imaging.

CHROMATIC PRECISION

Using newly designed CS2 optics and 405 nm laser

incoupling, Leica Microsystems pushes the edge of

chromatic correctness to a new level. This setup

enables you to make the most of multicolor imaging

and colocalization studies. After all, great imaging

requires great optics.

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Ready to Grow –Future-proof with the Leica TCS SP8Life sciences are continually advancing, and it may be difficult to know which direction

your research will take in the future. This is why the Leica TCS SP8 builds on a flexible

concept. No matter where you start, you can configure more functionality as your needs

evolve. Your investment in a Leica TCS SP8 will pay off – now and in the future.

COMPACT, WITHOUT COMPROMISE

What do you do if you are looking for a cost-efficient system

that does the job but does not limit you? With its new compact

supply unit, the Leica TCS SP8 allows you to buy into Leica

Microsystems’ high-end platform whilst starting small.

This supply unit, with its small-sized footprint, solid state lasers

and LIAchroic scan head, teams with the Leica DMI6000 CEL

microscope to give you everything you need for high-end

research. Your compact Leica TCS SP8 will grow with your

research to allow the addition of more imaging detectors, more

excitation lasers, and a range of multiphoton options.

MULTICOLOR FLEXIBILITY

There is always a reason to want more. More colors, more

flexibility, more light. This is why the Flexible Supply Unit is

equipped with a full complement of laser lines or the White

Light Laser. It enables the use of up to eight gas laser lines.

Atomic energy transitions deliver the most monochromatic light

available. For highest performance, especially when combined

with the AOBS, gas lasers still provide the best utilization of

the spectral detection window. Discover the benefit of

multicolor imaging.

COMPACT SUPPLY UNIT– ENTRY TO SP8 CLASS FLEXIBLE SUPPLY UNIT– MAXIMUM

SPECTRAL DETECTION WINDOW

19LEICA TCS SP8 – READY TO GROW

FULL SPECTRUM WITH THE WHITE LIGHT LASER CONFOCAL

Why limit yourself if you can have full spectral freedom?

The White Light Laser (WLL) provides custom selection of

wavelengths with multiple laser lines. In combination with the

AOBS and the SP Detector, the WLL leaves nothing to be

desired when it comes to visible light excitation. Multiple

optimizations in the scan head, for instance mirror coatings

and lens transmission, guarantee high image contrast. Now in

its second generation, the WLL can serve as a FLIM source,

optionally with pulse picker, and with the contrast enhanced

by Lightgate. Lightgate is a universal non-optical approach to

reflection suppression, which does away with any limitations

from filters.

Fill the white spots on the map with 200 colors – produced by

your White Light Laser.

› Grows as your research horizons expand

› Flexible when you need it

› Available with additional modules of your choice:

STED, MP, SMD, CARS, HCS A

WHITE LIGHT LASER – TRILLIONS OF LASER COMBINATIONS

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RAPID DETECTION FOR ALL PURPOSES

Unlike PMTs, which intrinsically have a

longer time-of-flight for photoelectrons,

HyDs™ generate ultra-short pulses.

In combination with rapid sampling

electronics at a 640 MHz rate, this allows

precise photon counting with everyday

samples. Quantitative imaging thus

becomes the standard for your research.

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21LEICA TCS SP8 – LEICA HyD™: ALL-PURPOSE SUPER-SENSITIVITY

Leica HyD™ – All-Purpose Super-SensitivityInnovation is a driving force for discovery. New areas can be uncovered by new

methodologies. Leica HyD™ sets a new standard in super-sensitive imaging.

It is no longer necessary to compromise. Photon counting or imaging? Low light or bright

fluorescence? High speed or crisp images? With Leica HyD™ you can do it all.

GO LIVE WITH HIGH DEFINITION

High speed and great image quality – in

the past these two conflicted with each

other. This is due to minimal light

collection at short pixel integration times

and PMTs producing a number of

artefacts leading to unclear images.

Introducing a highly sensitive detection

system (about 2x more quantum

efficiency at 500 nm compared to typical

photomultipliers) into confocal

instruments solves the problem. Being an

extremely responsive detection system,

the Leica HyD™ produces none of the

artifacts inherent to PMTs or GaAsP

photomulti pliers, such as after-pulsing or

pixel convolution. The result is sharp

images conveying every detail at high

fidelity.

A VIABLE SOLUTION

Live cell imaging tends to suffer from

inherent phototoxic effects. While many

of the underlying mechanisms have been

well understood, the effect of photo-

toxicity can be hard to pin down in the

biological system being studied. High

sensitivity directly translates into

reduction in light dosage delivered to the

sample. Live cell viability clearly benefits

from reduced free radical concentration

or photobleaching. Even delicate

systems, such as yeast or worms, are

accessible to Leica HyD™ Detection – at

full confocal resolution.

