MICC Cell Observatory Resources

Imaging

The Lightsheet enable fast and gentle fluorescence detection in 3D, and is intended for larger volumes with minimal photobleacing.

Suitable for cell cultures, plants, tissue sections or thicker samples. Enables Z compensation for even illumination.

Wide Field Microscopy (UK Bld, room 7)

Suitable fast imaging of fixed tissue cells. thin tissue sections or thin plant sections.

Fully integrated high-end automated live cell imaging system that enables high sensitivity and focus stability in live cell experiments (imaging period: days)

Offers fast and sensitive 3D imaging, utilizing WF imaging, confocal imaging with 2 different pinhole sizes (40µm for higher contrast/25 µm for higher resolution) or TIRF (total internal reflection) imaging

offers resolution up to 30nm lateral and ~100nm axial in 3D. combination of gated, spectral detection and white light laser provides excellent signal with full freedom in choice of fluorophores at the visible range

Sample Preparation
Commercial Software Licenses
Workstations
Data storage
Additional Information and Training Material

Imaging

 Light Sheet - 

Lavision “Ultra Microscope”

Suitable for morphological studies of large samples (max 10*10*50 mm) cleared using water or organic solvents (RI:1.33-1.56). provides fast 3D imaging with Z resolution of ~5µm.

  1. a. Objectives:
  2.        1. Zoom Body  (0.63 X-6.3X) WD 6mm (depends on the dipping cup). Equipped with an Olympus MVPLAPO 2X/0.5

                 Final magnification (1.3X – 12.6)

              2. Infinity corrected single lens
                  LVMI-fluor 4X/0.3 (Zoom optional to 8X) WD 5.7mm

               3.   1.3 air / dipping for overview

       b. sCMOS at 16bit

       c. Excitation/Emission filters:

Excitation filter

Emission Filter

470\40

525\50

514\10

595\40

545\25

595\40

560\40

630\75

640\30

690\50

710\75

810\90

 

Lightsheet Z1 - Zeiss

  1. Suitable for live (morphogenesis/embryogenesis, 3D culturs/ spheroids)  fixed, or water based cleared (CLARITY/ Scale /Pact)
  2. whole tissues (link to tissue clearing page).
    1. Objectives:
      excitation: 5X and 10X
      detection objectives: Air: 5X ; Water:10X-20X-40X ; Clearing:20X clearing for 1.45 Refractive index / 20X clearing for 1.38 Refractive index
    2. Multiview imaging for maximal 3D resolution, multiposition imaging
    3. Combines environmental control for prolong imaging with minimal perturbation.
  3.   d. Emission filter combinations covering fluorophores excited by the 405-633

        e. CCD at 16it

 

Leica SP8 confocal-2Photon (upright)

Suitable for cell cultures, plants, tissue sections or thicker samples. Enables Z compensation for even illumination.

      1. Excellent spectral separation and high sensitivity, combining Acuso-Optical-Tunable-Filter, spectral detection
      2. and 1 PMT and 2 HyD internal detectors. HyD are characterized with high sensitivity and low noise detection.  
      3. Chameleon Vision II coherent tunable laser, combined with 2 sensitive HyD external detectors (NDD) enables
      4. Second-Harmonic-Generation detection in 3D and 4D
      5. Objectives:
        1. HC PL FLUOTAR 10x/0.30 (air)
        2. HC PL APO 20x/0.75 CS2 (air)
        3. HC FLUOTAR L 25x/0.95 W VIS (water dipping)
        4. HC PL APO 40x/1.10 W CORR (water immersion)
        5. HC PL APO 63x/1.40 OIL CS2 (oil immersion)
        6.  HC FLUOTAR L 25x/1.00 IMM  Leica motCORR - ne=1.457 (for cleared samples)
      6. Large fields of view acquired in fast scanning mode, enable accurate navigation within the sample during high- resolution imaging
      7. Environmental control enables live imaging for longer periods, combined with fast resonant scanner, sensitive detectors and software solutions for lower phototoxicity
      8. LASX software pack, including integrated Deconvolution module
      9. Variety of optional sample holders for increased imaging stability of cleared or expanded sample

