Equipment in iCALM

The laser beam illuminates the sample from the side and excites fluorophores along a single line inside of the specimen. An optically sectioned image is recorded by rapidly scanning an entire plane in the specimen and detecting the fluorescence at a right angle to the illumination axis. This technique reduces photobleaching and phototoxic effects in live samples, allowing for long-term imaging in real time in 3D and 4D.

The system enables ultra-low bleaching and low phototoxicity with ~ 1000-10000 higher sensitivity than confocal Laser Scanning Microscopy. In addition, temperature and CO2 regulation allows live cell imaging.  Data processing is carried out by software version: ZEN 2014 SP1 (black edition).

Technical specifics

• Filter modules

- DAPI-GFP / DAPI-Cy3 / GFP-Cy3 / GFP-mCherry / LBF 405/488/561

• Solid statel lasers

- excitation at 405 nm (20 mW); at 488 nm (30 mW); at 561 nm (20 mW)

• Cameras

- 2 x Lightsheet detection module Z.1 “Standard” (D)

• Objective lenses

-10x/0.3 Air (illumination optics)

- 20x/0.8 Air (detection optics)

- 63x/1.0 Oil (detection optics)

SIM is an easily accessible super-resolution technology that uses standard fluorophores and yields up to 8-fold improvement in voxel resolution.  Single molecule localization techniques (such as PALM and STORM) generally require use of unusual labeling techniques, but can yield more than ten-fold lateral resolution improvement. Note that imaging is limited to the area close to the coverslip.

Technical specifics

• Microscope
  - Axio Observer for super-resolution microscopy (inverted)
  - Motorized stage: Scanning table 130x100 DC (D)
• Light source for fluorescence excitation
  - X-Cite 120 Q
• Filter set
  - GFP + mRFP + Alexa 633
• Diode lasers
  - exitation at 405 nm (50 mW) / at 488 nm (100 mW) / at 561 nm (100 mW) / at 642 nm (150 mW)
• Emission filters
  - BP 420-480 + LP 750  /  BP 495-575 + LP 750  /  BP 570-650 + LP 750  /  LP 655
• Cameras
  - sCMOS pco SIM
  - EM-CCD Andor PALM
• Objective lenses
  - EC Plan-Neofluar 10x/0.3 Air
  - C-Apochromat 63x/1.2 W Korr
  - Plan-Apochromat 63x/1.4 Oil DIC
  - α Plan-Apochromat 100x/1.46 Oil DIC
  - α Plan-Apochromat 100x/1.57 Oil DIC Korr Elyra

The Cell Observer is also equipped with temperature and CO2 regulation for live cell imaging of fixed and life cells. Sensitive, gentle live cell imaging is possible by combining fast switching LEDs ranging from UV (365 nm) to red (625 nm) with a sCMOS camera (Axiocam 702 Mono). Data processing is carried out by Software version: ZEN 3.2 (blue editon).

Technical specifics

• Microscope
  - Axio Observer.Z1
  - Yokogawa Spinning Disk unit CSU-X1A 5000 dual cam

• Light source for fluorescence excitation
  - Colibri LEDs 365 nm, 470 m, 555 nm, 625 nm
  - YHXP 120 V (metal halide)

• Filter sets
  - GFP: EX 450-490, BS 495, EM 500-550
  - DsRed: EX 538-562, BS 570, EM 570-640
  - DAPI: EX 359-371, BS 395, EM 397- ∞
  - DAPI//FITC/Rh/Cy5: EX 357-393/473-496/545-562/620-642, BS 400//503/569/650, EM 407-412/511-
  528/577-602/658-704
  - CFP/GFP/DsRed: EX TBP 406+489+561, BS TFT 427+503+578, EM TBP 459+525+608
  - BP 420-480 + LP 750  /  BP 495-575 + LP 750  /  BP 570-650 + LP 750  /  LP 655
• Lasers
  - Diode: 405 nm (50 mW) / 445 nm (20 mW)  / 514 nm (50 mW) / 561 nm (40 mW) / 638 nm (30 mW)
  - OPSL laser: 488 nm (100 mW)
• Emission modules
  - QDM dichroic 405/488/561/640
  - TDM dichroic 457/514//647
  - DM dichroic 560
  - BP 450/50 emission filter
  - BP 485/30 emission filter
  - FE01-520/35 emission filter
  - BP 527/54+645/60 emission filter
  - BP 562/45 emission filter
  - BP 629/62 emission filter
  - BP 690/50 emission filter
• Objective lenses
  - EC Plan-Neofluar 10x/0.3 Air
  - LD LCI Plan-Apochromat 25x/0.8 W/Glyc/Oil
  - C-Apochromat 63x/1,20 W Korr UV-VIS-IR
  - Α Plan-Apochromat 100x/1,46 Oil DIC (UV) VIS

