Leica TCS SMD User Manual

Leica TCS SMD Series

Single Molecule Detection Platform

Technical Documentation

General Specifications of SMD Prepared Leica TCS SP5

Microscopes

Microscope anti-vibration table

Z-drive

Continuous wave lasers

Upright

Inverted

Specifi cation For imaging

Vibration insulation Passive

SuperZ galvanometer stage 1500 m travel range/3 nm stepsize

Motorfocus (stand)

Laser type For imaging

VIS

UV Diode, 50 mW: 405 nm

Laser type For imaging

IR

Leica DM6000 CS
Leica DM6000 CFS
Leica DMI6000 CS

Leica DMI6000 CS bottom port

Travel range depending on mechanics
of microscope/15 nm step size

Diode, 40 mW
Ar, 65 mW: 458, 476, 488, 496, 514 nm
HeNe, 1 mW: 543 nm
HeNe, 2 mW: 594 nm
HeNe, 10 mW: 633 nm
DPSS, 20 mW: 561 nm

iSa 1.2 ps 1 W 690...1040 nm

T
(various ranges)

: 442 nm

Pulsed lasers

Excitation modulation

VIS

UV –

Modulation type For imaging

AOTF VIS Up to 8 channels
AOTF UV
EOM IR Yes
Pulsed laser driver Optional

Up to 3 channels

–

–

2

Additional SMD Specific Specifications

TCS SMD FCS TCS SMD FLIM TCS SMD FLCS

Yes Yes Yes
Yes Yes Yes
Yes, recommended Yes Yes, recommended

Yes Yes Yes

For FCS For FLIM For FCS and FLIM

Passive Passive Passive

1500 m travel range/3 nm stepsize
Travel range depending on mechanics

of microscope/15 nm step size

For FCS No cw laser excitation for FLIM For FCS

Diode, 40 mW: 442 nm – Diode, 40 mW: 442 nm
Ar

, 65 mW: 458, 476, 488, 496, 514 nm – Ar, 65 mW: 458, 476, 488, 496, 514 nm
HeNe, 1 mW: 543 nm – HeNe, 1 mW: 543 nm
HeNe, 2 mW: 594 nm – HeNe, 2 mW: 594 nm
HeNe, 10 mW: 633 nm – HeNe, 10 mW: 633 nm
DPSS, 20 mW: 561 nm – DPSS, 20 mW: 561 nm

–––

Pulsed excitation for FCS not required For FLIM For FLIM and FLCS

–

–

–

–

1500 m travel range/3 nm stepsize 1500 m travel range/3 nm stepsize
Travel range depending on mechanics

of microscope/15 nm step size

iSa 1.2 ps 1 W 680 ... 1040 nm

T
(various ranges)

Diode, up to 3 mW average @ 40 MHz,
470 nm (software controlled selection of pulse
frequency: 5, 10, 20, 40 MHz), < 90 ps ... < 500 ps
(depending on selected power level)

Diode, up to 4.5 mW average @ 40 MHz,
640 nm (software controlled selection of pulse
frequency: 5, 10, 20, 40 MHz), < 90 ps ... < 400 ps
(depending on selected power level)

Diode, up to 3 mW average @ 40 MHz,
405 nm (software controlled selection of pulse
frequency: 5, 10, 20, 40 MHz), < 70 ps ... < 300 ps
(depending on selected power level)

Travel range depending on mechanics
of microscope/15 nm step size

TiSa 1.2ps 1 W 680 ... 1040 nm
(various ranges)

Diode, up to 3 mW average @ 40 MHz,
470 nm (software controlled selection of pulse
frequency: 5, 10, 20, 40 MHz), < 90 ps ... < 500 ps
(depending on selected power level)

Diode, up to 4.5 mW average @ 40 MHz,
640 nm (software controlled selection of pulse
frequency: 5, 10, 20, 40 MHz), < 90 ps ... < 400 ps
(depending on selected power level)

Diode, up to 3 mW average @ 40 MHz,
405 nm (software controlled selection of pulse
frequency: 5, 10, 20, 40 MHz), < 70 ps ... < 300 ps
(depending on selected power level)

For FCS For FLIM For FCS and FLIM

Up to 8 channels – For FCS: 8 channels

–––
–
– For VIS and UV FLIM: up to 3 channels For VIS and UV FLIM: up to 3 channels

