STED RESOLFT

Confocal Widefield

ImagIng beyond barrIers From the inventors of

STED and RESOLFT

Our ConceptAbberior Instruments was founded in early 2012 as a spin-off from the Max-Planck Institute in Göttingen and the DKFZ in Heidelberg. The following standards guide our product design:

Achieve the highest resolution technically possible

Fast time-to-market; development cycles < 1 year

Custom adaptation possible e.g. STED/RESOLFT Upright system, combination with AFM

One platform across all microscope types

Open approach:

Interface for custom software/drivers

Optical hardware accessible

Electronics capable of driving additional hardware

Our speed of innovation

First publication Launch of Abberior product

RESOLFT with rsEGFP2011 2012

Parallel RESOLFT2013 2014

2-color STED @ 775 nm2013 2014

raw data

References:Specs:

2-color STED resolution typically < 40x40nm

3D upgrade optional

Combine with STED @ 775 nm and RESOLFTfor up to 6 superresolution channels

RESCue mode optional

Abberior multicolor STED @ 595 nm

Spectral properties and labels:

440 nm 488 nm 520 nm 595 nmExcitation wavelengths pulsed STED

Suitable labels:

Abberior STAR 440SXP, Abberior STAR 488, GFP, YFP, Oregon Green 488, ATTO 488,Chromeo 488, etc.

Confocal STED

Pulse/ pixel/ line/ frame interleaved acquisitionTønnesen, J. et al. “Two-Color STEDMicroscopy of Living Synapses Using ASingle Laser-Beam Pair“, Biophysical J.101, 2545 (2011)

Hein, B. et al. “Stimulated emission depletion(STED) nanoscopy of a uorescent protein-labeled organelle inside a living cell“, PNAS105, 14271 (2008)

Rankin, B.R. et al. “Nanoscopy in a LivingMulticellular Organism Expressing GFP“,Biophysical J. 100, L63 (2011)

Clausen, M.P. et al. “Pathways to opticalSTED microscopy“, NanoBioImaging 1, 2299 (2013)Pulsed STED laser and time gating

APD detection with highest detection sensitivity(>62% @ 680nm)

Change between objectives (water/ oil/ glycerol immersion)

References:Specs:

2-color STED resolution typically < 40x40nm

3D upgrade optional

Combine with STED @ 775 nm and RESOLFTfor up to 6 superresolution channels

RESCue mode optional

Abberior multicolor STED @ 595 nm

Spectral properties and labels:

440 nm 488 nm 520 nm 595 nmExcitation wavelengths pulsed STED

Suitable labels:

Abberior STAR 440SXP, Abberior STAR 488, GFP, YFP, Oregon Green 488, ATTO 488,Chromeo 488, etc.

Confocal STED

Pulse/ pixel/ line/ frame interleaved acquisitionTønnesen, J. et al. “Two-Color STEDMicroscopy of Living Synapses Using ASingle Laser-Beam Pair“, Biophysical J.101, 2545 (2011)

Hein, B. et al. “Stimulated emission depletion(STED) nanoscopy of a uorescent protein-labeled organelle inside a living cell“, PNAS105, 14271 (2008)

Rankin, B.R. et al. “Nanoscopy in a LivingMulticellular Organism Expressing GFP“,Biophysical J. 100, L63 (2011)

Clausen, M.P. et al. “Pathways to opticalSTED microscopy“, NanoBioImaging 1, 2299 (2013)Pulsed STED laser and time gating

APD detection with highest detection sensitivity(>62% @ 680nm)

Change between objectives (water/ oil/ glycerol immersion)

References:

Wildanger, D. et al. “Solid Immersion FacilitatesFluorescence Microscopy with NanometerResolution and Sub-Ångström EmitterLocalization“, Adv. Mater. 24, 309 (2012)

Specs:

2-color 2D STED resolution < 30x30 nm(25x25 nm typical)

3D STED resolution < 100x100x100 nm,80x80x90 nm typical (with easy 3D STEDmodule)

Change between objectives (water/ oil/ glycerol immersion)

APD detection with highest detection sensitivity(>62% @ 680nm)

Pulsed STED laser and time gating

Combine with STED @595nm and RESOLFTfor up to 6 superresolution channels

Pulse/ pixel/ line/ frame interleaved acquisition

Abberior multicolor STED @ 775 nm

Spectral properties and labels:

488 nm 594 nm 640 nm 775 nmExcitation wavelengths pulsed STED

Recommended labels:

Abberior STAR 520SXP, Abberior STAR 580, Abberior STAR 635P, Abberior STAR RED, ATTO 647N, ATTO 590, etc.