BROADEN YOUR SCOPE

Many other highly sensitive detectors,

such as traditional GaAsP photomultipli-

ers, can age quickly and lose sensitivity.

Due to its hybrid photo-detector design,

the Leica HyD’s photocathode and

downstream amplifying elements remain

sensitive. Techniques borrowed from

silicon chip manufacturing and a

simplified geometry, combine to produce

perfectly smooth internal surfaces that

are more robust. This long-term stability

ensures brilliant images without

compromise whenever you turn on your

confocal instrument.

› Multi-spectral detection for diverse applications

› Reduced light dosage improves cell viability

› Ideal for high-speed imaging

› Quantitative through single photon counting

› Descanned or non-descanned detector

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405 nm

488 nm

633 nm

Overlay

5 μm

Magnification / Numerical ApertureImmersionCorrection CollarConfocal Scanning

Correction CollarCorrects for variation in coverglass thickness and temperature

ImmersionWater ■■

Oil ■

Glycerin ■

Objective Class

Magnification

1x/1.25x ■

1.6x/2x ■

2.5x/3.2x ■

4x/5x ■

6.3x/8x ■

10x/12.5x ■

16x/20x ■

25x/32x ■

40x/50x ■

63x/80x ■

100x ■■

Leica objectives comply with ISO8038, ISO8039, ISO8578, ISO9345-2, ISO19012-1, ISO19012-2.

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23LEICA TCS SP8 – THE BEST OBJECTIVES FOR SUPERIOR IMAGES

The Best Objectives for Superior ImagesObjectives are the eyes of every microscope and are critical to determining the resolving

power of a confocal system. Transmission and color correction of an objective influence

excitation and detection efficiencies. Leica Microsystems offers a broad range of high-

end objectives specifically designed for the needs of different research applications.

All Leica objectives are produced on state-of-the-art high precision machinery, by

experienced optics engineers. The wealth of knowledge of objective assembly and

optics innovation come together to provide the finest optics for the Leica TCS SP8.

OBJECTIVES TAILORED TO THE NEEDS

OF YOUR RESEARCH

Only high-quality objectives allow the

image resolution to reach the diffraction

limit over a wide range of wavelengths.

For fluorescence imaging, all excitation

and detection wavelengths must be

included to ensure that image resolution

is as high as physically possible.

Perfect colocalization is mandatory in

many studies, e.g., interaction of

subcellular components. Therefore, the

best achievable color correction is desired.

Leica objectives cover all demands from

routine fluorescence imaging to

sophisticated confocal methods.

OUTSTANDING COLOR CORRECTION

FOR CONFOCAL SCANNING

Leica CS objectives match the highest

specifications for confocal microscopy.

The chromatic correction of the new

Leica CS2 objectives is perfect over the

whole field of view for precise colocaliza-

tion of different fluorophores. In addition,

numerical aperture and free working

distance are pushed to new limits.

The design of the Leica CS2 objectives

goes hand in hand with the innovative UV

optics of the Leica TCS SP8, to give the

most stable UV color correction.

SPECIAL APPLICATIONS REQUIRE

DEDICATED OBJECTIVES

Sophisticated methods such as super-

resolution or multiphoton microscopy

require specifically designed objectives

with excellent chromatic correction and

transmission in the specific wavelength

range. With the Leica HCX PL APO

100x/1.40 OIL STED orange objective and

the Leica IRAPO objectives, Leica

Microsystems offers the best solutions

for such applications.High-resolution

water immersion objectives with

correction collars are a prerequisite for

aberration-free imaging in aqueous

samples such as living cells. To provide

stable water immersion even at 37°C,

the Water Immersion Micro Dispenser

automatically adds water during an

experiment. With the motorized

correction collar, precise adjustment of

the optics is easy and reliable.

Leica (Semi-)Apochromatic Objectives (coming from Greek: without color)

HCX PL Fluotar affordable quality, suitable for fluorescence imaging

HC PL APO/HCX PL APO superior color correction and field flatness

HCX PL APO CS optimized for confocal scanning (CS)

HC PL APO CS2 new generation of CS objectives

HC PL IRAPO color correction and transmission optimal for MP and CARS

m

m

m

y

m

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25LEICA TCS SP8 – INTUITIVE SOFTWARE THAT MAKES YOUR LIFE EASIER

Intuitive Software that Makes Your Life EasierLAS AF 3 (Leica Application Suite Advanced Fluorescence) microscope software guides

the user step by step through data recording and evaluation. The workflow design helps

you to use the Leica TCS SP8 more efficiently. It offers full control over the microscope

hardware and provides all necessary information at a glance.

