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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