BioOptics - Light Microscopy

Inverse confocal microscope with two VSDs (Variable Secondary Dichroic) allowing for finer spectral adjustment and spectral Airyscan imaging. Two highly sensitive GaAsP-PMTs for high sensitivity + Airyscan 2 detector (32 channel GaAsP) for super-resolution imaging or high-speed acquisition (Multiplex mode with up to 4x parallelisation of detection), transmitted light PMT. AI Sample finder for automated sample recognition. Motorized XY stage with Z-Piezo top plate, fully incubated. Widefield illumination: 4-channel Zeiss Colibri LED, halogen lamp, Axiocam 305 for sample overviews. (room 1.223)

Objectives

• EC Plan-Neofluar 2.5x/0.085, working distance (WD) 8.8 mm
• Plan Apochromat 10x/0.45, WD 2.1 mm
• Plan-Apochromat 63x/1.4 Oil DIC, WD 0.19 mm
• LD LCI Plan-Apochromat 40x/1.2 Multi-Immersion, WD 0.41 mm
• LD C-Apochromat 63x/1.15 Water, WD 0.6 mm with Auto-Immersion

Lasers

• 405 nm
• 488 nm
• 561 nm
• 640 nm

Inverse confocal microscope with 2 PMT channels (up to 2048 x 2048 pixels), transmitted light PMT and motorized stage. Spectral imaging via sequential acquisition. Multipositioning, tiling and online stitching. Widefield illumination: halogen lamp, CoolLED pE-2 (room 1.223)

Objectives

• EC Plan-Neofluar 10x/0.3, working distance (WD) 5.2 mm
• Plan-Apochromat 20x/0.8, WD 0.55 mm
• LD LCI Plan-Apochromat 25x/0.8 Imm Corr (oil, glycerol, water immersion), WD 0.57 mm
• Plan-Apochromat 40x/1.3 Oil DIC, WD 0.21 mm
• Plan-Apochromat 63x/1.4 Oil DIC, WD 0.19 mm
• LD C-Apochromat 40x/1.1 W Corr (water immersion), WD 0.62 mm

Lasers

• 405 nm (5 mW)
• 488 nm (10 mW)
• 555 nm (10 mW)
• 639 nm (5 mW)

Technical specifications

Startup and shutdown guide

Inverse super-resolution microscope for lattice SIM, 3D SMLM (PALM/STORM), TIRF and laser widefield imaging. Apotome mode for optical sectioning of thick specimen. Dual-camera port with 2 sCMOS cameras for fast acquisitions. Lasers to image dyes from blue to far red. Multi-positioning (Piezo-driven XY stage with Z-Piezo insert) and hardware autofocus. Environmental box for live imaging with temperature and CO₂ control. Widefield illumination: Excelitas Xylis (white LED), halogen lamp. Processing PC for offline image processing (room 1.723)

Objectives

• EC Plan-Neofluar 10x/0.3 M27, working distance (WD) 5.2 mm
• Plan-Apochromat 40x/1.4 Oil DIC M27, WD 0.13 mm
• C-Apochromat 63x/1.2 W Corr M27, WD 0.28 mm
• Plan-Apochromat 63x/1.4 Oil DIC M27, WD 0.19 mm
• alpha Plan-Apochromat 63x/1.46 Oil Corr M27, WD 0.10 mm
• alpha Plan-Apochromat 100x/1.46 Oil DIC M27, WD 0.11 mm

Lasers

• 405 nm (50 mW)
• 488 nm (100 mW)
• 561 nm (100 mW)
• 642 nm (150 mW)

Technical specifications

Environmental controls

Start up Shut down

SIM Data Processing

Setting up TIRF

Setting up Lattice SIM

Data size, handling and transfer

System for high-throughput live imaging and screening. For multiwell plates (glass or plastic bottom), dishes and chamber slides. Hardware autofocus, autocorr objectives (50x with water immersion), magnification changer (up to 2x), sCMOS camera, 7-color LED for fluorescence, IR-LED for transmitted light and phase gradient contrast, temperature/CO₂ control. Python interface for real-time image analysis and custom workflows (room 1.320)

Objectives

• Plan-Apochromat 5x/0.35, working distance (WD) 5.1 mm
• Plan-Apochromat 20x/0.7 autocorr, WD 2.2 mm
• Plan-Apochromat 50x/1.2 W autocorr, WD 0.84 mm

Technical specifications

Startup and shutdown

Working with ibidi multiwell µ-slides

Upright point scanning microscope with 2 avalanche photodiode detectors for dual-channel 2D STED (orange, dark red) or conventional confocal imaging of 3 dyes (green, orange and dark red). Piezo-driven z-focus for 3D acquisition, manual XY stage for standard slides. Widefield illumination: halogen lamp, CoolLED pE-2 (room 1.723)