Leica DMI8 Wide-Field (Inverted)

  1. Suitable for fast imaging of fixed tissue cells, thin tissue sections or thin plant sections.
    1.  Motorized stage for large, accurate XY movements. Offers full navigation within the sample.
    2. High speed autofocus allows efficient 3D view of large areas
    3. 2 cameras optional: Leica DFC7000GT monochromatic, triggered camera (for fluorescent imaging) and DFC310FX color camera
    4. Several transmission modes: Bright Field, Differential-Interference-Contrast (DIC) imaging, Phase imaging
    5. Objectives:
      1. 5X/0.12 dry
      2. 10X/0.25 dry
      3. 20X/ 0.4 dry
      4. 20X/ 0.8 dry
      5. 40X/0.6 dry
      6. 100X/ 1.4 oil
    6. Equipped with 2 separate filters for excitation and emission wavelengths, therefore allowing different combinations of excitation (402-488-561-561-638-100%) and emission (450/525/600/700/100%).

        f. LASX software pack for analysis

 

Cell Discoverer 7-Zeiss (inverted)

Fully integrated high-end automated live cell imaging system that enables high sensitivity and focus stability in=

live cell experiments (imaging period: days)

      1. Allows automated recognition of single plate/multi-well plates and chamber slide`s parameters: material (glass/plastic),  thickness, skirt height (for maximal high-res lateral view)
      2. Objectives:
        *effective (optical) zoom 0.5X-1X-2X
        ZEISS Plan-APOCHROMAT 5x / 0.35
                                                      20x / 0.7 Autocorr Objective  
                                                      50x / 1.2 W Autocorr and Autoimmersion Objective
        *Automated water injection/removal allows smooth shift between magnifications during experiments
      3. Combination of hardware and software based focus strategies allow fast focus determination and stability over long periods
      4. Excitation LEDs : 385, 420, 470, 511, 567, 590, 625. The system is equipped with high efficiency multibandpass filtersets. Other contrast options include: bright field, DIC and phase gradient
      5. Two cameras: Zeiss Axiocam 506 mono and s CMOS 702

             6. Possibility for smart experimental design: Use Python scripts to customize and automate your workflows. • Integrate external image analysis applications into    
                 your workflows.
                 • Exchange image data with external programs like ImageJ, Fiji, MATLAB, KNIME or Python.
                 • Use feedback for smart experiments.

Aandor Dragonfly on a Leica DMi8 microscope (inverted)

Offers fast and sensitive 3D imaging, utilizing WF imaging, confocal imaging with 2 different pinhole sizes (40µm for higher contrast/25 µm for higher resolution) or TIRF (total internal reflection) imaging

    1. Excitation wavelengths (nm): 405-445-488-514-561-633
      Emission filters: 450-525-600-647
      *dichroic mirror deflects between GFP-RFP/CFP-YFP detection
    2. Objectives:
      1. 5x/0.15 air
      2. 10X/0.1 air
      3. 25X/0.95 w
      4. 40X/1.1 w
      5. 63X/1.3 G
      6. 63X/1.4 O
      7. 100X/1.47 TIRF
    3. Multi-color fast acquisition as well as simultaneous dual-color imaging for all the above modes
    4. Finite-burst experiment design allows 2D dual color simultaneous imaging at 100 fps (2048*2048 format)
    5. Enables fast, accurate navigation within large areas (16*14mm)
    6.  2 Zyla 4.2 camera with flexible AOI (area of interest) definition, binning (for enhanced sensitivity) and zoom options between 1X-1.5X-2X
    7. Mosaic for simultaneous, accurate illumination (photobleaching/photoactivation) of multiple areas on the sample
    8. Environmental control and auto-focus for live experiments, stage holder fitted to 35mm dishes, multi-well plates and slides

        i. Fusion software offers GPU-enhanced deconvolution and instant Imaris view

Leica SP8-STED (inverted)