CLEAR STRUCTURE SAVES TIME AND

LEARNING EFFORT

Clearly structured panels for the

configuration of each acquisition step

can be permanently opened or hidden,

providing exactly the information you

need. The ergonomic design of LAS AF 3

reduces the learning effort and allows

you to focus on your research instead of

learning to use the software. LAS AF 3 is

fully synchronized with the programmable

control panel, which allows interactive

and fast setup.

The interface of LAS AF 3 is optimized

for operation in dark rooms, reducing

stray light from computer screens. For

better visibility, the complete user

interface can be magnified. Now,

imaging facilities can efficiently teach

larger groups of users, reducing the time

spent on training. LAS AF 3 has the same

architecture across all Leica microscope

platforms, giving you familiarity and

confidence for fast and accurate results.

CUSTOMIZED SOFTWARE TO MEET

YOUR NEEDS

In addition to basic image acquisition,

LAS AF 3 can be extended with a range

of additional software packages.

Software wizards reduce the time spent

on configuring tools, and even less

experienced users can perform complex

experiments within a short time.

The LAS AF Microlab package consists

of several wizards optimized for fast

photomanipulation experiments (FRAP,

FLIP, photoconversion) and FRET.

LAS AF Live Data Mode is a tool for the

easy setup of complex, interactive

time-lapse experiments. LAS AF

Electrophysiology combines Live Data

Mode with the recording of electrophysi-

ological data. The extremely flexible

Leica HCS A provides all the tools for

easy automation of high-content

screening (see page 35).

IMAGE ACQUISITION, PROCESSING, AND

QUANTIFICATION IN ONE PACKAGE

A variety of processing tools, including

deconvolution and advanced 3D

visualization bring out the most

important details of your data without

the need for external software. The new

3D visualization tool offers GPU-based

processing of 3D datasets with novel

manipulation options and a movie maker

to create astonishing animations.

Guided evaluation steps within the

wizards help with the quantification of

complex experiments for quick and

accurate analysis. All processing and

quantification packages are fully

integrated in LAS AF 3 for fast and

intuitive data handling. When used

offline, they free up time at the

microscope itself. For custom analysis

and documentation, data can be

exported in common image and movie

formats, including OME-TIFF.

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Excitation wavelength λ

Exci

tatio

n in

tens

ity

Continuously tunable excitation:

best image quality and sample protection.

Lambda Square Mapping: full spectral

information from your sample by

excitation-emission correlation.

Tunable wavelength and pulse

picking: true FLIM results.

Up to 8 laser lines simultaneously – freely

tunable in wavelength and intensity.

Tuning range of 470 to 670 nm in 1 nm

intervals.

Illumination regimes can be applied

within microseconds.

Non-S1 phase

S1 phase

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27LEICA TCS SP8 – FREELY TUNABLE CONFOCAL IMAGING

Freely Tunable Confocal ImagingIs there an optimal excitation source to image any kind of dye combination – suitable

even for FLIM measurements? Yes – the Leica White Light Laser. The Leica TCS SP8 X

based on the White Light Laser (WLL) offers this level of flexibility. The acousto-optical

beam splitter (AOBS) is the key to selecting any wavelength from the white spectrum.

Completed by the tunable spectral detector (SP), the Leica TCS SP8 X represents the only

filter-free versatile confocal microscope.

BACKGROUND QUENCHING WITH TIME

GATED DETECTION USING LIGHTGATE

LightGate imaging

Time gated imaging is an ingeniously

flexible approach that excludes

unwanted light from emission collection.

It switches off data collection during the

White Light Laser pulse; the desired

photons are harvested only during an

adjustable LightGate window. This way,

the highest image contrast is obtained.

› Improvement of image quality in

reflection-sensitive samples

› Maximizing fluorescence harvest when

working with short Stokes-shift dyes

› Dye discrimination by fluorescence

decay time using light gate adjustment

SEPARATION OF FLUOROPHORES IN

SPECTRALLY COMPLEX MIXTURES

Lambda Square Mapping

The lambda square map uncovers the

sample dependent relationship between

excitation and emission spectra. An auto-

mated acquisition process collects the

related spectral data, which is summa-

rized in a two-dimensional intensity map.

› Best image quality by optimization of

spectral instrument parameters

› Characterization of new fluorescent

markers

› Selection of optimal specific stains that

sidestep auto-fluorescence

OPTIMIZED EXCITATION WAVELENGTH

AND REPETITION RATE

White Light Laser FLIM

The White Light Laser allows you to set

the FLIM excitation wavelength as

desired. The repetition rate is variable

due to a built-in pulse picker from 10 to

80 MHz. Pulse picking and wavelength

selection are fully integrated into the

LAS AF 3 software.

