PoL – Physics of Life | Confocal & widefield microscopy

Confocal & Widefield Microscopy

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Zeiss LSM 980 with Airyscan 2 Nikon Spinning Disk with CSU-W1 SoRa

Zeiss LSM 980 with Airyscan 2

Zeiss LSM 980 is a point scanner confocal on a fully motorized Axio Observer 7 inverted microscope stand with a WSB scanning piezo stage (130 × 100 mm) and Z-Piezo 500 µm (1 nm resolution). The microscope is enclosed in a light-tight PECON enclosure (XLmulti S2 DARK) with temperature control (heating). It is also equipped with a stage top environmental chamber from PECON allowing temperature, CO2 and humidity control. Inserts for slides, dishes and multiwell plates are available.

Typical applications

  • Live-cell imaging and fixed samples
  • 2D and 3D cell culture
  • Zebrafish and fruit fly development
  • Long term timelapses
  • Cleared samples (RI: 1,33; RI: 1.47; RI: 1,5), using water or multi-immersion objectives.

Imaging modalities

  • Imaging of fixed sample with stitiching of large areas (tiling)
  • Widefield/Confocal/Super-resolution imaging
  • FRAP, Photo-activation
  • Spectral imaging (linear unmixing; FRET)
  • Fast imaging with Airyscan multiplex modes

Illumination

Lasers: 405/445/488/514/561/594/639/730 nm

Colibri 7 LED illumination (UV – 385/30 nm; V – 423/44 nm; B – 469/38 nm; C – 511/44 nm; G – 555/30 nm; Y – 590/27; R – 631/33 nm; FR – 735/40 nm).

Detection

Objectives

Objective Immersion Working Distance (mm) Cover Glass Thickness (mm) Best for
EC Plan-Neofluar 2.5x/0.085 air 8.8 0.17 BF, Fluorescence (VIS, UV)
EC Plan-Neofluar 5x/0.16 air 18.5 0.17 BF, DIC, Fluorescence (VIS, UV)
Plan-Apochromat 10x/0.45 air 2 0.17 BF, DIC, Fluorescence (VIS, NIR-I)
EC Plan-Neofluar 10x/0.3 air 5.2 0.17 BF, DIC, Fluorescence (VIS, UV)
Plan-Apochromat 20x/0.8 air 0.55 0.17 BF, DIC, Fluorescence (VIS)
LD LCI Plan-Apochromat 25x/0.8 multi-immersion 0.57 0 - 0.17 BF, DIC, Fluorescence (VIS, NIR-I)
C-Achroplan 32x/0.85 water 1.1 0 - 0.17 BF, Fluorescence (VIS, NIR-I, 2-P)
Plan-Apochromat 40x/0.95 air 0.25 0.13 - 0.21 BF, DIC, Fluorescence (VIS)
LD LCI Plan-Apochromat 40x/1.2 multi-immersion 0.41 0.15 - 0.19 BF, DIC, Fluorescence (VIS, NIR-I)
C-Apochromat 40x/1.2 water 0.28 0.13 -0.20 BF, DIC, Fluorescence (UV, VIS, NIR-I, 2-P)
LD C-Apochromat 40x/1.1 water 0.62 0.14 - 0.19 BF, DIC, Fluorescence (UV, VIS, NIR-I, 2-P)
C - Plan-Apochromat 63x/1.4 oil 0.14 0.17 BF, DIC, Fluorescence (UV, VIS, NIR-I, 2-P)
LD C-Apochromat 63x/1.15 water 0.60 0.14 - 0.19 BF, DIC, Fluorescence (UV, VIS, NIR-I, 2-P)

 

Detectors

  • 2x Multi-Alkali PMTs
  • Spectral GaAsP PMT detector (32 channel)
  • Extended NIR GaAsP-PMT
  • T-PMT
  • Airyscan  2 (GaAsP)
  • Axiocam 712 mono camera (CMOS)

Add-ons

AI Sample Finder – automated sample and carrier identification, facilitating sample navigation and imaging.
Autoimmersion Module – automatic application of immersion media for water immersion objectives, streamlining the workflow, allows long term imaging with water objectives.
Definite focus 3 – focus stabilization and Z-drift compensation, allows for long term imaging.

