Leica SP8 WLL MP laser scanning confocal microscope with supercontinuum “white light” laser and multiphoton excitation

 

Microscope stand: inverted Leica DMi8 stand with binocular, motorized condenser, side port for camera for image acquisition in bright field (BF), DIC and fluorescence, motorized scanning table (with 127 x 83 mm scanning range, image acquisition for ROIs, TileScan, Mark&Find), with Super Z Galvo insert (z-range: 1500 µm)

 

Basic set of objectives:

    1. HC PL FLUOTAR (FLUOR) 5x/0.15 NA, WD=10 mm;

    2. HC PL FLUOTAR (FLUOR) 10x/0.30 NA, WD= 11 mm;

    3. Multiimmersion objective HC PL APO 20x/0.75 NA, IMM CORR CS2, WD=0.67 mm, DIC;

    4. HC PL APO 63x/1.20 NA W CORR CS2, WD=0.30 mm, DIC;

    5. HC PL APO 63x/1.40 NA OIL CS2, WD=0.14 mm, DIC.

(other objectives from the same producer can be used if agreed with the manager)

 

Scanning head UV-VIS-IR: Acousto-optical beam splitter (AOBS), conventional scanner (7 fps @ 512 x 512 pixels unidirectional scan) and fast 8 kHz resonant scanner (14 fps @ 512x512), spectral detector with tunable emission range 350 – 800 nm.

 

Lasers:

1. DMOD laser 405 nm (50 mW),

2. WLL2  Ar multiline laser 458 - 476 - 488 - 496 - 514 nm (65 mW), pulsed supercontinuum “white light” laser of 2nd generation with wavelengths range 470 - 670 nm (laser power at each wavelength approx. 1.5 mW), with possibility to use up to 8 lines simultaneously by means of AOBS (tunable with a step of 1 nm), with independent laser power control, with fast modulation of the laser intensity by acousto-optical transmission filter (AOTF);

3. Chameleon Ultra I (Coherent Inc., CA): Ti-Sapphire infra-red (IR) pulsed laser, tunable in the range of 690 - 1040 nm (step 1 nm), maximum output power 4 W (@ 800 nm), pulsing frequency 80 MHz, pulse width ~140 fs and with laser power controlled by attenuator and electro-optic modulator (EOM).

 

Detectors:

1. Internal (confocal or descanned) – 4 detectors:

a) 2 highly sensitive HyD spectral detectors (with maximum QE ~45% @ 500 nm) with detection range of 400 - 720 nm, with low noise and high dynamic range, photon-counting and gating capabilities, independent gain for each detector;

b) 2 PMT sensitive internal detectors (with maximum QE ~30% @ 500nm), detection range 400 – 800 nm, indepedendent offset and gain for each detector, with tunable detection range for each detector (step 1 nm, minimum detection window width: 1 nm);

2. External (non-descanned) – 3 detectors:

a) 2 reflected light detectors (RLD) - highly sensitive HyD spectral detectors (with maximum QE ~45% @ 500 nm) with detection range of 400 - 720 nm, with low noise and high dynamic range, photon-counting and gating capabilities;

b) 1 transmitted light detector (TLD)  - PMT detector for bright field mode (BF), DIC, or forward scattered SHG, suitable for image acquisitionsimultaneously with other channels (descanned or non-descanned detection);

 

On-stage incubator:

Chamber for live cell imaging (Oko-Lab) with controlled temperature by H301-T-UNIT-BL-PLUS (including objective heater), controlled concentration of CO2 (0-20%) and O2 (1-95%, hyperoxic conditions only if agreed with the manager concerningadditional O2 supply) by CO2-O2 Unit-BL [0-20;1-95], humidifier and  a H301-K-FRAME with perfusion capability, GS35-M insert (for one 1x 25x76 mm slide and up to two 35 mm Petri dishes with glass bottom) and with magnetic sample holders;

 

FLIM module:

One-channel system for time correlated single photon counting (TCSPC) Simple-Tau-150-D1 (Becker & Hickl GmbH, Berlin, Germany) – ultrafast system for single photon counting based on SPC-150 module with Magma PCI box, detector controller DCC-100 and cooled ultrafast PMC-100-1 detector based on PMT (for detection in 300 – 820 nm). Also, standard non-descanned Leica HyD detectors (2x) can be used for FLIM and PLIM data acquisition by a dedicated SPC cards for simultaneous 2-channel FLIM and PLIM (kindly loaned by the Becker & Hickl GmbH).

 

Software: LAS X for microscope stand control as well as automatized image acquisition (including single images, time, z-series as well as spectral series and their combinations) using different laser lines including sequential scans with laser re-tuning.

SW modules: Dye Finder, Live Data Mode, 3D Visualisation, Co-Localisation, MicroLab (for FRAP, FRET, FLIP experiments and ROI), FRAP Zoomer (for FRAP utilizing tandem scanner).