Abberior Facility Line STED System
The microscope uses Stimulated Emission Depletion (STED) technology to achieve higher spatial resolution beyond the diffraction limit of standard light microscopy(30nm in XY and 70nm in Z) . This makes it possible to visualize structures at the nanoscale. Additionally, the integration with Fluorescence Lifetime Imaging Microscopy (FLIM) allows for the measurement of the exponential decay rate of the fluorescence from a fluorescent sample, providing not just structural but also functional information about the molecules being observed. This combination of STED and FLIM opens up a range of possibilities for in-depth analysis in cellular biology, neurobiology, and materials science.
Comparison of Confocal(Top) and STED(Bottom) resolution.(Live cell mitochondria, scale bar 1 micron).
Specification
Imaging Laser:
405nm (cw, 20mW)
485nm (pulsed, 1 mW@ 40 MHz out of laser head)
561nm (pulsed, 0.2 mW@40 MHz out of laser head)
640nm (pulsed,1 mW@ 40 MHz out of laser head)
STED Laser:
775 nm > 2750mW @40Mhz
Easy3D for 775 nm STED
Objectives:
| Objective | NA | WD | Immersion | Notes |
|---|---|---|---|---|
| UPLXAPO60XO | 1.42 | 0.15mm | Oil | STED and Confocal Compatible |
| UPLSAPO60XS | 1.3 | 0.3mm | Silicon Oil |
STED and Confocal Compatible |
| UPLAPOL60XW | 1.2 | 0.2mm | Water | STED and Confocal Compatible |
| UPLXAPO40X | 0.95 | 0.18mm | Air | Confocal Compatible |
| UPLXAPO20X | 0.8 | 0.6mm | Air | Confocal Compatible |
| UPLXAPO10X | 0.4 | 3.1mm | Air | Confocal Compatible |
Detectors:
Transmitted Light Detector (PMT)
For fluorescence detection, there are two types of detectors:
a. Four Single-Photon-Counting APD Detectors thats compatible with Spectral Detection (QE around 65% at 500nm,low readout noise)
b. MATRIX Spectral APD array (FLIM, STED and Confocal Compatbile) (QE around 50% at 500nm,low readout noise)
Traditional PMT detectors on point scanning confocals have QE around 30% (Ref)
The MATRIX detector can also be used for Image Scanning Microscopy (same technique as the Zeiss Airyscan) and can achieve higher resolution(around 160nm) with regular fluorophores.
FLIM (Fluorescences Lifetime Imaging Microscopy)
The system can function as a single-detector FLIM (MATRIX Detector) that can be used to perform TCSPC based FLIM imaging (FLIM-STED, FLIM-Matrix(Imaging Scanning Microscopy, similar to Airyscan), FLIM-FRET) with temporal resolution of around 50 ps.
Common dye's life time plot (author: Dominik Frei)
Other Modules
FLEXPOSURE: Adaptive Illumination Package for Highest Resolution and Live cell
Imaging
ADAPTIVE OPTICS: Adaptive optics to correct optical distortion when imaging deeper into the sample.
Image Processing
The system is equipped with a Huygens Pro license for image deconvolution.
Sample Preparation
Special secondary antibody should be used for STED imaging (usually fluoresohpore that has some emission wavelength overlapping with the STED laser 775nm works). The NorMIC facility has an array of Abberior STAR secondary antibodies available for platform users to test. Please consult the platform staff to obtain an aliquot.
STED Sample Preparation Guide(Abberior)
STED Sample Preparation Guide(Leica)
Multi-color STED Imaging(Abberior)
5 Color STED antibody suggestion
Contact: xian.hu at medisin.uio.no