› One laser for all: versatile and

convenient illumination selection for

imaging and FLIM

› Filter-free FLIM: fluorescence lifetime

measurements at tunable excitation and

emission wavelengths

› True FLIM data by adapting the

repetition rate to the fluorescence

lifetimeLightGate window

Background

Pho

tons

Time/ns

Exci

tatio

n

Fluorescence

28

Dual Color STED

Dual Color Confocal

Excitation and fluorescence emission

detected

S1

S0

hνexc. hνem.

filtered out

Excitation and stimulated emission

hνexc. hνSTED

S1

S0

The focal spot in

STED microscopy

9

29LEICA TCS SP8 – OPENING THE GATE TO SUPER-RESOLUTION

Opening the Gate to Super-ResolutionStudying subcellular architecture and dynamics can be challenging, as structures smaller

than 200 nm are lost in the blur. The Leica TCS SP8 STED allows you to discover more

and provides fast, intuitive, purely optical access to structural details smaller than

50 nm – even inside living specimens.

PERFECT MATCH: CONFOCAL AND STED

Stimulated emission depletion (STED)

microscopy is a stunningly simple way to

overcome the diffraction limit. The spot

where fluorescence is generated is

scaled down to sub-diffraction size.

Stimulated emission switches off the

ability of the dye to fluoresce in the

periphery of the excitation spot. This

yields confocal super-resolution, pixel by

pixel. Even details smaller than 50 nm

are resolved, unravelling details never

before elucidated, for example in

membrane patterning.

The Leica TCS SP8 is the ideal platform

for super-resolution. Superior optics

combined with multispectral Leica HyD™

detectors, maximize the number of

collected photons. This gives super-

resolved images of optical sections, even

from weakly stained samples. The

modular design of the Leica TCS SP8

means that Leica STED is an affordable

upgrade for almost all configurations.

SUPER-RESOLUTION – FAST AND DIRECT

Just one mouse click instantly switches

from confocal to super-resolution, even

during a live scan. Within seconds,

imaging parameters can be optimized

and super-resolved images obtained.

The 12 kHz resonant scanner, able to

record up to 420 fps at 512 x 16 image

format, yields super-resolution at

maximum speed. STED microscopy does

not rely on mathematics. What you see is

what you get. Of course, results can be

further improved by deconvolution.

Gated STED – the latest milestone in

confocal super-resolution – substantially

extends the functionality of the proven

STED CW.

Combining the superior Leica HyD™

detectors and White Light Laser, it

improves resolution and contrast while

distinctly reducing laser intensity. This

enhances photo-stability, meaning that

gated STED provides considerable

benefits to super-resolution for live cell

imaging.

APPLICATIONS BEYOND THE LIMITS

STED microscopy works with standard

fluorophores such as Alexa 488, Oregon

Green 488 and FITC and does not rely on

oxygen depletion or reducing agents in

the embedding medium. Also eYFP, Citrin

and eGFP yield convincing results. This

allows you to stay as close as possible to

routine protocols, saving time and money.

Multi-color STED imaging empowers

colocalization studies at the nanoscale

without the need to compensate for

chromatic aberrations, as the same STED

doughnut is used for all colors.

STED microscopy combines intrinsic

optical sectioning capability, fast image

acquisition and robustness against

sample drift. The suitability of fluores-

cent proteins further qualifies STED

imaging to reveal fast dynamic processes

inside living cells, or even organisms,

with unmatched precision.

D

l

9

300

100

200

300

400

500

600

z [μm]

10

31LEICA TCS SP8 – LOOK DEEPER INTO TISSUES WITH MULTIPHOTON MICROSCOPY

Look Deeper into Tissues with Multiphoton MicroscopyImaging deep within biological specimens requires efficient multiphoton excitation.

Leica TCS SP8 MP makes full use of the most advanced infrared lasers up to 1300 nm,

including prechirped femtosecond lasers and Optical Parametric Oscillator (OPO).

Super-sensitive Leica HyD™ non-descanned detectors record even the faintest structures

from deep tissue sections. Combined with the high speed 12 kHz scanner and the largest

field of view, the Leica TCS SP8 MP is the perfect tool for multiphoton microscopy.

SUPERIOR OPTICS AND DETECTION FOR

BRIGHTER, HIGH-CONTRAST IMAGES

Leica’s broadband anti-reflective

coatings provide highest transmission in

the visible and infrared for scan optics

and objectives resulting in optimal

excitation. Super-sensitive HyD™

detectors, combined with Leica IRAPO

objectives featuring transmission greater

than 85% from 470–1200 nm, collect the

maximum number of emitted photons.

Efficient excitation and detection results

in brighter images from deeper tissue

sections. There is less need for high laser

powers, thus protecting the specimen

from photodamage. Leica IRAPO high NA

objectives are color corrected in the

infrared up to 1300 nm to minimize the

axial shift in colocalization experiments

and localized photomanipulations.