Objectives

• EC Plan-Neofluar 10x/0.3, working distance (WD) 5.2 mm
• EC Plan-Neofluar 40x/1.3 Oil, WD 0.21 mm
• alpha Plan-Apochromat 100x/1.46 Oil DIC, WD 0.11 mm

Lasers

• 488 nm
• 561 nm
• 640 nm
• 775 nm STED depletion laser (1.25 W)

Recommended dyes (maximum resolution in brackets)

• Abberior STAR RED (41 nm, a very reliable dye), ORANGE (54 nm), 580 (60 nm), 600 (72 nm), 635 (42 nm), 635P (41 nm),
• Alexa Fluor Plus 594 (60 nm), 647 (30 nm)
• ATTO 590 (60 nm), 633 (42 nm), 647N (42 nm)

Technical specifications

Quick guide

Inverse spinning disk microscope unit for fast pseudo-confocal imaging, preferably for live applications at ambient temperature. Fast sCMOS and high-sensitivity EM-CCD camera. Motorized XY stage with Piezo Z-drive and real time controller for fast 3D imaging, point scanner for FRAP/photoactivation with all available laser lines, stage-top temperature control and a CherryTemp heater/cooler for fast temperature shift experiments (10-45°C). Widefield illumination: SpectraX LED lamp, white LED (room 1.223)

Objectives

• A-Plan 5x/0.12, working distance (WD) 10.1 mm
• EC Plan-Neofluar 20x/0.50, WD 2.0 mm
• LD LCI Plan-Apochromat 25x/0.8 Imm Corr (oil/glycerol/water), WD 0.57 mm
• Plan-Apochromat 63x/1.4 Oil DIC, WD 0.19 mm
• LCI Plan-Neofluar 63x/1.3 Imm Corr DIC (glycerol/water), WD 0.17 mm
• EC Plan-Neofluar 100x/1.30 Oil Iris, WD 0.2 mm

Lasers

• 405 nm laser diode (120 mW)
• 488 nm laser diode (100 mW)
• 561 nm solid state laser (200 mW)
• 640 nm laser diode (150 mW)

Technical specifications

Startup/shutdown guide

Hardware autofocus

Relative stage positions

CherryTemp user guide

Importing images in ImageJ

Importing ROIs in ImageJ

Inverse spinning disk microscope (Nikon Ti2E double-deck stand) for fast pseudo-confocal imaging, preferably for long-term live applications. Fast high-resolution sCMOS or sensitive EM-CCD camera, multi-positioning and full environmental control. Pulsed 355 nm laser for nanodissection, all lasers available for photomanipulation (FRAP, photoactivation). Widefield illumination: 8-color SPECTRA-III LED, white LED for transmitted light (room 1.318).

Objectives:

• CFI Plan Apo Lambda 20x/0.75, working distance (WD) 1.00 mm
• CFI Plan Apo 40x/1.25 LambdaS, WD 0.3 mm (silicone immersion)
• CFI Plan Apo Lambda 60x/1.42 Oil, WD 0.15 mm
• CFI Plan Apo VC 60x/1.20 WI, WD 0.28-0.31 mm (coverslip thickness correction; water immersion)
• CFI Plan Apo Lambda 100x/1.45 Oil, WD 0.13 mm
• CFI Plan Fluor 100x/1.30 Oil Iris, WD 0.16 mm (UV-transmissive for use with the 355 nm laser)

On request:

• CFI Plan Apo Lambda 40x/0.95, WD 0.25-0.16 mm (coverslip thickness correction)

Lasers:

• 405 nm laser diode (140 mW)
• 488 nm laser diode (150 mW)
• 561 nm solid state laser (200 mW)
• 640 nm laser diode (200 mW)
• 355 nm passively Q-switched pulsed ablation laser (16 mW average power)

Technical specifications

Startup and shutdown guide

Hardware autofocus

Relative stage positions

Selection of the VisiView file format (STK or OME-TIFF)

Inverse widefield microscope unit with solid state LED light source and filter sets to image all conventional dyes. 3-color TIRF and single-point 405 nm FRAP option. Equipped with a 4 Megapixel sCMOS camera for fast high-resolution imaging, sensitive EM-CCD for TIRF and low-light applications, or color CMOS camera. Motorized stage for 5D-data acquisition, full environmental control for long-term live imaging. Microfluidics device for mammalian cells, yeast and bacteria (room 1.223)

Objectives

• UPlanSApo 4x/0.16, working distance (WD) 13.0 mm
• UPlanSApo 10x/0.4, WD 3.1 mm
• UPlanSApo 20x/0.75, WD 0.6 mm
• UApo/340 40x/1.35 Oil Iris, WD 0.1 mm
• PlanApo N 60x/1.42 Oil, WD 0.15 mm
• UApo N 100x/1.49 Oil TIRF Corr (cover glass thickness correction), WD 0.1 mm

Lasers

• 405 nm (100 mW laser diode, TIRF or FRAP)
• 488 nm (100 mW laser diode, TIRF)
• 561 nm (100 mW DPSS laser, TIRF)