Confocal STED

RESCue mode optional

Göttfert, F. et al. “Coaligned Dual-Channel STED Nanoscopy and Molecular DiffusionAnalysis at 20 nm Resolution“, Biophysical J.105, L01 (2013)

Harke, B. et al. “Three-Dimensional Nanoscopyof Colloidal Crystals“, Nano Lett. 8, 1309(2008)

Berning, S. et al. “Nanoscopy in a LivingMouse Brain“, Science 335, 551 (2012)

561nm520 nm

Aberration corrections for 2D and 3D STED

Booth, M. et al. “Aberrations and adaptive optics in super-resolution microscopy”,Microscopy 64 (4), 251 (2015)

Gould, T.J. et al. “Adaptive optics enables 3DSTED microscopy in aberrating specimens”,Opt Expr. 20, 20998 (2012)

Abberior easy3D STED

References:Harke, B. et al. “Three Dimensional Nanoscopyof Colloidal Crystals”, Nano Lett. 8, 1309(2008)

Willig, K.I. et al. “STED Microscopy with continuous wave beams”, Nature Meth. 4,915 (2007)

Specs:

Based on a freely programmable SLM(Spatial Light Modulator)

2D STED resolution: < 30x30 nm (25x25 nm typical)

3D STED resolution: < 100x100x100 nm (80x80x90 nm typical)

Prepared to correct for sample aberrations/distortions

Upgrade kit for all Abberior STED systems

easy3D STED with a single beam

STED-Laser

Variable2D/3D selection(half wave plate)

Microscope

Vortex Phase ring

Beamshaper 1(s-pol)

Beamshaper 2(p-pol)

Single beam

Vortex(donut)

Phasering

easy3D STED2D STED2D STED

z-range 900 nmz-range 900 nm

ConfocalConfocal

80x80x90 nm

Confocal easy3D STED

STED light can be split between 2D (vortex) and 3D (ring) mode in any proportion

Abberior RESCue STED

References:

T. Staudt, A. et al. “Far-field optical nanoscopy with reduced number of state transition cycles”Opt. Express 19, 5644 (2011)

Specs:RESCue STED reduces the light dose on the sample down to 4% compared to gated cw STED without compromising the resolution

Beneficial for volume imaging with 3D STED

Can similarly be applied to confocal imaging

RESCue STED designed for live-cell applications

where no signal arises in the first microseconds of the pixel dwell timewhere already enough signal has been collected

Neighboring pixels are NOTpre-stressed/ pre-bleached

Basic idea: Shut-off excitation & STED light

Light dose

gate

d cw

STE

D

100%RESCue

STEDLiving cell

RE

SC

ue S

TED

<4%

Beneficial for time-lapse STED imaging

Avoids unnecessary excitation and de-excita-tion cycles (conventional STED applies thesame light dose to any pixel)

Reduction of photobleaching of any fluorescent marker

Reduction of the light dose enhances live-cellimaging conditions in general

STED(conv.)

RESCueSTEDRESCueSTED

60th STED image

Booth, M. et al. “Aberrations and adaptive optics in super-resolution microscopy”,Microscopy 64 (4), 251 (2015)

Gould, T.J. et al. “Adaptive optics enables 3DSTED microscopy in aberrating specimens”,Opt Expr. 20, 20998 (2012)

Abberior easy3D STED

References:Harke, B. et al. “Three Dimensional Nanoscopyof Colloidal Crystals”, Nano Lett. 8, 1309(2008)

Willig, K.I. et al. “STED Microscopy with continuous wave beams”, Nature Meth. 4,915 (2007)

Specs:

Based on a freely programmable SLM(Spatial Light Modulator)

2D STED resolution: < 30x30 nm (25x25 nm typical)

3D STED resolution: < 100x100x100 nm (80x80x90 nm typical)

Prepared to correct for sample aberrations/distortions

Upgrade kit for all Abberior STED systems

easy3D STED with a single beam

STED-Laser

Variable2D/3D selection(half wave plate)

Microscope

Vortex Phase ring

Beamshaper 1(s-pol)

Beamshaper 2(p-pol)

Single beam

Vortex(donut)

Phasering

easy3D STED2D STED2D STED

z-range 900 nmz-range 900 nm

ConfocalConfocal

80x80x90 nm

Confocal easy3D STED

STED light can be split between 2D (vortex) and 3D (ring) mode in any proportion

Abberior RESCue STED

References:

T. Staudt, A. et al. “Far-field optical nanoscopy with reduced number of state transition cycles”Opt. Express 19, 5644 (2011)

