Lighthouse Update

Lighthouse Core Facility

Marie Follo, Ph.D.

• 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

Cell Sorters BD FACSAria III / BD FACSAria Fusion

• 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

Cell Sorters MoFlo Astrios EQ (CCI)

• 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)

Flow Cytometers BD LSR Fortessa Analysers

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

• Installed mid-2017

• Lasers: 405nm, 488nm, 638nm

• Spectral unmixing

• 20+ colors

• User-friendly software

Flow Cytometers Sony SP6800 ZE Spectral Analyzer

Flow Cytometers Sony SP6800 ZE Spectral Analyzer

• As long as spectra differ in shape - colors can be distinguished

• Possibility of 20+ colors simultaneously

Flow Cytometers Sony SP6800 ZE Spectral Analyzer

• GaAsP detectors - very sensitive, single photon counting

• 32 channel spectral scans - fast multicolor imaging

• Motorized XY stage - tiled scans, multipositional time series

• AiryScan detector

Microscopes Zeiss LSM 880 AiryScan

Folie 9 05.03.2018 Korobchevskaya, 2017

Zeiss LSM 880 AiryScan

Microscopes

Resolution:

XY: 120 nm

Z: 350 nm

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

Microscopes Zeiss LSM 880 with AiryScan

• 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)

Microscopes Zeiss LSM 710 Confocal (CCI)

Microscopes Olympus Scan^R

High Content Screening Station

• Widefield system

• Flexible choice of fluorochromes

• Fully automated

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

• 5 Lasers (405,488,546,594,633)

• Filter-free

• Flexible choice of colors

Microscopes

Leica SP2 AOBS Confocal (retiring)

Zeiss AxioImager Upright Microscope

• New and sensitive color and mono cameras

• IHC or fluorescence imaging

• 1.25x, 5x, 10x, 20x, 40x, 63x, 100x

• Slides

Microscopes

Zeiss AxioObserver / Apotome (CCI)

Zeiss Discovery v.8 Stereo Microscope

• Inverse Microscope

• Fluorescence and brightfield

• Mini climate chamber (live cell imaging)

• Dishes, plates or slides

• Now with digital color camera and Zen software

• Brightfield / darkfield / transmitted light

• Dishes, plates, slides

• Overview / larger objects

• Fluorescence?

Microscopes Image Analysis Workstations

• AutoQuant (Deconvolution)

• Metamorph (Image Analysis)

• Zen blue / black

• ScanR Analysis software, Spotfire

• Cell Profiler, Fiji/Image J

• Imaris (new) - 3D Reconstruction and Analysis

Deconvolution

3D Analysis

• 384-well plates

• Low sample volumes (10-12 µL)

• Up to 3 reference genes simultaneously

QPCR Roche LightCycler 480 (LC480) - 2 machines

Analysis Software • Beacon Designer - licensed primer/probe design software

• LightCycler 480 offline software

• QuantaSoft - ddPCR offline software

Droplet Digital PCR (ddPCR) Bio-Rad QX100 ddPCR System

• Absolute quantification

• Rare event detection

• Detects small-fold changes (+/-10%)

• No standard curve required

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

Lighthouse - UKL/Med I Website http://www.core-facility.de

Lighthouse UKL Website - Internet http://www.core-facility.de

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

Lighthouse Website - Internet University Website

Our Team

Marie Follo

Klaus Geiger

Dieter Herchenbach

Jan Bodinek - CCI

Alexandra Schwierzok - CCI

Folie 24 05.03.2018