Institute of Molecular Genetics of the Czech Academy of Sciences
The inverted microscope DMi8 with laser scanning confocal head Leica TCS SP8 has differential interference contrast available for all objectives. The confocal head is equipped with two fluorescence PMT detectors and two highly sensitive HyD detectors with time resolved gating function. System is equipped with STED 3X module and 660 nm continual and 775 pulse depletion lasers, and enables super-resolution imaging by stimulated depletion of emission method (STED) with up to 35 nm lateral, 130nm axial resolution.
| Methods | Confocal scanning fluorescence imaging with STED superresolution module Transmitted light imaging Brightfield Time-lapse experiments Multi-positions experiments |
| Illumination | lamp for transmitted light Leica EL6000 with HXP 120W/45C VIS Hg lamp – for fluorescence excitation lasers: – pulsed white light laser (WLL2) – 470-640 nm with 1nm step, 8 parallel laser lines, 1.5 mW per each – continuouse 405 nm DMOD Flexible laser – 50 mW depletion lasers: – 660 nm CW STED, >1.5 W 775 nm pulse STED, >1.5 W |
| Objectives | HC PL APO 10x/0.40 DRY CS; FWD 2.2; CG 0.17 | BF, POL, DIC HC PL APO 100x/1.4 OIL STED WHITE CS2; FWD 0.13; CG 0.17 | BF, POL, DIC |
| Eyepiece filtercubes | DAPI (A) (Ex: 360/40; DM 400; Em: LP 425) FITC (I3) (Ex: 470/40; DM 510; Em: LP 515) RHOD (N2.1) (Ex: 537/45; DM 580; Em: LP 590) Cy5 (Y5) (Ex: 620/60; DM 660; Em: 700/75) |
| Detectors | 2x photomultiplier tube (PMT) 400-800 nm 2x supersensitive hybrid detectors (HyD) 400-720 nm 1x transmitted light detector |
| Confocal head | acousto-optical tunable filter (AOTF) acousto-optical beam splitter (AOBS) standard scanner (1-1800 Hz line frequency) maximum scanner resolution 8192×8192 pixels spectrally tunable detection hardware zoom 0.75x-48x |
| Stage | motorized microscope stage with Super Z-galvo scanning insert for fast and precise Z movement HW autofocus control |
| Additional equipment | antivibration table incubation chamber Okolab (CO2, temperature, humidity) |
| Software | LAS X 64bit – with LAS AF SP8 Dye Finder – 3D visualization, deconvolution and colocalization module |
| Location | room no. 0.174 (Red) |
| Phone | ext. 2433 |
| Booking | Calpendo (“STED”) |
The DeltaVision OMX imaging platform is an advanced multi-mode, super-resolution inverted microscope system which offers super-resolution imaging using 3D Structured Illumination (3D-SIM) and Dense Stochastic Sampling Imaging (DSSI) – Localization Microscopy. The Blaze SIM Module offers dynamic high speed 3D-SIM suitable for live cell super-resolution imaging. Blaze incorporates a proprietary, ultra-fast, structured illumination module, advanced optical platform design and the latest high-speed camera technologies. The system can be used also for super-fast widefield acquisition and photo-kinetic experiments (FRAP).
Methods
Illumination
lamp for transmitted light
InsightSSI (Lumencore) illumination module – for WF imaging; standard shutters, open/close time ~ 1.8 ms excitation lasers – for 3D SIM, TIRF and localization; high speed galvanometer controlled shutters, open/close time ~ 200 μs
3D-SIM resolution at different wavelengths
| Laser | Type | Power (mW) | Expected XY resolution | Expected Z resolution |
| 405 nm | diode | 100 ± 10 | 110 ± 5 nm | 340 ± 10 nm |
| 445 nm | diode | 100 ± 5 | 115 ± 5 nm | 340 ± 10 nm |
| 488 nm | OPSL | 100 ± 4 | 120 ± 5 nm | 340 ± 10 nm |
| 514 nm | OPSL | 100 ± 6 | 120 ± 5 nm | 350 ± 10 nm |
| 568 nm | OPSL | 100 ± 4 | 135 ± 5 nm | 350 ± 10 nm |
| 642 nm | diode | 110 ± 10 | 160 ± 5 nm | 380 ± 20 nm |
Objectives
PLAN APO N 60x/1.42 OIL; FWD 0.15; CG 0.17 | BF, POL, DIC (optimized)
U APO N 60x/1.49 CORR OIL TIRF; FWD 0.1; CG 0.13-0.19 & 23/37°C | BF, POL, DIC
U APO N 100x/1.49 CORR OIL TIRF; FWD 0.1; CG 0.13-0.19 & 23/37°C | BF, POL, DIC
U PLAN S APO 60x/1.3 CORR SILICONE; FWD 0.3; CG 0.15-0.19 & 23/37°C | BF, POL, DIC