MORE DATA IN LESS TIME – LARGEST

FIELD OF VIEW AT HIGHEST SPEED

At 22 mm, the exceptionally large field of

view of the Leica TCS SP8 allows large

areas of tissue sections to be scanned in

one go. The new 12 kHz scanner in

combination with the SuperZ Galvo stage

acquires 3D stacks up to 50% faster than

comparable systems. Even at those high

speeds, no compromise in resolution,

sensitivity or contrast is experienced,

thanks to high-numerical aperture

objectives with long working distance

and the super-sensitive Leica HyD™

non-descanned detectors. For fast

set-up, all multiphoton lasers, including

the precompensation, are fully controlled

via LAS AF microscope software.

INTRAVITAL IMAGING AND ELECTRO-

PHYSIOLOGY WITH LEICA DM6000 CFS

The Leica DM6000 CFS fixed stage

microscope provides the best mechanical

stability and lowest electronic noise for

your sensitive experiments. The

motorized, remote controlled 2-fold

objective changer allows smooth,

vibration-free switching and dipping of

objectives to avoid disturbing complex

experiment set-ups. Dedicated objectives

with inert ceramic fronts and color

correction in the infrared are ideal for

simultaneous multiphoton imaging and

recording of electrophysiological data.

The LAS AF Electrophysiology software

package saves time during data

evaluation by directly correlating voltage

recordings with fluorescence intensity

data.

0

32

Video rate and high resolution in one system.

Easy experiment setup with a fully

integrated system.

Excellent imaging with VIS, UV, IR, SHG

and CARS.

Leica TCS SP8 upgrade to CARS anytime.

11

12

13

33LEICA TCS SP8 – IMAGING NATIVE SPECIMENS WITHOUT DYES

Imaging Native Specimens without DyesCARS (Coherent Anti-Stokes Raman Scattering) microscopy is an analysis method that

does not require staining. An infrared laser source generates the CARS signal, which is

non-toxic, non-destructive, and almost non-invasive to living samples. The integrated

Leica TCS SP8 CARS extends fluorescence microscopy to new horizons.

WHY CARS?

Conventional microscopy techniques

depend on structures providing

auto-fluorescence or structures that can

be labeled by fluorophores. In most cases

staining processes imply modification of

cells or tissues and destruction of the

natural environment. With the

integration of CARS technology into the

confocal system Leica TCS SP8 CARS,

Leica Microsystems offers an efficient

way to overcome these drawbacks.

CARS IN A NUTSHELL

CARS microscopy utilizes molecule-

specific vibrational energies to generate

contrast. To obtain a signal, two pulsed

laser beams simultaneously arrive at the

specimen. If the difference between the

photon energies of those wavelengths

matches the vibrational energy of the

molecule, a strong CARS signal is

obtained. This signal caused by the

molecule itself is used for imaging.

REAL LIVE CELL IMAGING

The CARS signal is generated by infrared

excitation. This 3rd order non-linear

process can only occur in the focal plane,

providing excellent optical sectioning.

The result is highly sensitive imaging at

sub-cellular resolution – the sample

remains almost unaffected. CARS

technology perfectly matches the

requirements for analyzing living cells or

small animals without modifications

under almost natural conditions.

CARS AT THE TURN OF A KEY

With Leica TCS SP8 CARS, scientists can

focus on their experiments rather than on

technical setup. Two infrared sources in

a single box are controlled by LAS AF

system software. Turning on or off and

tuning to the desired wavelength or

wave number, is done by a single click.

Once set up, Leica TCS SP8 CARS needs

no further calibration of laser or beam

path.

FOCUS ON APPLICATIONS

Metabolic disorders and obesity studies

involving lipids and their metabolism are

two lifestyle diseases currently in the

focus of life science research. For

analysis of the distribution and content

of lipids in cells and tissues, fast lipid

tracking in metabolic pathways,

membranes, and interactions between

different compounds, CARS is the

method of choice.

FULLY INTEGRATED SYSTEM

Leica TCS SP8 CARS is a true confocal

system that can be combined with up to

eight VIS laser lines and a 405 nm laser.

It provides scanning speeds from 1 line/

sec to more than 420 images/sec with

512 x 16 pixels. The image sizes from 256

x 256 pixels up to 8000 x 8000 pixels

allow a brief overview and deeper

insights of small structures. This

flexibility makes the Leica TCS SP8 CARS

an ideal device for multi-user facilities.

1

34

Zoom = 0.5 x

T = 0 h T = 7 h T = 10 h T = 15 h

Zoom = 2.3 x Zoom = 5.0 x 15

16

14

35LEICA TCS SP8 – AMPLIFY THE POWER OF SCREENING

OVERVIEW AND DETAIL IN ONE

For fast, efficient generation of highly

qualitative and quantitative data, Leica

HCS A offers high resolution and perfectly

stitched images in one and the same

experiment. Dedicated wizards guide you

through the experiment and offer various

scanning templates.

