| Table of Contents | ||
|---|---|---|
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Lasers
| Excitation Lasers | ||
|---|---|---|
| Wavelength | Type | Fluorophores |
| 405 nm | CW | DAPI, Hoechst, etc |
| White Light Laser† | Pulsed | All visible dyes from green to far red |
| STED Depletion Lasers‡ | ||
| 592 nm | CW | STED with green dyes |
| 660 nm | CW | STED with red dyes |
| 775 nm | Pulsed | STED with far red dyes |
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| Objective lenses | |||||
|---|---|---|---|---|---|
| Magnification | Type | Immersion | NA | Working Distance (mm) | Coverslip correction |
| 10x | HC PL FLUOTAR | Air | 0.3 | 11 | 0.17 |
| 20x | HC PL APO CS2 | Imm (Oil, Glycerol and Water) | 0.75 | 0.68 | 0/0.17 |
| 25x | HC FLUOTAR L VISIR | Water | 0.95 | 2.4 | 0.17 |
| 40x | HC PL APO CS2 | Oil | 1.3 | 0.24 | 0.17 |
| 63x | HC PL APO CS2 /1.4 (WD = 0.14 mm) | Oil | 1.4 | 0.14 | 0.17 |
| 100x | HC PL APO CS2 (dedicated STED objective) | Oil | 1.4 | 0.13 | 0.17 |
NOTE: There is only space for 6 objective lenses on the objective turret, so if the lens you would like to use is not on there please contact lmcb-lm-help@ucl.ac.uk
MICROSCOPE: DMI6000, inverted
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STED RESOLUTION
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XY
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~ 50 nm
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Z
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same as confocal
IMAGING MODES:
- Laser scanning confocal imaging (multi-colour, multi-point, live cell)
- STED with 592 nm depletion, up to 3 colours
FLUORESCENCE FILTER SETS: This microscope uses an Acousto-Optical Beam Splitter (AOBS) with a freely selectable emission detection range rather than fluorescence emission filter cubes.
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DETECTORS
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3x HyD, high sensitivity, 3 operating modes (standard, counting, BrightR) + time-gating
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3x PMT, one of which for transmitted light imaging
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A
INCUBATION: The microscope is fully enclosed and equipped with The Cube chamber heater and The Brick CO2 gas mixer.
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SOFTWARE
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Acquistion
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LAS X
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Deconvolution
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Huygens Professional Version 15.10.1p2
A
ADDITIONAL INFORMATION:
Microscope stand specifications and filters
The microscope is a fully motorised DMI 6000 CS inverted model with AFC auto focus, a Märzhäuser motorised scanning stage and a high speed Z Galvo focus in addition to the stand's internal focus motor. The microscope is full enclosed by a blacked-out chamber and is equipped with a Life Imaging Services Cube chamber heater and Brick CO2 gas mixer. Differential Interference Contrast (DIC) is available for the 10x, 20x and 40x lenses. Fluorescence can be viewed using the EL6000 metal halide lamp and the filter sets below.
| Note |
|---|
The EL6000 lamp and the filters in the table below are only for viewing the specimen down the eyepieces. For imaging the confocal/STED system users lasers to illuminate the specimen and a spectral detection system to filter fluorescence. |
| Filter sets | ||||
|---|---|---|---|---|
| Name | Excitation Filter | Dichroic Mirror | Emission Filter | Typical Fluorophores |
| I3 | BP 450-490 | 510 | LP 515 | FITC, GFP, Alexa 488 |
| N2.1 | BP 515-560 | 580 | LP 590 | TRITC, RFP, Alexa 568 |
| POL | - | - | - | Polariser for DIC |
| BF | Empty | Empty | Empty | Confocal or Brightfield |
| A | BP 340-380 | 400 | LP 425 | DAPI, Hoechst |
| Empty | - | - | - | Spare position |
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Detectors
The confocal/STED scanning system has three HyD high-sensitivity hybrid detectors and two standard photo-multiplier (PMT) detectors for fluorescence. It also has a transmitted light detector (TLD) for bright-field or DIC imaging. The HyD detectors are very sensitive and have a time-gating function that is critical for gated STED, so they should always be used for STED imaging.
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Additional Information
- resonant scanner (8000 Hz) for fast imaging
- anti-vibration optical table
- fully enclosed
- autofocus
- motorized XY stage
- Z-galvo