Specs:RESCue STED reduces the light dose on the sample down to 4% compared to gated cw STED without compromising the resolution

Beneficial for volume imaging with 3D STED

Can similarly be applied to confocal imaging

RESCue STED designed for live-cell applications

where no signal arises in the first microseconds of the pixel dwell timewhere already enough signal has been collected

Neighboring pixels are NOTpre-stressed/ pre-bleached

Basic idea: Shut-off excitation & STED light

Light dose

gate

d cw

STE

D

100%RESCue

STEDLiving cell

RE

SC

ue S

TED

<4%

Beneficial for time-lapse STED imaging

Avoids unnecessary excitation and de-excita-tion cycles (conventional STED applies thesame light dose to any pixel)

Reduction of photobleaching of any fluorescent marker

Reduction of the light dose enhances live-cellimaging conditions in general

STED(conv.)

RESCueSTEDRESCueSTED

60th STED image

Quad scanner design with 4 galvo mirrors

Abberior QUAD scanner

Features

Patent protected scanning technology

Novel four-mirror design

Compact and robust scan head

:deriuqersnelnacsoNminimal wavefront distortions

Beam alignment by software

4 degrees of freedom: beamposition & angle independently addressable

Scan eld: > 80 x 80 µm

Line frequency: up to 2 kHz

References:

microscopy“, NanoBioImaging 1, 2299 (2013)

Bingen, P. et al. “Parallelized STED fluores-cence nanoscopy“, Opt. Expr.19, 23716 (2011)

(100x/1.4 oil immersion lens)

Clausen, M.P. et al. “Pathways to optical STED

4 Degrees of FreedomPerfect donut overfull field of view

Controlling platformfor Abberior microscopes

Abberior Patchpanelfor synchronized Multi-I/O

72 con gurable I/O connectors

4 ultrafast laser trigger outputs, 2 SYNC inputs, 6 gateable photon counting inputs

Typical Abberior con guration leaves 40+connectors for your custom application

Abberior software platformImspector

Microscope control:

RESOLFT and STED image acquisition

QUAD scanner control

pixel/ line/ frame interleaved multiplexing

mirrors, ipper, shutters, microscope body, …)

optomechanics

Image analysis:

Analysis toolbox for quantitative microscopy

noitulovnoced)LR(raenil-nondnaraeniLalgorithms

Lifetime analysis (STED FLIM)

Ready to control your own lasers and

Laser timing and data collection for pulse/

Control of all motorized components (pinhole,

RESCue mode

FRAP (Fluorescence Recovery After Photobleaching)

Quad scanner design with 4 galvo mirrors

Abberior QUAD scanner

Features

Patent protected scanning technology

Novel four-mirror design

Compact and robust scan head

:deriuqersnelnacsoNminimal wavefront distortions

Beam alignment by software

4 degrees of freedom: beamposition & angle independently addressable

Scan eld: > 80 x 80 µm

Line frequency: up to 2 kHz

References:

microscopy“, NanoBioImaging 1, 2299 (2013)

Bingen, P. et al. “Parallelized STED fluores-cence nanoscopy“, Opt. Expr.19, 23716 (2011)

(100x/1.4 oil immersion lens)

Clausen, M.P. et al. “Pathways to optical STED

4 Degrees of FreedomPerfect donut overfull field of view

The software platform Imspector offers full flexibility in the design of experiments

Connectable hardware Gating & fast-signal timing with 90ps accuracy

n time windows per pixel with 10ns accuracy

• 4 SPC detectors, e.g. APD‘s

• 8 lasers triggers • Shutter triggers, etc…

8 x gating channels

8 x fast-signal triggering

Typical configuration

Det1

Laser1

Laser2

Det1

Det2

on off off

on off off

Get full live-time functionality with adding TCSPC hardware

…

…

…

…

Multiple gates for one detector

Source: Abberior Instruments GmbH, www.abberior-instruments.com

t

t

t t

Time interleaved channel separation per pixel and/or line/frame

150 cm

200 cm

100 cm

Abberior Instruments is the winner of the 2014Innovation award of the German Economy

Abberior Instruments GmbH

Hans-Adolf-Krebs-Weg 137077 GöttingenGermanyPhone +49 551 30724-170Fax +49 551 30724-171info@abberior-instruments.comwww.abberior-instruments.com

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理化学機器営業部〒 105-0014　東京都港区芝 3 丁目 5 番 1 号　コーンズハウスTel. 03-5427-7568E-mail:ctl-science@cornes.jp