Leica SP5 HyD MP Multiphoton/Confocal Microscope

The Leica SP5 HyD MP microscope combines the flexibility of confocal imaging with the deep tissue imaging capability of multiphoton (or two-photon) microscopy. The Leica SP5 HyD MP is equipped with a tuneable titanium-sapphire infrared laser capable of emitting between 700 and 1040nm. Multiphoton microscopy uses better penetrating infrared light to excite standard fluorescent markers and fluorescent proteins such as GFP. Multiphoton excitation allows fluorescence to be visualised at depths much greater than the ~100 microns typically achievable using standard fluorescence techniques. Multiphoton microscopy can be used to view complex living tissues such as whole model organisms or tissue biopsies. In addition, multiphoton excitation has the advantage of only exciting a defined volume at the focal plane, which reduces photobleaching and improves the precision of techniques such as FRAP and photoactivation. Multiphoton microscopy also has many applications in material science, nanotechnology, photonics and physics.

External HyD detectors for Multiphoton

The Leica SP5 HyD MP is the first system in Spain to be fitted with dual super-high sensitivity external NDD HyD detectors for Multiphoton improving yet further its sensitivity and its ability to perform deep tissue imaging even with weakly expressing fluorescence.

Full confocal functionality

In addition to its multiphoton capabilities, the Leica SP5 HyD MP is also a fully equipped confocal microscope with 7 different excitation laser lines in the visible range, including a 405nm violet laser, and the ability capture up to four fluorescence channels simultaneously. Researchers also have the option of using multiphoton excitation in conjunction with the internal PMT detectors for dynamically adjustable spectral fluorescence detection. Moreover, both conventional and high-speed resonant scanning options can be used with confocal and multiphoton modalities.


  • Inverted Leica DMI 6000 fully motorised microscope.
  • Confocal detectors: 4 x PMT + 1 transmitted light detector.
  • External NDD detectors: 2 x HyD Hybrid detectors
  • Tandem Scanner – HQ conventional scanner and high-speed Resonant scanner (28 frames/sec @ 512 x 512)
  • Dichroic filter-based beamspliter
  • Optical zoom up to 64x (maximum resolution is dependent on the objective used).
  • Choice of objectives:
    • Plan Apo Oil immersion: 100x (1.4 NA), 63x (1.4 NA Blue), 40x (1.3 NA)
    • Plan Apo 20x (0.7 NA) multi-immersion (water, glycerol, oil) 0.26mm WD
    • Plan Apo 10x (0.4 NA) dry.
  • 7 Laser lines in the visible range:
    • Violet 405nm (50mW), Blue Argon 458nm, 476nm, 488nm, 514nm (65mW), Green DPSS 561nm (20mW), Red HeNe 633nm.
  • Microscope enclosure with temperature, humidity and CO2 control.
  • High precision Super Z motorized stage with multi-point and multi-tile software control. 3nm resolution galvometric z-axis control.


Helpful information

  • Open Leica format files (.lif) with Fiji or ImageJ and the Bioformats plugin. Also available to local users via in the GGI Software folder on the intranet.
  • LASAF lite – limited version of Leica LASAF software – able to open .lif files, perform basic operations and extract metadata. Also available to local users via in the GGI Software folder on the intranet.
  • Multiphoton introduction (Nikon)
  • Two-photon Microscopy (Wikipedia)

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