Software

ZEN 3.7.4 – Deconvolution, FRAP analysis, Experiment Designer, Tiles / Positions, Guided Acquisition, Direct Processing, Advanced Processing, Colocalization, Macro Environment.

www.zeiss.com/microscopy/en/products/light-microscopes/confocal-microscopes/lsm-980-with-airyscan-2.html

Gallery

Zeiss LSM 980 with Airyscan 2 – full view

Nikon Spinning Disk with CSU-W1 SoRa

The Nikon Spinning Disk is a microlens-enhanced Nipkow disk scanning confocal microscope on an inverted Nikon Ti2 stand, with an additional microlensed emission pinhole SoRa disk, a Mad City Labs Z-piezo (Nano-Z500) with 500 µm travel range and a motorized XY stage. The microscope is enclosed in an transparent OKO Lab H201-enclosure with temperature control (heating), and a stage top incubator with temperature, CO2 and humidity control. This system is further equiped with a photomanipulation scanner module (FRAP, Photoswitching/-conversion) and a OMS - UV 355 nm pulsed 15uJ Laser Cutting and FRAP Unit.

Typical applications

  • Fixed and live samples, 2D and 3D
  • Fast acquisition
  • Long-term live imaging
  • Large field of view (600 x 600 µm2 for 25x magnification objective)

Imaging Modalities

  • Widefield Epifluorescence
  • Spinning Disk Confocal
  • High Resolution in SoRa mode with up to 140 nm lateral resolving power (Super Resolution by Optical Pixel Reassignment.
  • Photomanipulation: FRAP, photoconversion, ablation, cutting

Illumination

Lasers: 405, 445, 488, 515, 561, 638 nm

CoolLED pE4000: 16 selectable LED wavelengths (365, 385, 405, 435, 460, 470, 490, 500, 525, 550, 580, 595, 635, 660, 740, 770 nm)

­­Detection

Objectives

Objective Immersion Working Distance (mm) Cover Glass Thickness (mm) Best for
CFI Plan Apochromat Lambda D 2X/0.1 air 8.5 0 - 0.17 BF, Fluorescence (VIS, NIR-I)
CFI Plan Apochromat Lambda D 4X/0.2 air 20 0 - 0.17 BF, Fluorescence (VIS, NIR-I)
Plan Apo λ 10x/0.45 air 4 0.17 BF, DF, DIC, Fluorescence (VIS)
CFI Plan Apo VC 20x/0.75 air 1 0.17 BF, DF, DIC, Fluorescence (VIS)
CFI Plan Apo λD 20x/0.8 air 0.8 0.17 BF, DF, DIC, Fluorescence (VIS, NIR-I)
CFI Plan Fluor 20x/0.75 multi-immersion 0.51 - 0.33 0 - 0.17 BF, DF, DIC, Fluorescence (UV, VIS, 2-P)
CFI Apo LWD λS 20x/0.95 water 0.99 - 0.9 0.11 - 0.23 BF, DF, DIC, PhC, Pol, Fluorescence (VIS, NIR-I)
CFI Plan Apo λS 25x/1.05 silicon 0.55 0.11 - 0.23 BF, DF, DIC, Pol, Fluorescence (VIS)
Plan Apo l 40x/0.95 air 0.21 - 0.16 0.11 - 0.23 BF, DF, DIC, Fluorescence (VIS)
CFI Plan Fluor 40x/0.75 air 0.66 0.17 BF, DF, Fluorescence (UV, VIS)
CFI Plan Apo λS 40x/1.25 silicon 0.3 0.13 - 0.21 BF, DF, DIC, Pol, Fluorescence (VIS, NIR-I)
CFI Apo λS 40x/1.25 water 0.2 - 0.16 0.15 - 0.19 BF, DIC, PhC, Pol, Fluorescence (UV, VIS)
CFI Apo LWD λS 40x/1.15 water 0.61 - 0.59 0.15 - 0.19 BF, DF, DIC, PhC, Pol, Fluorescence (VIS, NIR-I)
CFI Apo TIRF 60x/1.49 oil 0.16–0.07 0.13–0.19 BF, DIC, PhC, Pol, Fluorescence (VIS)
CFI SR Plan Apo IR 60x/1.27 water 0.18–0.16 0.15–0.19 BF, DIC, PhC, Pol, Fluorescence (VIS, NIR-I, 2-P)
CFI Plan Apo VC 60x/1.2 water 0.31–0.28 0.15–0.18 BF, PhC, Fluorescence (UV, VIS, 2-P)
CFI SR HP Plan Apo λS 100x/1.35 silicon 0.31–0.28 0.15–0.19 BF, DIC, Pol, Fluorescence (VIS)

 

Detectors

  • Two Hamamatsu Fusion BT sCMOS cameras (high quantum efficiency and extremely low read-out noise)

Filters

DAPI, FITC, Cy3, Cy5, Cy7 Penta LED HC Set (Semrock)

Add-ons

  • Automatic Water Immersion Dispenser

 

Software