The Zeiss LSM 780  is also equipped with a motorized stage for screening slides, tile scanning and point visiting. Imaging of cells is carried out typically with UV and visual fluorophores. The microscope is equipped with a fluorescence unmixing mode to separate emission from overlapping or removal of autofluorescence. Data processing is carried out by Software version: ZEN 2012 FP3 (black) Version 14.0.0.0

Technical specifics

• Microscope
  - Axio Observer Z.1 (inverted)
  - Motorized stage: Scanning table 130x100 DC (D)

• Light source for fluorescence excitation
  - HXP 120 V (D)

• Filter sets
  - EX G 365, BS FT 570, EM BP 445/50
  - EX BP 470/40, BS FT 495, EM BP 525/50
  - EX BP 545/25, BS FT 570, EM BP 605/70
• Lasers
  - Diode: 405 nm / 561 nm (30 mW) / 633 nm
  - Argon, exciting at 458, 488, 514 nm (25 mW)
  - HeNe, exciting at 594 nm
• Spectral detection LSM 780 GaAsP 32+2 PMT of fluorescence light emission
  - Selection of appropriate dyes dependent only on available laser excitation lines
• Objective lenses
  - Plan-Apochromat 10x/0.3 Air
  - Plan-Apochromat 20x/0.8 Air
  - EC Plan-Apochromat 40x/1.3 Oil DIC
  - Plan-Apochromat 63x/1.4 Oil DIC

The Zeiss Axio Imager is also equipped with a motorized stage allowing multipoint imaging, while also allowing Z-sectioning. The microscope is equipped with temperature and CO2 regulation for live cell imaging and Zeiss AxioCam (HRm + HRc). Data processing is carried out by Software version: ZEN (blue edition)

Technical specifics

• Microscope
  - Axio Imager.Z2 (upright)
  - Motorized stage: Märzhäuser SCAN 130 x 85 mm

• Light source for fluorescence excitation
  - Colibri LEDs 365 nm, 470 nm, 555 nm, 590 nm
  - HXP 120 C (metal halide)

• Filter sets
  - EX BP 470/40, EM BP 525/50
  - EX BP 530-585, EM LP 615
  - EX BP 545/25, EM BP 605/70
  - EX BP 550/25, EM 605/70
  - EX BP 370/40, EM 474/28, EX 585/35, TBP 425+527+615
• Objective lenses
  - EC Plan Neofluar 1x/0,025 Air
  - EC Plan-Neofluar 1,25x/0,03 Air
  - C-Apochromat 40x/0,45 W
  - Plan-Apochromat 63x/1.4 Oil DIC
  - Various others

The Zeiss Axio Imager is also equipped with a motorized stage allowing multipoint imaging, while also allowing Z-sectioning. The microscope is equipped with temperature and CO2 regulation for live cell imaging. Data processing is carried out by Software version: AxioVision 4.8.2

Technical specifics

• Microscope
  - Axio Observer.Z1 (inverted)
  - Motorized stage: Märzhäuser SCAN 130 x 100 mm

• Light source for fluorescence excitation
  - Colibri.2 LEDs 365 nm, 470 nm, 590 nm
  - HXP 120 C (metal halide)

• Filter sets
  - G 365, FT 395, BP 445/50
  - BP 470/40, FT 495, BP 525/50
  - BP 550/25, FT 570, BP 605/70
  - BP 640/30, FT 660, BP 690/50
  - BP 370/40, 474//28, 585/35, TFT 395+495+610, TBP 425+527+615
• Objective lenses
  - Plan-Apochromat 5x/0.16 Air
  - Plan-Apochomat 10x/0,3 Air
  - LD EC Epiplan-Neofluar 20x/0,22 Air DIC
  - LCI Plan-Neofluar 63x/1,3 DIC Imm Korr
  - Plan-Apochromat 100x/1.4 Oil
  - Various others