Lighthouse Core Facility Lighthouse Update

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1 Lighthouse Core Facility Lighthouse Update Marie Follo, Ph.D.

2 Cell Sorters BD FACSAria III / BD FACSAria Fusion Sort speeds over 20,000 total events/sec. 4-way sorting Cell / well cloning (up to 96 wells) Index sorting (post-sort phenotyping) Aerosol evacuation system Biosafety Hood (Fusion) Multicolor Flow Cytometry: 4-5 Lasers : 375 nm / 405 nm (colinear) 488 nm 561 nm 633 nm Up to 17 colors / 19 total parameters Cuvette-based systems - high optical sensitivity

3 Cell Sorters MoFlo Astrios EQ (CCI) Sort speed >30,000 total events/sec Jet-in-air System 6-way sorting Cell / well cloning (up to 1536 wells) Index Sorting (post-sort phenotyping) Small particle detector Lasers / Parameters: 4 Lasers : [405nm, 488nm, 561nm, 633nm] 14 colors / 16 total parameters Fee for Service (MoFlo): 50 /h + 25 /set-up

4 Flow Cytometers BD LSR Fortessa Analysers Up to 40,000 total events/sec From 14 to 17 colors Different Laser/Color Combinations: 405nm, 488nm, 640nm (Frida - now with 14 colors) 351nm, 405nm, 488nm, 640nm (Dalí - CCI) 405nm, 488nm, 561nm, 640nm (Leonardo) 405nm, 488nm, 561nm, 640nm + Plateloader (Vincent) Analysis Workstations FlowJo (x2) Sony SP6800 Software - offline Kaluza (Beckman), Diva (BD) FCAP Array (BD Bead Array) ModFit (Cell Cycle) Fee for using LSR Dalí (CCI): 5 / hour

5 Flow Cytometers Sony SP6800 ZE Spectral Analyzer Installed mid-2017 Lasers: 405nm, 488nm, 638nm Spectral unmixing 20+ colors User-friendly software

6 Flow Cytometers Sony SP6800 ZE Spectral Analyzer

7 Flow Cytometers Sony SP6800 ZE Spectral Analyzer As long as spectra differ in shape - colors can be distinguished Possibility of 20+ colors simultaneously

8 Microscopes Zeiss LSM 880 AiryScan GaAsP detectors - very sensitive, single photon counting 32 channel spectral scans - fast multicolor imaging Motorized XY stage - tiled scans, multipositional time series AiryScan detector

9 Microscopes Zeiss LSM 880 AiryScan AiryScan Detector: 1.7x better resolution in X,Y,Z (~5x smaller volume) beyond the diffraction limit ("superresolution") higher sensitivity / detection (equiv. to 1.25 AU) higher resolution (equiv. to 0.2 AU) improved signal to noise low phototoxicity/bleaching compatible with all colors/dyes Resolution: XY: 120 nm Z: 350 nm Folie Korobchevskaya, 2017

10 Microscopes Zeiss LSM 880 with AiryScan

11 Microscopes Zeiss LSM 710 Confocal (CCI) Live Cell Imaging - full climate chamber XY Motor - multiwell / multipositional time series, tiled scans 32 channel spectral scans - fast, multicolor imaging Zen Blue/Black (widefield and confocal)

12 Microscopes Olympus Scan^R High Content Screening Station Widefield system Flexible choice of fluorochromes Fully automated

13 Microscopes Olympus Scan^R High Content Screening Microscope Combines aspects from: Automated microscopy Image analysis Flow cytometry-type analysis Objective and quantitative analysis Applications: Screening assays/large-scale experiments (96-well, 384-well) Slides, multi-well chambers, dishes Adherent or suspension cells Fluorescence (or IHC) Time-lapse, live cell imaging Chemotaxis Apoptosis in situ Cell cycle Morphometry Translocation/compartmentalization Tissue sections or arrays

14 Microscopes Leica SP2 AOBS Confocal (retiring) 5 Lasers (405,488,546,594,633) Filter-free Flexible choice of colors Zeiss AxioImager Upright Microscope New and sensitive color and mono cameras IHC or fluorescence imaging 1.25x, 5x, 10x, 20x, 40x, 63x, 100x Slides

15 Microscopes Zeiss AxioObserver / Apotome (CCI) Inverse Microscope Fluorescence and brightfield Mini climate chamber (live cell imaging) Dishes, plates or slides Zeiss Discovery v.8 Stereo Microscope Now with digital color camera and Zen software Brightfield / darkfield / transmitted light Dishes, plates, slides Overview / larger objects Fluorescence?

16 Microscopes Image Analysis Workstations Deconvolution AutoQuant (Deconvolution) Metamorph (Image Analysis) Zen blue / black ScanR Analysis software, Spotfire Cell Profiler, Fiji/Image J Imaris (new) - 3D Reconstruction and Analysis 3D Analysis

17 QPCR Roche LightCycler 480 (LC480) - 2 machines 384-well plates Low sample volumes (10-12 µl) Up to 3 reference genes simultaneously Droplet Digital PCR (ddpcr) Bio-Rad QX100 ddpcr System Absolute quantification Rare event detection Detects small-fold changes (+/-10%) No standard curve required Analysis Software Beacon Designer - licensed primer/probe design software LightCycler 480 offline software QuantaSoft - ddpcr offline software

18 Lighthouse Core Facility DFG RIsources Database Lighthouse is recognized as an official core facility by the DFG - listed on the DFG RIsources web page - easier for researchers to apply for grant money for facility usage

19 Lighthouse - UKL/Med I Website

20 Lighthouse UKL Website - Internet

21 Lighthouse Intranet Page MRBS Calendar Link - also accessible from outside UKL

22 Lighthouse Website - Internet University Website

23 Our Team Dieter Herchenbach Jan Bodinek - CCI Marie Follo Alexandra Schwierzok - CCI Klaus Geiger

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