Equipment

Leica TCS SP8 Microscopes

The SP8 systems can provide visible light laser excitation at 405, 488, 514, 552 and 638nm. Their stands are DMi8 CS Trino research inverted microscopes with fluorescence, polarization and DIC capabilities. They are outfitted with a series of objectives spanning the range of magnification and numerical aperture 10x/0.3 to 40x/1.3, to perform wide-field/low resolution imaging for initial sample assessment and narrow-field/high-resolution imaging with optimal refractive index matching (water and oil immersion objectives) and long free working distances for imaging thick specimens. Images can be collected by three distinct internal descanned detectors within wavelength ranges set arbitrarily by the user in the 400-800 nm range. Full emission spectral images can also be collected over these ranges in wavelength steps defined by the user and optimized for the specific application. In addition, external non-descanned detectors with specialized filters are available for nonlinear microscopy covering a broad range of chromophores and various imaging modalities (TPEF, SHG, CARS and FLIM). Use of highly sensitive, low-noise Leica HyD (hybrid) detectors significantly enhances the detection of weak optical signals and provides photon-counting capabilities.

 

(i) FLIM SP8 system

  • Imaging modalities: Confocal, TPEF, SHG & FLIM
  • VIS lasers, cw (Leica): 405, 488, 514, 552 and 638nm
  • NIR, fs-pulsed (Insight DS+): 680-1,300nm; rep rate=80MHz, avg power=2W, pulse width <100fs
  • Descanned detectors: 2PMTs & 1 HyD (spectral range 400-800nm, continuously tunable bandwidth)
  • Non-descanned detectors: 2 PMTs (@624±20 nm & 685±20 nm), 2 HyDs (@460±25 nm and 525±25 nm)
  • Objectives: 10x/0.40, 20x/0.75 (air); 25x/0.95 2.5mm working distance, 40x/1.10 (water); 40x/1.30 (oil)
  • FLIM modality: <1ps resolution, Picoquant SymPhoTime analysis software

 

(ii) CARS SP8 system

  • Imaging modality: CARS
  • VIS lasers, CW (Leica): 405, 488, 552 and 638nm
  • NIR, fs pulsed (MaiTai HP): 690-1,040nm; rep rate=80MHz, avg power=2W, pulse width <100fs NIR, ps pulsed (A.P.E. picoEmerald): CARS Pump=750-940nm, Stokes=1,064nm; ∆Evib=1,200-3,900cm-1, spectral bandwidth=2-3cm-1; rep rate=80MHz, avg power=600mW
  • Descanned detectors: 2PMTs (spectral range 380-780nm, continuously tunable bandwidth)
  • Non-descanned detectors: 4 PMTs (forward- and epi-CARS & SHG)
  • CARS filters: 750±105 nm (∆Evib=1,250-2,000 cm-1) & 650±105 nm (∆Evib=2,000-3,900 cm-1)
  • SHG filter: 465±85 nm
  • Objectives: 20x/0.75, 40x/1.1 (water);  40x/1.3 (oil)