Centre for Molecular Imaging at NT (MINT)

Leica TCS SP5

– Confocal microscope

The Leica TCS SP5 is a confocal microscope system with a broad range of scan speeds and is well suited for live-cell imaging and high-resolution morphological imaging, as well as multispectral imaging. The emission bands in front of the detectors are fully tunable due to a high efficiency prism spectrometer.

The microscope

Leica DMI 6000 CS AFC Bino inverted microscope with motorized XY-stage and a touch control panel

Focus drives

Motorized objective nosepiece.

Super Z Galvo stage (max 1500 um travel range) for higher speed and accuracy.

 Incubation system and accessories

  1. Large incubator covering the entire microscope, for warm air incubation and CO2-control.
  2. Incubator-kit for Super Z Galvo stage, including different inserts and mask sets for the stage.
  3. Micro dispenser for automated supply of water immersion during long term experiments.
  4. Hardware-based focus stabilizer.

Objectives

Magnification

NA

 Type

Immersion

Coverslip

WD (mm)

      5x 0.15 HCX PL Fluotar Air - / 0.17 12
   10x 0.40 HC PL Apo CS Air - / 0.17 2.20
   20x 0.75 HC PL Apo CS2 Imm/Corr - / 0.17 0.68
    40x 1.1 HC PL Apo CS2 Water 0.14 - 0.18 0.65
   25x 1.95 HCX IRApo Water 0.17 2.4
   63x 1.2 HC PL Apo CS2 Water 0.14 - 0.18 0.30
  63x 1.4 HC PL Apo CS2 Oil 0.17 0.14

The 20x immersion objective is for water, glycerine and immersion oil

The 40x and the 60x water objectives have motorized correction collars and can be used with the micro dispender

Condensor top

  • NA 0.55 (S28), air, working distance 0.28 mm
  • NA 0.90 (S1) air, working distance 1 mm
  • NA 1.40 (S1) oil, working distance 1 mm 

Fluorescence filter sets

Name Excitation filter Emission filter Dichroic
A BP 340 - 380 nm LP 425 nm 400 nm
I3 BP 483 - 498 nm LP 515 nm 500 nm
Y3 BP 525 - 656 nm BP 572 - 647 nm 565 nm

Lamps

Halogen: 12V - 100W.

Mercury shirt arc lamp HXP 120W

Lasers

Type                Wavelenght (nm)   Output power (mW)     Pulse frequency
UV 405 3 5, 10, 20 or 40 MHz
Argon 458, 476, 488, 496,  514 65 (all lines) continuous wave
WLL 470 - 670 1.5 (per line) variable, max 80 MHz
MP laser 690 - 1050 2500 (average at peak) 80 MHz
  1. The MP laser is the TiSp-laser Chameleon Vision-S from Coherent
  2. An acousto-optical tunable filter (AOTF) controls the intensity of the visible excitation laser light.
  3. An electro-optical modulator (EOM) is used to control the intensity of the MP laser.
  4. An acousto-optical beam splitter (AOBS) is used to split excitation light and emission light instead of filter blocks.

Scanners

Conventional scanner: 10-1400 Hz (2-3 frames/sec at 512 x 512 pixels image format)

Resonant scanner: 8000 Hz (25 frames/sec at 512 x 512 pixels image format)

Internal detectors

Reflection/fluorescence mode (spectral detection range 400-800 nm)

  • 2 x HyD (GaAsP detectors)
  • 2 x PMT (standard photomultiplier tube)

The SMD platform from PiqoQuant for FLIM and FCS

  1. The pulsed lasers: the UV, WLL and MP laser.
  2. External detectors: 2 x SPAD (single photon avalanche diodes).
  3. Filtercube for FITC and TRITC: 500-550 and 565-605 nm (changeable).
  4. A SMD computer with the software SymPhoTime version 5.3.

The MP system

  1. The MP laser for two-photon or SHG imaging
  2. External NDD detectors
    1. 2 x HyD (GaAsP detectors) in the reflection mode.
    2. 2 x PMT in the transmission mode
  3. One filter cube for each mode with a number of different dichroic mirrors and emission filters that are easily changeable.

Software on the SP8 computer

Leica LAS AF version 3.1.2 with software modules

  1. FRAP Wizard
  2. FRAP XT Wizard
  3. FRET AB Wizard
  4. FRET SE Wizard
  5. FLIM Wizard
  6. FCS Wizard
  7. Matrix Screener
  8. Live Data Mode

Software on the SMD computer

SymPhoTime version 5.3 from PiqoQuant.

Mon, 31 Oct 2016 11:06:13 +0100

Technical support

Optical/confocal microscopy
Image analysis

Contact: Astrid Bjørkøy


Atomic force microscopy (MultiMode8)

Contact: Gjertrud Maurstad


Flow cytometry

Contact: Kristin Grendstad Sæterbø

Astrid Bjørkøy and the confocal microscope Leica TCS SP5. Photo: Irene Aspli