Confocal LEICA SP5 microscope (Villejuif)



The system is installed on a DMI6000 inverted microscope, fully motorised and computer-controlled.
– 10x / 0.30 HCX PL FLUOTAR Dry (11506505) – Working distance: 11.0mm – Maximum field strength: 1550×1550μm (confocal)
– 20x / 0.70 PL APO Multi-imm (oil-glyc-coveg:0.17-w(water salted)-0(water)(11506514) – Working distance: 0.17mm → 0.26mm – Maximum field strength: 775×775μm (confocal)
– 40x / 1.25-0.75 HCX PL APO Oil (11506253) – Working distance: 0.1mm – Maximum field strength: 387.5×387,5μm (confocal)
– 63x /1.40-0.60 HCX PL APO Oil (11506192) – Working distance: 0.1mm – Maximum field strength: 246×246μm (confocal)
– 100x /1.40 HCX PL APO Oil (11506038) – Working distance: 0.09mm – Maximum field strength: 155×155μm (confocal)
Model S28 – numerical aperture of 0.55 with a minimum working distance of 28mm.
Fluorescence excitation: provided by a Leica EL 6000 fibre light source (no alignment required – no heating of the preparation – lifetime >2000h) with 5 mechanical attenuation levels: 0, 12.5, 25, 50 and 100%.
>>Domaines spectraux d’observation (non confocale)
– filter cube A (exc. 340-380/ dichr. 400 / emission LP 425) for Dapi or Hoechst.
– filter cube I3 (exc. 450-490/ dichr. 510 / emission LP 515) for FITC, Alexa 488, eGFP, etc.
– N2.1 filter cube (exc. 515-560/ dichr. 580 / LP 590 emission) for TRITC, Cyanine 3, Alexa 546, 555 and 568, etc.
Composite turntable
– an XY motorised stage allowing the acquisition of a mosaic of images in order to reconstitute an enlarged view of the samples, or to image different positions of a sample with the possibility of repositioning on these points during a time-lapse.
– an over-platinum plate that guarantees fast movements and precise Z (Z-galvo) positioning (accuracy of 40 nm over a thickness of 1.5 mm). This over-platinum can be equipped with different racks: for blade, petri dish, well plate and culture chamber (for the installation of these racks, please contact the person in charge).
Thermostatically controlled enclosure : the entire stand is placed in an enclosure with temperature control and CO2 circulation.

The confocal system

>>Laser excitation sources
– 405 nm diode (<7mW in the focal plane)
– Argon laser 458, 476, 488, 496, 514 nm (<50mW in the focal plane)
– DPSS diode 561nm (<12mW in the focal plane)
– 633nm He/Ne laser (<5mW in the focal plane)
The excitation intensities are controlled by an AOTF system.
>>Fluorescence emission: instead of dichroic filters, an AOBS (Acousto Optical Beam Splitter) device selects the emission cut-off with a gain in speed, absence of optical distortions and high spectral flexibility. The light is then directed onto a prism which allows very fine selection of the ranges of wavelengths imaged (between 400 and 800 nm) on 4 PMT detectors.
This device also makes it possible to collect the emission spectra for each voxel.
1 transmission channel (Brightfield, DIC)
>>Conventional scanner 
acquisition speeds: 10Hz = 10 lines per second) – 100Hz – 200Hz – 400Hz (default – zoom min 1) – 700Hz (zoom min 2.5) – 1000Hz (zoom min 2.5) – up to 1400Hz (zoom min 6)
Modes of acquisition :
– xyz – xzy – xt – xyt – xzt – xzyt – xyλ – xzλ – xyλz – xyλt – xzλt – xyzλt
Image definition :
Up to 8192².
Image dynamics
8, 12 or 16bits