Offers resolution up to 30nm lateral and ~100nm axial in 3D. combination of gated, spectral detection and white light laser provides excellent signal with full freedom in choice of fluorophores at the visible range

  1. Lasers: 405, Ar, WLL (adjustable exc), STED dep lasers (594,660,775)
  2. Objectives:
    1. 10x/0.4 air
    2. 20x/0.75 air
    3. 40x/1.1w
    4. 63X/1.2w
    5. 63/1.4o
    6. 86x/1.2 w (STED)
    7. 93X/1.3 glycerol  (STED)
    8. 100X/1.4 o (STED)
  3. 3HyD Detectors , 2PMTs
  4. Scanners- galvo/resonant
  5. Falcon -Life time imaging

Sample Preparation

Active clearing

  1. Tissue Clearing
    we provide CLARITY based protocol suitable for different samples, reagents, suitable imaging platforms and guidance during experimental set-up
      1. Active Clearing: SmartClear II- Lifecanvas
        1. Based on stochastic electrotransport (Kim et al, PNAS, 2015) providing shorter clearing times without tissue damage or fluorescence quenching
        2. Pre-prepared clearing buffers, non-toxic, allow controlled and comparable process
        3. All clearing parameters (time, temperature, voltage, cycle shift) can be controlled and adjusted
        4. Various holder configuration for optimal clearing penetration for different sample size and composition
      2. Passive clearing: EasyClear
        1. Allows up to 10 independent samples (50ml tube size) to be cleared at different temperature (range: 30-90 degC)
        2. Gentle shaking to maintain morphology of delicate samples, with adjustable shaking angle and speed
      3. Index matching solution- EasyIndex
        Suitable for clarity based protocols and others, matches the refractive index of the sample to RI=1.46, allowing good fluorescence preservation

Commercial software licenses

Booking is done through Internal Services => Adavanced Light Microscopy.

All the above commercial software are available on the IA workstations, as well as: Fiji, Matlab and selected open-source software (eg. Ilastik, ICY, CellProfiler, Vaa3D). Other open-source software can be installed on demand.

Workstations

  • 2 IA workstations are available at Levine #201 . Spec: Intel Xeon CPU E5-2620 @ 2.0 GHz, 64 GB of memory, NVIDIA Quadro 4000 graphic card. Booking is done through Internal Services => Biomaging Queue.
  • 3 very high performance IA Workstations dedicated to processing of Lightsheet and other very big datasets at Levine #209.
  • Analysis 30T: 2*Intel Xeon E5-2690 CPU @ 2.6 GHz, 256GB of memory, Nvidia GeForce GTX TITAN X graphic card
  • Analysis 80T: 2*Intel Xeon E5-2690 CPU @ 2.6 GHz, 256GB of memory, AMD Radeon R9 200 graphic card
  • Analysis 20T: 2*Intel Xeon E5-2667 CPU @ 3.2 GHz, 512GB of memory, Nvidia Quadro M6000 24 GB graphic card, dedicated fast SSD disk for program cache

Booking is done through Internal Services => Adavanced Light Microscopy.

All the above commercial software are available on the IA workstations, as well as: Fiji, Matlab and selected open-source software (eg. Ilastik, ICY, CellProfiler, Vaa3D). Other open-source software can be installed on demand.

Data storage

In order to enable imaging data backup and availability the cell observatory maintains two types of data servers -

BioImg Server – Central long-term Storage Server for raw imaging data. Automatically collect imaging data from multiple campus wide imaging platforms. Users can document, search, access and share data from anywhere on campus. (BioImg step by step guide)

ESS server - A specially defined large storage with fast communication for data retention analysis and archiving, Supports the large data sets produced by lightsheet microscopes