KEEPING THE FOCUS FOR SHARP IMAGES

Five autofocus modules identify the focal

plane, create a topological focus map,

adjust the z-position of interest, and keep

it over the required time period. Freely

configurable parameters such as scan

patterns or individual z-positions of

selected scan fields, comfortably adapt

the instrument to the requirements of

high content screening. Predefined or

individually configured setups can be

freely chosen.

SCREENING CONTENT

Scan settings from frequently used slides

can be recalled and adjusted if

necessary. This saves valuable time in

experimental setup. Applying drift

compensation and single object tracking

keep living cells in the center of focus in

lateral direction even during a long-term

experiment, and so provides images of

valuable content.

FROM CELL TO CONCLUSION

Recorded metadata are of great

importance for drawing the correct

conclusions from an experiment. Leica

HCS A links metadata to the image and

provides individual results of each well

at any time. With a connection to

LAN-attached network devices (NAS),

the data is easily shared with colleagues

or stored on servers. Leica HCS A offers

fast and secure data exchange within a

facility, between labs or with colleagues

all over the world.

OPEN EXPORT

A high-performance storage and analysis

tool provides full flexibility for data

analysis of huge numbers of images.

Unlimited data streams can be routed

into the target server. New data export

formats are compatible with the Open

Microscopy Environment by OME.TIFF.

Standard and TIFF-files can be read by

any modern image analysis software.

FLEXIBLE CUSTOMIZATION

Computer Aided Microscopy (CAM)

provides free adaptation of the software

to the experiment. Detection of rare

events such as mitosis phases are

possible. On-the-fly data analysis and

remote control of the system during the

ongoing scanning process ensure highest

efficiency of experiment performance.

Fast switching from pre-scan to

high-resolution zoom-in mode for 3D

image recording offers excellent

perspectives for new experiments.

Amplify the Power of ScreeningHigh content screening is a growing discipline in life science research, as the number of

complex experiments is increasing, and statistically relevant data becomes a must for

scientific publications. Combining the strength of the high-resolution point scanner with

the power of the Leica HCS A software platform, Leica TCS SP8 delivers a highly flexible

tool for automated multi-dimensional imaging.

5

6

4

36

Binding studies using FLIM FRET. Shorter donor fluorescence lifetime indicates binding.

Multiparametric data analysis of dynamic

processes using FCS:

The autocorrelation curve reveals information on the

molecular scale such as diffusion coefficient, particle mass,

and concentration. An increase of the cross correlation

amplitude (FCCS) signifies binding processes.

Specimen: double stranded DNA labeled with Alexa 488 and Cy5.

0,8

0,7

0,6

0,5

0,4

0,3

0,2

0,1

00,0001 0,001 0,01 0,1 1 10

lag time [ms]

G(t

)

GreenRedCrosscorr.

Leica TCS SP8 SMD:

An integrated platform for FCS, FLIM,

FLCS, and imaging methods.

User-friendly implementation of all

Leica and PicoQuant components.

Dedicated application wizards for fast,

reliable experimental setup.

17

37LEICA TCS SP8 – LEARN MORE FROM SINGLE MOLECULES

Learn More from Single MoleculesQuantitative characterization of biological phenomena is gaining increasing importance

in modern life science. The Leica TCS SP8 SMD offers all you need for quantification –

at the push of a button. Established confocal image acquisition procedures combined

with sophisticated single molecule tools are opening up entirely new areas of research.

The Leica TCS SP8 SMD provides global control of an experiment through the full integration of SMD-specific hardware and

software from PicoQuant GmbH. A wide variety of lasers – from UV to IR and the unique pulsed White Light Laser – are

available. Specific single photon counting detectors for external or internal spectral data acquisition fit all applications.

ANALYSIS OF MOLECULAR

NANO- ENVIRONMENT AND

FLUOROPHORE PROPERTIES

FLIM/Spectral FLIM

Fluorescence Lifetime Imaging (FLIM)

measures the average lifetime that

molecules reside in the excited state.

The resulting images show the

fluorescence life span in each pixel.

Modern biosensors use that property to

very sensitively probe molecular

environmental parameters and binding by

FRET-FLIM. New dyes are precisely

characterized with FLIM methods.

› Deep tissue MP-FLIM with non-

descanned Leica HyD™ for large

z-stacks with high signal-to-noise ratio

› PIE (Pulsed Interleaved Excitation) at

the push of a button

QUANTIFICATION OF MOLECULAR

MOVEMENT AND INTERACTION

FCS/FCCS

Fluorescence Correlation Spectroscopy

(FCS) analyzes the intensity fluctuations

caused by the entry and exit of

fluorescent molecules in a fixed

diffraction limited laser focus. An

autocorrelation of the signal provides

quantitative insight into transport

properties and dye distribution.

