Confocal & Widefield Microscopy
Source: https://physics-of-life.tu-dresden.de/research/core-facilities/pol-microscopy/technologies-systems/confocal Parent: https://physics-of-life.tu-dresden.de/research/core-facilities/pol-microscopy
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.\ FCS and FCCS – analysis of concentration and dynamics in the low molecular concentration regime.\ Dynamics Profiler – analysis of concentration and dynamics, including flow directionality in the low–medium molecular concentration regime.\ Spectral RICS – Image correlation based analysis of molecular dynamic at the cellular level.
Software
ZEN 3.10 – Deconvolution, FRAP analysis, Experiment Designer, Tiles / Positions, Guided Acquisition, Direct Processing, Advanced Processing, Colocalization, Macro Environment.
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, O2 and humidity control. This system is further equipped 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 – automatic application of immersion media for water immersion objectives, streamlining the workflow, allows long term imaging with water objectives.
- Enclosure with temperature control
- Stage-top incubator – control of temperature, humidity, CO2 and O2 (including hypoxia conditions)
- Perfect Focus System – focus stabilization and Z-drift compensation for long term imaging
Software
NIS 5.42 and NIS 6.10 – JOBS, ND Acquisition, Deconvolution, General Analysis 3, 3D Tracking, Calcium FRET, Illumination Sequence