InsightSSI excitation filters (for widefield applications only)
DAPI 395.5/29
FITC 477/32
mCherry/Alexa Fluor 568 571/19
Cy5 645.5/15
CFP 438/24
YFP 512.5/15
Emission filters
DAPI 435.5/31
FITC 528/48
mCherry/Alexa Fluor 568 609/37
Cy5 683/40
CFP 477.5/35
YFP 541/22
Cameras
4x pco.edge 5.5 sCMOS, Readout speeds 95 MHz, 286 Mhz, 15 bit, pixel size: 6.5 μm camera, 80 nm 60x, 48 nm 100x
Stage
XYZ nanomover and fast Z-piezo
Additional equipment
antivibration table
sample holders:
Software
OMX Acquisition control software running on OMX Master computer under Windows 7
SoftWoRx – image reconstruction, deconvolution and analysis running on the SI workstation running under the CentOS v6 (Linux)
Dense Stochastic Sampling Imaging (DSSI) algorithm for localization microscopy images
Location: room no. 0.174 (Red)
Phone: ext. 2433
Booking: Calpendo (“OMX”)
The Elyra 7 is a motorized inverted microscope equipped with a super-resolution system that uses a specialized lattice illumination pattern for 3D structured illumination (SIM) images and single-molecule localization methods (SMLM) like STORM and PALM. The lattice illumination allows for faster, gentler, and deeper imaging of live samples, and can reach up to 255 FPS during time-lapse acquisition. The unit has an incubation chamber for live imaging with controlled CO2 and temperature. The signal detection is assured by 2 sCMOS cameras which enable simultaneous detection of two different fluorophores.
| Methods | Lattice SIM, SIM2 Single Molecule Localization Microscopy (3D PALM / dSTORM) TIRF Apotome (optical sectioning) mode Transmitted light imaging Brightfield and Nomarski contrast (DIC) (automatic) Time-lapse experiments (live-cell imaging) Multi-positions experiments |
| Illumination | Halogen 12 V/100W lamp – for transmitted light Metal halide HXP 120 V lamp – for fluorescence Excitation lasers (for TIRF, SIM, SMLM): – 405 nm diode, 50 mW – 488 nm OPSL, 500 mW – 561 nm OPSL, 500 mW – 642 nm OPSL, 500 mW |
| Objectives | EC Plan-Neofluar 10x/0.3 M27 DRY | BF, POL C-Apochromat 40x/1.2 Water Corr M27 | BF C-Apochromat 63x/1.2 Water Corr M27 | BF, DIC α Plan-Apochromat 63x/1.46 Oil Corr M27 | BF, TIRF α Plan-Apochromat 100x/1,46 Oil DIC M27 | BF, DIC, TIRF |
| Eyepiece filtercubes | Laser Blocking Filter for 405/488/561/642 named: – DAPI – FITC – mCherry – Cy5 Simultaneous two-channel shooting: – GFP + mCherry – GFP + Cy5 Sequential four-channel shooting: – DAPI and mCherry + GFP and Cy5 – DAPI and GFP + mCherry and Cy5 |
| Cameras | DuoLink for a parallel imaging on two cameras: – 2x pco.edge sCMOS (version 4.2 CL HS) – thermal stabilization at 0°C / +5 °C / +7 °C – 16-bit depth – high resolution: 2048 x 2048 px – pixel size 6.5 x 6.5 µm – quantum efficiency up to 82% – exposure times from 100 µs to 20 s – maximum frame rate 100 fps |
| Superresolution modules | Lattice SIM, SIM2 Single Molecule Localization Microscopy (3D PALM / dSTORM) |
| Stage | XY Scanning Stage Piezo 130 x 100 (D): – Movement range 130 mm x 100 mm (adjustable) – Max speed 100 mm/s – 0.2 μm resolution – Repeatability +/- 1 μm – Absolute accuracy +/- 5 μm Z-piezo insert: – Movement range at least 100 μm – < 5 nm resolution – Suitable for: Standard microscopic slip: 76 x 26 mm, Petri dish 30 – 35 mm in diameter, LabTek II chamber, 55 x 24 mm |
| Additional equipment | Antivibration table 120 x 90 cm Definite Focus – holding focus to compensate Z drift Dark incubation unit (CO2, temperature < 40°C, humidity) – Imaging chamber for Z-piezo inserts PC for storage and data analysis |
| Software | ZEN 3.0 (black edition) 64-bit Modules: – Lattice SIM2/SIM2 Apotome – SMLM (PALM/dSTORM) module – 3D-PALM – Measurement – Multi Channel – Panorama – Manual Extended Focus – Image Analysis – Time Lapse – Tiles & Positions – Z-stack – Extended Focus – Autofocus – Colocalisation – Connect Entry (ZEN blue edition) |
| Location | room no. 0.174 (Red) |
| Phone | ext. 2433 |
| Booking | Calpendo (“Elyra 7”) |