Molecular binding becomes evident in

Fluorescence Cross-Correlation

Spectroscopy (FCCS).

› Profile-scan positioning for precise

aiming at membrane-associated targets

› Automated control image acquisition

during FCS sequences

EVALUATION OF MOLECULAR DYNAMICS

IN DIFFICULT SAMPLES

FLCS/FLCCS

Fluorescence Lifetime Correlation

Spectroscopy (FLCS) combines FCS and

lifetime measurement, where lifetime-

filtered data are correlated. This

approach extracts significant fluctuation

data from unwanted noise signals.

Concentration measurements at low

signal levels or high background reveal

true data.

› Inherent cross-validation of FCS and

FLIM data

› Clearly enhanced cross-correlation by

seamless integration of PIE illumination

0

rr.

38

Applications Methods Solutions

Colocalization studies STED super-resolution Leica TCS SP8 STED

Confocal imaging Leica TCS SP8

FRET Leica TCS SP8

FLIM-FRET Leica TCS SP8 SMD FLIM

Multiphoton Leica TCS SP8 MP

Live cell imaging CARS Leica TCS SP8 CARS

Multiphoton Leica TCS SP8 MP

FLIM Leica TCS SP8 SMD FLIM

FCS Leica TCS SP8 SMD FCS

FLCS Leica TCS SP8 SMD FLCS

High content screening Leica HCS A

STED super-resolution Leica TCS SP8 STED

FRAP/FLIP/photoconversion Leica TCS SP8

Super-continuum imaging Leica TCS SP8 X

Low light imaging Leica TCS SP8 with HyD™

Time-lapse recording Leica TCS SP8

Fast tracking Leica HCS A

Quantitative imaging Photon counting Leica TCS SP8 with HyD™

FLIM Leica TCS SP8 SMD FLIM

FCS Leica TCS SP8 SMD FCS

FLCS Leica TCS SP8 SMD FLCS

High content screening Leica HCS A

Super-resolution STED super-resolution Leica TCS SP8 STED

Deep tissue imaging CARS Leica TCS SP8 CARS

Multiphoton Leica TCS SP8 MP

Electrophysiology Leica TCS SP8 MP + CFS

3D imaging STED super-resolution Leica TCS SP8 STED

Multiphoton Leica TCS SP8 MP

Confocal imaging Leica TCS SP8

Confocal imaging Leica TCS SP8 with HyD™

Super-continuum imaging Leica TCS SP8 X

High content screening Leica HCS A

Label-free imaging CARS Leica TCS SP8 CARS

FLIM Leica TCS SP8 SMD FLIM

Spectral characterization Leica TCS SP8 X

39LEICA TCS SP8 – CAPTURES AND ACKNOWLEDGEMENTS

Image Captions and Acknowledgements:

Title page:

First: Mouse retina. Sample: courtesy of Dr. Frank Müller, Institute of Complex Systems 4, Cellular Biophysics, Research Center Jülich GMBH,

Jülich, Germany

Second: Mouse diaphragm. Green: nerve fiber, Alexa 488. Red: synapses, Rhodamin. Blue: muscle fiber, myosin. DODT contrast.

Sample: courtesy of Ulrike Mersdorf, Max Planck Institute for Medical Research, Heidelberg, Germany

Third: Food moth (Plodia interpunctella), larval silk gland. 10x magnification, overlay image. Red: fat cells under the cuticula. Green: cuticula.

Leica Microsystems

Fourth: Dual color STED image. Green: histone 3. Red: microtubules. Both visualized in HeLa cells with Chromeo 505 and BD HorizonV500,

respectively. Leica Microsystems

[1] Larvae of Terebratalia transversa. Sample: courtesy of Prof. Andreas Wanninger, Vienna, Austria

[2] Platynereis dumerillii, (2 month). Blue: nuclei, DAPI. Green: tubulin, FITC. Grey: phalloidin, Rhodamin. Red: serotonin, Cy5.

3D maximum projection of image taken with a 10x objective fully zoomed out at 5000 x 5000 scan format.

Sample: courtesy of Dr. Antje Fischer and Dr. Detlef Arendt, Heidelberg, Germany

[3] Coronar section of mouse nasal cavity. Mosaic compiled from 48 single images acquired using a high NA objective lens.

Leica Microsystems

[4] Zebrafish muscle – SHG. Sample: courtesy of Dr. Patrick Kölsch, Karlsruhe Institute of Technology, Karlsruhe, Germany

[5] Rat primary culture. Leica Microsystems

[6] HeLa cells. Blue: Nucleus, DAPI. Green: Tubulin, Alexa 488. Red: F-actin, TRITC-phalloidin, Excitation wavelengths are indicated

for each channel. Leica Microsystems

[7] Detail of mouse embryo heart. Blue: nuclei stained with DAPI. Green: Cy2 labelled actin. Red: uncharacterized protein labeled with Cy5.

