Stimulated emission depletion (STED) microscopy
13 Sep 2016
Yes
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Stimulated emission depletion (STED) microscopy

 

Image of a T lymphocyte crawling taken with a Leica SP8 g-STED

 
LEICA SP8 3X SMD
Contact: Dr. Jorge Bernardino de la Serna

Capabilities

  • Confocal laser scanning microscope. (Simultaneous 5-color imaging, time lapse imaging, 3D Imaging, time lapse and 3D animations (movies).
  • DIC imaging (independent and simultaneous to fluorescence imaging)
  • Spectral imaging. Lambda scans both at the excitation and emission.
  • Large size specimen imaging by tiling several images. (mosaic stitching)
  • Fluorescence Recovery after Photobleaching (FRAP)
  • Foster Resonance Energy Transfer (FRET) intensity-based
  • Spectral imaging. Lambda scans both at the excitation and emission.
  • Time correlated single photon counting module for Fluorescence Lifetime Imaging (FLIM), point and scanning Fluorescence (Cross-, dual colour) Correlation Spectroscopy (FCS and FCCS).
  • Fluorescence
  • Lifetime (Cross-, dual colour) Correlation Spectroscopy (FLCS, and FLCCS), single photon FRET-FLIM.
  • Gated cwSTED and pulsed STED XY lateral nanoscopy (enables super-resolution imaging to ~50nm XY). Simultaneous 3-4 color super-resolution imaging. STED FCS and Scanning STED FCS, both single and dual color (FCCS)
  • 3D STED XYZ lateral and axial nanoscopy- ( enables super-resolution imaging to ~50nm XY and ~150nm Z resolution)

Technical Specifications

  • Microscope: LEICA SP8 3X SMD inverted
  • This system is a AOBS confocal laser scanning microscope on a Leica DM6000 inverted epifluorescence microscope with “Adaptative Focus Control” to correct focus drift during time lapsed experiments.
  • Fluorescence filter cubes for FITC and TRITC for visual inspection.
  • Stage: Motorized Märzhäusser stage with Leica Galvo stage – enables multi-position acquisition and tiled imaging. Tandem Scanner with Galvanometric scanning unit (1800-and 3600Hz bidirectional)Incubation: box type temperature incubation (Cube) with CO2.
  • Excitation range (470-680nm): Suitable for a wide range of cyan, green, red and far-red fluorophores.
    • Equipped with a pulsed super continuum laser (white light laser, WLL) with 2.5 mW/nm and a pulse picker to select 80,40,20 or 10 MHz rep rates
    • AOTFs (Acoustic-Optical Tuneable filters) for the WLL to allow rapid attenuation and switching between up to 8 different wavelengths ( 470-680nm)
    • 3 STED lasers lines with automatic spatial (X,Y and Z) alignment to the excitation laser, two continuous wavelength 592nm and 660nm, and one 775nm pulsed laser with additional temporal alignment.
    • Notch filters block reflection signal, enabling clearer imaging close to coverslips
  • Detection range (480-780nm): Suitable for a wide range of cyan, green, red and far-red fluorophores
    • AOBS (Acoustic-Optical Beam Splitter) automatically adjusts to selectively reflect each excitation line and allows optimisation of detection close to (and overlapping-for reflexion mode)) excitation lines.
    • 2x high-sensitive internal gated HyD photon-counting detectors (SMD GaAsP)
    • 2x spectral PMT
    • 1 TLD (Transmitted light - brightfield)
    • Combining spectral detection with WLL enables excitation/emission scanning (lambda-mode 2).
  • Single Molecule Detection: Time-correlated single photon counting (TCSPC). Allows TCSPC fluorescence lifetime data (FLIM) acquisition, fluorescence (cross-) correlation spectroscopy, and fluorescence lifetime (cross-) correlation spectroscopy (FCS, FCCS, and FLCS, FLCCS measurements.
    • PicoHarp 300 (Pico Quant TCSPC Module)
    • 4 Channel Detector Router (PHR 800)
  • Hyvolution.

Objectives

Magnification

Objective type NA (1) Immersion Working Distance (mm) Resolution @ 525 nm in XY(3) Resolution @ 525 nm in Z(4)
10x HC PL APO CS 0.40 - 2.2 0.37 1.58
20x HC PL APO 0.70 IMM(2)

0.26 (Water)

0.17 (Oil)

0.21 0.90
40x HCX PLAN APO

0.85

- 0.24 0.17 0.74
63x HCX PL APO

1.40  - 0.60

OIL 0.10 0.11 0.45
100x STED white HC PL APO 1.40 OIL   0.13 0.45

(1) NA - Numerical Aperture. For more information, follow this link.
(2) Requires manual adjustment of the correction collar; works with oil, glycerol or water.
(3) This value is the theoretical optical resolution limit depending on the NA. To achieve optimal lateral (XY) resolution and Nyquist Rate, please speak with the personnel for optimal pixel size.
(4) For optimal Axial (Z axis) resolution, divide this value by 2 to get Nyquist Rate.

Analysis Software

  • Leica LAS X SP8 and offline Leica LAS AF for Image processing with a good 3D volume renderer capable of reconstructing 3D images from confocal planes, and a simple imaging processing and quantification module
  • Additional licence for STED, Life data mode, Dye separation, 3D rendering
  • SVI software package. Huygens Deconvolution via CUDA GPU, capable of performing high-end deconvolution, and image surface rendering, image quantification, Object analysis, Co-localisation analysis, Single particle Tracking analysis, Movie generator…PSF distiller
  • FLIM/FCCS/FLCS Software: SymPho Time 32 and FLIM SymPho Time 64 1.6 (Picoquant)
  • SIM-FCS software for FCS, RICS, Number and Brightness, Pair correlation.

Useful links

Case Studies

Contact: Bernardino De La Serna, Jorge (Imperial Coll.,RAL,RCAH)