Optional for MP FLIM Optional for MP FLIM

3

General Specifications of SMD Prepared Leica TCS SP5

Features For imaging

Number of laser ports Up to 3 (UV - VIS - IR)

Optics

Scanner

Number of lasers

Excitation – emission splitting

Detection range 400...800 nm

UV and IR imaging Sequential or simultaneous

Field upgradable To IR: yes

UV correction

Pinhole Alignment stable single pinhole

Pinhole diameter control

Scanner design For imaging

Scanning concept Optically correct scanning at low inertia

Switch conventional – resonant scanner

Conventional scanner For imaging (PMT and APD)

Maximal line frequency 2800 Hz
Minimal line frequency
Scan speed granulation 1400
Maximal frame rate 512 x 512 5 Hz
Maximal frame rate 512 x 16 50 Hz
Beam park Yes
Maximal frame resolution 8192 x 8192 pixel
Scan zoom 1.0 ... 64 x
Panning Yes
Field rotation 200° optical
Field diameter 22 mm

Up to 8

Acousto Optical Beam Splitter (AOBS
or dichroic beam splitters

Individual precise correction optics
(up to 6 positions)

Motorized by software,
automatic mode available

Conventional and resonant scanner
in one system (optional)

1 Hz

®

)

4

Additional SMD Specific Specifications

TCS SMD FCS TCS SMD FLIM TCS SMD FLCS

For FCS For FLIM For FCS and FLIM

1 (VIS) 2 (UV & IR, or UV & VIS)

Up to 8

AOBS For pulsed lasers: dichroic mirrors

Depending on fi lter cube used

––

To FCS: yes To FLIM: yes –

UV not required for FCS Required for UV FLIM To FLCS: yes

Alignment stable single pinhole Alignment stable single pinhole Required for UV FLIM
Motorized by software,

automatic mode available

For FCS For FLIM For FCS and FLIM

Beam park Optically correct scanning at low inertia

Conventional scanner required

Up to 3

Internal SP FLIM: 400 … 800 nm
External FLIM: Depending on fi lter cube used

Motorized by software,
automatic mode available

Conventional and resonant scanner in one
system (optional)

For FCS: 1 (VIS)
For FLIM: 2 (UV & IR, or UV & VIS)

For FCS: up 8,
For FLIM: up to 3

For FCS: AOBS,
For pulsed lasers: dichroic mirrors

Depending on fi lter cube used

Internal SP FLIM: 400 … 800 nm
External FLIM, FCS, and FLCS: Depending on
fi lter cube used

Alignment stable single pinhole

FCS: Beam park
FLIM: optically correct scanning at low inertia

Conventional scanner required, resonant
scanner optional

No scanning during FCS measurement For FLIM data acquisition For FLIM data acquisition

– 1400 Hz 1400 Hz
–
– 1400 1400
– 5 Hz 5 Hz
– 50 Hz 50 Hz
– Yes Yes
– 512 x 512 pixel 512 x 512 pixel
– 1.0 ... 64 x 1.0 ... 64 x
–no no
– 200° optical 200° optical
– 22 mm 22 mm

1 Hz 1 Hz

5

General Specifications of SMD Prepared Leica TCS SP5

Resonant scanner For imaging

Maximal line frequency 16000 Hz

Scanner

Scan modes

Minimal line frequency
Scan speed granulation 1
Maximal frame rate 512 x 512 28 Hz
Maximal frame rate 512 x 16 290 Hz
Beam park No
Maximal frame resolution 1024 x 1024 pixel
Scan zoom 1.7 ... 64 x
Panning Yes
Field rotation 200° optical
Field diameter 15 mm

Scan options For imaging

xt Yes
xy Y
xyt Yes
xyλ
xz Yes
xzλ
xyz Yes
xyzλ
xyt Yes
xzt Yes
xyzt Yes
xytz Yes

8000 Hz

es

Yes

Yes

Yes

Beam park positioning

6

Automated brightness control of FLIM images –

Maximum number of FLIM images –

Beam park options For spot bleach (in general software)

xy Yes
xyz
xz No
xzy No
Maximum number of FCS/FLCS measurements –
Maximum number of distinct

measurement points
Maximum number of measurement

repetitions at a point
Maximum number of measurement cycles –

No

–

–

Additional SMD Specific Specifications

TCS SMD FCS TCS SMD FLIM TCS SMD FLCS

No scanning during FCS measurement For FLIM data acquisition For FLIM data acquisition

