COVID-19 Updates

Visit UC San Diego's Coronavirus portal for the latest information for the campus community.

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 (only BSL1 cell lines)
  • 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