Technical specifications

Startup and environmental control

TIRF guide

Focus maps

Inverse widefield microscope in Sedat setup equipped with a 4-Megapixel sCMOS camera for fast fluorescence and brightfield imaging. 7-color solid state light source for fluorescence imaging with Köhler and critical illumination, respectively, white LED for brightfield, DIC optics. Motorized XY stage, hardware autofocus and environmental chamber for temperature and CO₂ control, onboard deconvolution (room 1.320)

Objectives

• UPlanSApo 10x/0.4, working distance (WD) 3.1 mm
• UPlanXApo 40x/0.95 Corr (cover glass thickness correction), WD 0.20 mm
• UPlanXApo 40x/1.4 Oil, WD 0.13 mm
• UPlanSApo 60x/1.3 Silicone Corr (cover glass thickness/temperature correction), WD 0.3 mm
• UPlanXApo 60x/1.42 Oil, WD 0.15 mm
• UPlanSApo 100x/1.4 Oil, WD 0.13 mm
   
• UApo/340 20x/0.75, WD 2.15-2.89 mm (on request)  

Technical specifications

32-bit deconvolutions

Flat-field correction

Trainee's package

This microscope is no longer at the facility, if you need any information on its usage, contact Alexander Dammermann.
 

Inverse widefield epifluorescence microscope unit with 1.3 Megapixel CCD camera, 7-color LED for fluorescence, white LED for transmitted light. XYZ-scanning stage for 5D acquisitions, onboard deconvolution algorithms fitted to the optical path of the system. Optional: critical fluorescence illumination for dim samples (room 1.320)

Objectives

• UPlanSApo 20x/0.85 Oil, working distance (WD) 0.17 mm
• UApo N 340 40x/1.35 Oil, WD 0.1 mm
• PlanApo N 60x/1.42 Oil, WD 0.15 mm
• UPlanSApo 100x/1.4 Oil, WD 0.13 mm

Technical specifications

Deconvolution with border rolloff

Five microscopes (one on each floor and a teaching microscope) in the Perutz main building have been upgraded to state-of-the-art technology. They host high-end CCD cameras, they are fully equipped for standard fluorescence/brightfield imaging and are motorized to a large extent. Irmgard Fischer is the responsible person for these microscope. She helps in training, troubleshooting and maintenance/service. It is mandatory to follow the "in-house"-specific administrative rules!

Irmgard Fischer, room 4.621/5.506, phone ext. 52866, mobile 0660/7681381

2^nd floor (room 2305) - Zeiss Observer Z1, inverted
3^rd floor (room 3517) - Axio Observer Z1, inverted
4^th floor (room 4407) - Axio Imager Z2, upright
5^th floor (room 5423) - Axio Imager M2, upright
Teaching (room 5519) - Axio Observer Z1, inverse

Acquifer HIVE acquistion server with 10 TB storage space (RAID 5), Intel Xeon E5-1650 v3 CPU (3.50 GHz), 128 GB RAM and PNY NVIDIA Quadro P1000 GPU (4 GB RAM). Connected microscopes (10 Gb/s):

• Zeiss LSM 980
• Zeiss LSM 700
• Zeiss Celldiscoverer 7
• Zeiss Elyra 7 with processing PC
• Visitron Spinning Disk
• Visitron Live Spinning Disk/Nanodissection Unit
• Olympus cellSens

Access instructions

4U Intel Dual-CPU RI2424 server with 24 TB storage space (RAID 5), 2x Intel Xeon Bronze 3104 (1.70 GHz), 64 GB RAM. Connected microscopes:

• Deltavision Epifluorescence Microscope (10 Gb/s)
• Deltavision ULTRA Epifluorescence Microscope (1 Gb/s)

Secure central repository for microscopic images for research groups. Allows to organize, share, search for, view and analyze data using a web interface. Over 140 image file formats are supported

OMERO documentation

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Fiji/ImageJ timestamp reader plugin for Bio-Formats metadata
Imports timestamps from the following file formats into ImageJ/Fiji:

• Visitron VisiView (version 4.0 or higher, .nd/.stk/.tif)
• Olympus cellSens (version 2.0 or higher, .vsi),
• Deltavision Acquire Ultra (version 1.0 or higher, .dv)
• Zeiss ZEN Blue (version 3.0 or higher, .czi)

Download plugin | Instructions | Source code

GUV Profiler
Fiji plugin to analyze the fluorescence intensities of giant unilamellar vesicles (GUVs)

Download plugin | Instructions | Source code

• Stereomicroscope Zeiss Stemi 2000 (room 1.320)
• Stereomicroscope Leica S6E (room 1.318)
  • Tissue culture incubator (room 4.616; Vbc5-level E4) - Tissue Culture Rules
• Yeast 30°C incubator (room 1.320)

Please contact the facility staff: lightmicroscopy@mfpl.ac.at​​​​​​​