– 8000 Hz 8000 Hz
–
–1 1
– 28 Hz 28 Hz
– 290 Hz 290 Hz
– No No
– 512 x 512 pixel 512 x 512 pixel
– 1.7 ... 64 x 1.7 ... 64 x
–No No
– 200° optical 200° optical
– 15 mm 15 mm

No scanning during FCS measurement For FLIM data acquisition For FLIM data acquisition

–No No
–
– Yes Yes
– Yes (SP FLIM) Yes (SP FLIM)
– Yes Yes
– Yes (SP FLIM) Yes (SP FLIM)
– Yes Yes
– Yes (SP FLIM) Yes (SP FLIM)
– Yes Yes
– Yes Yes
– Yes Yes
–No No

8000 Hz 8000 Hz

Yes Yes

– Yes Yes

– 1000 1000

For automated FCS, FCCS, FLCS data
acquisition series (in FCS wizard)

Yes Yes Yes

s No Yes

Ye
Yes No Ye s
Yes No Ye s
1000 – 1000

100 – 100

100 – 100

100 – 100

For single point lifetime measurement
(in general software)

For automated FCS, FCCS, FLCS data
acquisition series (in FCS wizard)

7

General Specifications of SMD Prepared Leica TCS SP5

Detection features Up to 5 PMT for confocal imaging

Emission separation Highly sensitive prism spectral detector

5

2
APDs from PE (SPCM-AQRH series)

or MPD (PDM series)
PE APD: wavelength dependent,

typ. 65% @ 670 nm
MPD APD: wavelength dependent,
typ. 45% @ 550 nm

PE APD: < 250 cps
MPD APD: < 250 cps

Internal confocal detection

External confocal detection

Maximum number of confocal channels
Tunability of emission bands Yes
Spectral detection range 400 – 800 nm
Tuning steps of emission bands 1 nm
Minimal detection range 5 nm

Sensors High sensitivity low noise PMT: R 9624

Dark current –

TTS FWHM –

Detection features 2 APDs for confocal imaging

Emission separation User-exchangeable beam splitting fi lter cubes
Confocal channels

Sensors

Quantum effi ciency

Dark counts

Non-confocal detection

Electronics

Jitter FWHM Not relevant

Dead time Not relevant

Detection types For Imaging

Transmitted light detector Optional, allowing BF, DIC, Ph etc.
Non descanned transmitted light channels Up to 4 channels (MP)
Non descanned refl

Devices

Scanner control Digitally at high performance

Trigger in/out functions Yes
Auxiliary data input channels Up to 2
Max channels in parallel 12

Computer High performance PC workstation

Integration of third party software –

ected light channels Up to 4 channels (MP)

For imaging

eld programmable gate arrays)

(FPGA, fi

–

Programmable control panel with
LCD function & value display

8

Additional SMD Specific Specifications

TCS SMD FCS TCS SMD FLIM TCS SMD FLCS

Not for FCS data acquisition Up to 2 FLIM-PMTs for spectral FLIM Up to 2 FLIM-PMTs for spectral FLIM

– Highly sensitive prism spectral detector Highly sensitive prism spectral detector
–
– Yes Yes
– 400 – 800 nm 400 – 800 nm
– 1 nm 1 nm
– 5 nm 5 nm

–

– < 300 cps @ 15 °C < 300 cps @ 15 °C

–

2 external detectors for FCS & FCCS 2 external detectors for FLIM 2 external detectors for

User-exchangeable beam splitting fi lter cubes User-exchangeable beam splitting fi lter cubes User-exchangeable beam splitting fi lter cubes
222
APDs from PE (SPCM-AQRH series)

or MPD (PDM series)
PE APD: wavelength dependent,

typ. 65% @ 670 nm
MPD APD: wavelength dependent,
typ. 45% @ 550 nm

PE APD: < 250 cps
MPD APD: < 250 cps

Not relevant

Below typical count rate (40 ... 70 ns) Below typical count rate (40 ... 70 ns) Below typical count rate (40 ... 70 ns)

