Leica TCS SPE and SPE2
The Leica TCS SPE and SPE2 are almost identical basic "personal" confocal microscopes mounted on DM2500 upright microscope stands. They are designed principally for scanning samples that have been fixed and mounted on slides but they can also be used to image living samples if they can be mounted using gaskets and imaged through coverslips (e.g. Drosophila pupae). Alternatively we have a set of water dipping lenses for use with samples that require open chambers. Incubation at 37°C can be done using either a Warner Instruments perfusion system or Okolab instrumentation designed for use with dipping lenses on upright microscopes. Please email lmcb-lm-help@ucl.ac.uk well in advance if you want to use CO2 as a bottle of the right percentage will have to be supplied. Both machines have four lasers and a single detector so all multichannel imaging must be done sequentially.
Training & Booking
Specifications
Objective Lenses
None of the objectives are planar lenses (i.e. they do not have a flat field). Leica recommends zooms ≥1.6 to avoid out-of-focus regions around the edge of the field.
| Standard Objective Lenses | |||||
| Magnification | Type | Immersion | NA | DIC | Coverslip Correction |
|---|---|---|---|---|---|
| 10x | ACS APO | Air | 0.3 | C | None |
| 20x | ACS APO | Multi | 0.6 | no | 0 - 0.17 |
| 40x | ACS APO | Oil | 1.15 | E | 0.17 |
| 63x | ACS APO | