Leica SP8 Confocal with White Light Laser, Falcon (FLIM), STED and Lightning Deconvolution

What it’s good for

  • STED Super Resolution <50nm in XY
  • Tunable excitation source for hand picking excitation wavelengths of up to 8 simultaneously (470nm-670nm)
  • FLIM acquisition with Falcon module
  • Incubation for live cell imaging
  • Determining colocalization for multiple signals
  • Acquisition of up to 5 color channels simultaneously
  • Acquisition of multiple color channels sequentially
  • Acquisition of a transmitted light image with DIC
  • Fixed cells or tissues on a slide with a coverslip (#1.5) or in a dish
  • Spectral Deconvolution to correct for overlapping signals
  • On the fly image deconvolution for super resolution of 120nm in X and Y and 200nm in Z

Principles of operation

Laser light of specific wavelengths is scanned across the sample and filtered before detection to produce a high resolution image composed of a small optical slice of the sample. STED enahances resolution by shrinking fluorescence to a small spot with a depletion laser. Deconvolution further enhances resolution, including STED images.

Technical Information

  • Microscope: Leica DMi8 Inverted
  • Spectral for all channels
  • PMT and HyD detectors
  • STED - Super Resolution depletion lines 
  • White Light Laser tunable to any desired excitation between 470 and 670nm

Microscope objectives

  • 10x (.40 NA)
  • 20x Oil / Glycerol / Water (.75 NA)
  • 40x Water (1.1 NA)
  • 93x Oil (STED)
  • 100x Oil (STED)
Sample Preparation Guidelines
Choice of Samples:
  • For Live samples: Media should be clear and contain no phenol red or other color additives
  • For Fixed Samples: RI of mounting medium should match RI of immersion used (prolong glass or diamond)
  • DAPI should be AVOIDED, especially when using green fluorophores
  • Autofluorescence should be extremely low
  • It's important that the sample doesn't absorb 592nm, 660nm or 775nm
  • Use only #1.5 coverglass, including for glass bottom dishes
Recommended Dyes:
  • Single color for 592nm depletion line
  • DyLight 488 or 514
  • Oregon Green 488 or 514
  • AlexaFluor 488 or 514
  • ATTO 488 or 514
  • Single color for 660nm depletion line
  • Alexa 532
  • ATTO 532 or 550
  • TMR/TRITC
  • Cy3
  • Alexa 555
  • Single color for 775nm depletion line
  • ATTO 647N
  • Alexa 633
  • Alexa 594
  • ATTO 590
Recommended Fluorescent Proteins
  • eGFP (484nm ex / 592nm depletion)
  • EmGFP (487nm ex / 592nm depletion)
  • eYFP (514nm ex / 592/660nm depletion)
  • Venus (515nm ex / 592/660nm depletion)
  • mCitrine (516nm ex / 592/660nm depletion)
  • dsRed (558nm ex / 660nm depletion)
  • mStrawberry (574nm ex / 660nm depletion)
  • mKate2 (588nm ex / 775nm depletion)
  • Fluorescent Proteins to AVOID: mCherry, CFP, tagRFP

STED_Sample preparation guide-AppLetter_EN.PDFSTED Sample Prep Oct 18.pptx

LIGHTNING_WhitePaper.pdf

STED 3X Sample Prep 2018.pdf _ Powered by Box.html