None for FCS None for FLIM None for FLIM and FCS

–––
–––
–––

For FCS For FLIM For FCS and FLIM

Digitally at high performance
(FPGA, fi

eld programmable gate arrays)
Not required Required Required
Used for APD imaging Optional: Used for APD imaging Used for APD imaging
12 12 15
Second workstation for FCS

data acquisition and analysis
Client server network connection between work-

stations for full system control and data transfer

2 for FLIM + 3 for imaging 2 for FLIM + 3 for imaging

SP FLIM PMT (Hamamatsu R7400U series,
active cooled)

MP FLIM: < 300 ps
UV & VIS FLIM: < 400 ps

APDs from MPD (PDM series)

Wavelength dependent, typ. 45% @ 550 nm Wavelength dependent, typ. 45% @ 550 nm

MPD APD: < 250 cps MPD APD: < 250 cps

MPD APD:
400 – 500 nm: typ. 200 ps
> 500 nm: down to 50 ps

Digitally at high performance
(FPGA, fi eld programmable gate arrays)

Second workstation for FLIM
data acquisition and analysis

Client server network connection between work­stations for full system control and data transfer

SP FLIM PMT (Hamamatsu R7400U series,
active cooled)

MP FLIM: < 300 ps
UV & VIS FLIM: < 400 ps

FCS, FCCS, FLCS, and FLIM

APDs from MPD (PDM series)

MPD APD:
400 – 500 nm: typ. 200 ps
> 500 nm: down to 50 ps

Digitally at high performance
(FPGA, fi eld programmable gate arrays)

Second workstation for FCS and FLIM
data acquisition and analysis

Client server network connection between work­stations for full system control and data transfer

Required Required Required

9

General Specifications of SMD Prepared Leica TCS SP5

Devices For imaging

Fast ROI-spectrometer Optional

Extensions

Software (LAS AF)

Software options (LAS AF)

Auxiliary emission port

Environment accessories Various options

General Intuitive and guiding user interface

Context sensitive online help system Included
Multi-dimensional data acquisition
Region of interest (ROI) scan Included
Excitation line/frame sequential scan Included
Emission spectrum recording Included
Quantifi cation tools Included
Multi-color restoration, spectral unmixing Included
General time lapse experiment control

tile scanning (mosaic scan)

Dedicated application wizards For imaging

Live Data Mode

Advanced Mark & Find

3D visualization

Colocalization

Deconvolution

MicroLab

Optional

Included

Included

Interactive data recording also allowing
job sequencing and online evaluation

Combines Mark & Find with sophisticated 3D
recordings, Live Data Mode etc.

Maximum and other projections, simulated
fl

uorescence process, rotation animations,
stereo pairs, red-green anaglyphs, height color
coded extended depth of focus images etc.

Histogram based colocalization
and area measurements

Deconvolution option for widefi eld
and confocal images

FRAP wizard, FRAPxt wizard, FLIP wizard,
FRET SE wizard, FRET AB wizard etc.

SMD FCS wizard –

SMD FLIM wizard –

10

Additional SMD Specific Specifications

TCS SMD FCS TCS SMD FLIM TCS SMD FLCS

For FCS For FLIM For FCS and FLIM

Not possible Optional with internal SP FLIM

Not possible with external FLIM

Not possible

Various options Various options various options Various options

Included Included Included

Included Included Included
Included
included included included
Included Included Included
Included Included Included
Included Included Included
Included Included Included

Included Included Included

For FCS/FCCS For FLIM For FCS/FCCS/FLCS and for FLIM

Optional with internal SP FLIM
Not possible with external FLIM

Included Included

Not possible

Not possible

–––

–––

–––

–––

–––

–––

FCS wizard for optimization and automation
of FCS and FCCS measurement series

–

–

FLIM wizard for optimization and
automation of FLIM measurement stacks
and time series

FCS wizard for optimization and automation
of FCS, FCCS and FLCS measurement series

FLIM wizard for optimization and
automation of FLIM measurement stacks
and time series