Courtesy of Dr. Elisabeth Ehler, King‘s College, London, UK

[8] Cells expressing a cell cycle stage marker. Green: non-S1 phase. Red: S1 phase. Yellow: intermediate state.

Courtesy of Dr. Malte Wachsmuth and Dr. Lars Hufnagel, EMBL, Heidelberg, Germany

[9] Dual color confocal and STED image. Green: histone 3. Red: microtubules. Both visualized in HeLa cells with Chromeo 505 and BD

Horizon V500, respectively. Leica Microsystems

[10] Adult Thy1-EYFP H line mouse, in vivo (cranial window). Excitatory pyramidal neurons in Layer 5 partly express EYFP.

Courtesy of Dr. Masahiro Fukuda and Prof. Haruo Kasai, Center for Disease Biology and Integrative Medicine, Faculty of Medicine,

The University of Tokyo, Tokyo, Japan

[11] Drosophila melanogaster, living animal. Red: fat cells imaged at a wave number of 2850 cm-1 (at 816 nm). Green: two different structures

displayed in green autofluorescence (at 1064 nm): Long tubes are parts of the tracheal system, striped matter in the background are

muscles. Objective: Leica HCX IR APO L25x/0.95 W 0.17. Leica Microsystems

[12] Structural information of a food moth larvae Plodia interpunctella. Red: CH2 stretches imaged at a wave number of 2850 cm-1

(at 816 nm). Green: parts of cuticula displayed in green autofluorescence, green filaments are the bristles of the larvae (at 1064 nm).

Leica Microsystems

[13] Distribution of fatty components in potato crisps. Maximum projection. Red: lipid components in all regions of a potato chip.

Green: structural information provided by autofluorescence. Leica Microsystems

[14] Mouse diaphragm muscle stained against neurofilament 150. Mosaic: xyz: 5 x 5 x 101 images. Green: secondary antibody coupled

to Alexa Fluor 488 and acetylcholine receptors. Red: alpha-bungarotoxin coupled to Alexa Fluor 647.

Courtesy of Dr. Rüdiger Rudolf, Cellular Signaling in Skeletal Muscle, Karlsruhe Institute of Technology, Karlsruhe, Germany

[15] Zebrafish brain, in vivo imaging. Transgenic embryo, Danio rerio. GFP, epithalamus, optic tectum. Courtesy of K. Palma, N. Guerrero,

L. Armijo, ML. Concha, Laboratory of Experimental Ontogeny (LEO), S. Härtel, Laboratory of Scientific Image Analysis (SCIANLAB).

Anatomy and Developmental Biology Program, ICBM, Faculty of Medicine, University of Chile, Santiago, Chile

[16] Zebrafish, novocord development, Danio rerio. Tracking the development of life over time.

Red: Rhodamine-dextran. Green: GFP, labeling of the novocord.

Courtesy of Sophie Dal-Pra, Team B&C Thisse, Imaging Centre of IGBMC, IGBMC, Illkirch, France

[17] Nuclei of HeLa cells. Polymerase B labeled with Alexa 488 and Alexa 555 as a positive control for FRET, and donor bleached cells as

negative control. Courtesy of Pascal Kessler and Yves Lutz, IGBMC, Strasbourg, France

www.leica-microsystems.com

Order no.: English 1593003000 ∙ VIII/12/BX/Br.H. ∙ Copyright © by Leica Microsystems CMS GmbH, Mannheim, Germany, 2012.

Subject to modifications. LEICA and the Leica Logo are registered trademarks of Leica Microsystems IR GmbH.

1 Visible line lasers or White Light Laser

2 Acousto-optical tunable filter (AOTF)

3 Infrared (IR) lasers *

4 Electro-optical modulation (EOM)

5 Ultraviolet (UV) lasers *

6 AOTF or direct modulation (DMOD)

7 STED laser *

8 Monitoring diode for Setlight

9 Acousto-optical beam splitter (AOBS), other options available

10 FRAP Booster *

11 IR laser incoupling

12 UV laser incoupling with CS2 UV optics

13 STED laser incoupling

14 FOV scanner with tandem scanner option

15 Scan optics with alternative UVIS, HIVIS or VISIR coating

16 Scan field rotation (Abbe-König rotator)*

17 Reflected light detection (RLD) in non-descanned position *

18 Objective lens (different options available)

19 Transmitted light detection (TLD) in non-descanned position *

20 Square confocal pinhole

21 Fluorifier disc *

22 Outcoupling with X1 port *

23 External detection *

24 Prism-based dispersion

25 SP detection with spectrophotometer arrangement

26 Up to five photomultipliers (PMT) or up to four hybrid

photodetectors (HyD™)

*optional

Leica TCS SP8 Scan Head

visible and ultraviolet radiation:

infrared radiation: