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Center for Microscopy and Image Analysis

Spinning Disk Nikon CrestOptics X-Light V3 (Irchel)

Nikon CrestOptics X-Light V3

The Nikon CrestOptics X-Light V3 is a spinning disk confocal system optimized for fast, high-resolution fluorescence imaging. This system provides high-speed optical sectioning while minimizing phototoxicity, making it well-suited for live-cell imaging and volumetric imaging of fixed specimens. With a large field of view (25 mm) and dual-camera setup, the system facilitates efficient multi-channel acquisition for quantitative imaging.

Responsible Person

responsible person

Location

University Zurich, Irchel Campus, Room Y42F66.

Training Request

 Follow this link to apply for an introduction to the microscope

The Nikon CrestOptics X-Light V3 features the following modalities:

Wide-field microscopy

 Fluorescence wide-field microscopy is a technique where the entire specimen is illuminated uniformly, causing fluorophores within the sample to emit light. This emitted fluorescence is then captured by a detector (camera). The technique offers high sensitivity and allows for real-time imaging of live cells.

Spinning disk confocal microscopy

 Spinning disk confocal microscopy enhances optical sectioning while maintaining high acquisition speeds. Spinning disk systems use a rapidly rotating disk with multiple pinholes to selectively excite and detect fluorescence from a thin focal plane. This reduces out-of-focus light, improving contrast and resolution, especially in thick specimens. The technique is particularly well-suited for live-cell imaging, as it minimizes phototoxicity and photobleaching while enabling fast multi-channel and 3D imaging. 

Differential interference contrast microscopy (DIC)

Differential interference contrast (DIC) microscopy is a microscopy technique that enhances contrast in unstained, transparent specimens. DIC utilizes polarized light split into two beams that pass through the specimen. When these beams recombine, the differences in phase shift cause interference, creating an image with high contrast and pseudo-3D effect. 

 

Technical Specifications

Microscope body

  • inverted Ti2-E body

  • Motorized encoded xy-stage and motorized encoded z-drive

  • Perfect Focus System 4

  • Large field of view - 25 mm

  • Motorized fluorescence turret, filter-wheel, condenser turret, light path switcher

  • Piezo Z-stage with 600µm travel

  • Incubator dark (temperature and humidity control)

  • Auto-Water Immersion Dispenser for all water immersion objectives

    • The Water Immersion Dispenser enhances long-term imaging with PFS and water immersion objectives by automatically maintaining the correct water level, preventing drying and overflow. 

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Software

 NIS Elements 6.1 and JOBS

Spinning disk

CrestOptics  X-Light V3:

  • Disk speed: 15000 RPM
  • Scan rate: > 1000 fps 
  • Spinning disk geometry: 50 µm diameter and 250 µm spacing

Light Sources
  • Halogen lamp for transmitted light
  • Lumencor Celesta (7 independently controlled laser light sources with ~800 mW output per laser line) 
Common Fluorophores Excitation Laser (nm)
DAPI 405 nm 
CFP 446 nm
GFP, Alexa488, Cy2 FITC 476 nm 
YFP 518 nm
TRITC, Cy3 545 nm
mCherry (on demand) 575 nm
DRAQ5, Cy5, Alexa639 637 nm
Cy7, Alexa 750 748 nm

Camera System

  • 2x Teledyne Photometrics Kinetix

  • 16 bit cooled monochrome back-illuminated sCMOS camera

  • 3200 x 3200 pixel (6.5 x 6.5um pixel size)

  • QE: 96 %

  • dynamic range 16 bit

  • Full Frame Speed 88fps (max 498fps)
  • Quality Scan 83 fps @ 16bpp
  • Readnoise 1.6e-,
  • WellDepth 15000e
  • Dark Current 1.03e-/p

Environmental control

  • Okolab Cage Incubation with Temperature, and Humidity Control

    • Temperature Range: from 3°C above ambient to 60°C
    •  At 37°C relative humidity can be regulated in the range 50% to 95%

  • CO2 control and Oxygen Controller for Hypoxia

    • CO2 range: 0-10%; O2 range: 0-1.0%
      • CO2 Accuracy: ± 0.1
      • O2 Accuracy: ± 0.02% at 1% O2

Accessories
  • Okolab Sample Holders for Wellplates, 35 mm dishes, 1x and 4x Slides

Available Optics
Name Magnification NA Immersion WD (mm)
CFI Plan Apochromat Lambda D   2x                            0.1 Air 8.5
CFI Plan Apochromat Lambda D  10x 0.45                Air 4.0
CFI Apochromat LWD Lambda S 20x 0.95 Water 0.99–0.9
CFI Apochromat Lambda S 40x 1.25 Water 0.2–0.16
CFI Plan Apochromat VC  60x 1.2 Water 0.31–0.28
CFI SR HP Apochromat Lambda S  100x 1.35 Silicone Oil 0.31–0.28

DIC = Differential Interference Contrast

Available Filters

 

S-416906 penta-bandpass: Single EM-Celesta DA/FI/TR/Cy5/Cy7 (DAPI / FITC / TRITC / Cy5 / Cy7)

S-416976 V3-Celesta CFP/YFP

  
Fluorochromes EXCITATION FILTERS in the X-Light v3 scanhead DICHROIC MIRRORS in the X-Light v3 scanhead EMISSION FILTERS in front of the camera
DAPI 391 441 438/24
CFP 449 471 485/20
GFP, Alexa488, Cy2 FITC 477 511 511/20
YFP 520 539 560/25
TRITC, Cy3 549 593 595/31
DRAQ5, Cy5, Alexa639 639 684 685/40
Cy7, Alexa 750 741 817 multiband 421/511/593/684/817

User Guide

 Guides - Nikon Spinning disk

Links & Literature

Sample preparation

Resolution calculator
ibiology - Optical sectioning and confocal microscopy

Spinning Disk Confocal Microscopy

Spectra viewer

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