11

General Specifications of SMD Prepared Leica TCS SP5

General For imaging

Supported time-resolved analysis methods –

Software features SymphoTime

One-line visualization for time-resolved data
acquisition

FLIM/FCS options For imaging

Data acquisition method –

TCSPC channel width

Resolvable lifetime range –

FLIM analysis For imaging

Data processing –

Fitting models

Optimization methods –

Error test/assessment –

Error analysis –

FCS analysis For imaging

Correlation method –

–

–

–

Fitting model

Optimization methods –

Error test/assessment –

Error analysis –

12

–

Additional SMD Specific Specifications

TCS SMD FCS TCS SMD FLIM TCS SMD FLCS

For FCS/FCCS For FLIM For FCS/FCCS/FLCS and for FLIM

FCS, FCCS, FRET, Scripting language
for user

-defi ned analysis routines

Auto- or Crosscorrelation,
Intensity time-trace

For FCS/FCCS For FLIM For FCS/FCCS/FLCS and for FLIM

Time-Tagging of photon arrival times using
T

ime-Correlated Single Photon Counting

(TCSPC) electronics

– min. 4 ps min. 4 ps

–

For FCS/FCCS For FLIM For FCS/FCCS/FLCS and for FLIM

– Whole image or ROIs (arbitrary shape) Whole image or ROIs (arbitrary shape)

–

–

–

– Asymptotic standard errors Asymptotic standard errors

For FCS/FCCS For FLIM For FCS/FCCS/FLCS and for FLIM

Software correlation
(auto- and crosscorrelation)

Pure diffusion, triplet-state, conformational,
protonation, 2D/3D Gaussian PSF

Least squares, Marquardt-Levenberg,
Monto Carlo

Chi-Square, distribution weighted
residuals

Asymptotic standard errors, bootstrap
and support plane analysis

FLIM, FLIM-FRET, Lifetime Histogram,
Scripting language for user-defi ned analysis
routines

FLIM image, Intensity-time trace,
TCSPC histogram

Time-Correlated Single Photon Counting
(TCSPC)

< 100 ps to some µs (depending on system
confi guration and experimental conditions)

1 to 4 exponentials, iterative
reconvolution or tail fi

Least squares, MLE,
Marquardt-Levenberg, Monto Carlo

Chi-Square, distribution weighted
residuals

tting

–

–

–

–

–

FLIM, FLIM-FRET,FCS, FCCS, FLCS, FRET, Life­time Histogram, Fluorescence Time/Lifetime
Traces, Scripting language for user-defi ned
analysis routines

Auto- or Crosscorrelation, FLIM image,
Intensity-time trace, TCSPC histogram

Time-Correlated Single Photon Counting
(TCSPC)

< 100 ps to some µs (depending on system
confi guration and experimental conditions)

1 to 4 exponentials, iterative
reconvolution or tail fi tting

Least squares, MLE,
Marquardt-Levenberg, Monto Carlo

Chi-Square, distribution weighted
residuals

Software correlation
(auto- and crosscorrelation)

Pure diffusion, triplet-state, conformational,
protonation, 2D/3D Gaussian PSF

Least squares, Marquardt-Levenberg,
Monto Carlo

Chi-Square, distribution weighted
residuals

Asymptotic standard errors, bootstrap
and support plane analysis

13

Installation Requirements

VIS Max. 320 kg

Weight base system

Heat load max.

Separate cooling IR laser IR laser, air-cooled heat exchanger (chiller)

Electric supply

Environment

IR Optical bench 900 x 1500 mm: + ca. 280 kg

IR laser system: + ca. 100 kg

SMD specifi c components + up to 160 kg
VIS 3.2 kW
IR 6.2 kW

VIS lasers
( with or without 405 nm cw laser)

IR laser +1x 100 … 120 or 200 … 240 V AC, 15 … 10 A, 50/60 Hz
Chiller for IR laser +1x 100 … 120 or 200 … 240 V AC, 10 A/6 A, 50/60 Hz
SMD +1x 100 … 120 or 200 … 240 V AC, 20 A/16 A, 50/60 Hz
Room temperature +18 to + 25°C, avoid proximity to air conditioning equipment!
Temperature for optimal optical behavior +21 to + 23 °C
Vibration velocity

@ Frequency range 5 Hz–30 Hz
@ Frequency range 5 Hz–30 Hz

Pollution degree Class 2, protect from dust!
Relative humidity 20 – 80% (not condensing)
Illumination Room darkening recommended!
Load carrying capacity 200 kg/m
Door width > 1.00 m

3 x 100 … 120 or 200 … 240 V AC. 1600 VA, 50/60 Hz

< 30 µm/s (effective value)
< 60 µm/s (effective value)

2

visible and ultraviolet radiation:

14

infrared radiation:

Leica TCS SMD at Inverse Microscope

Leica TCS SMD at Upright Microscope

15

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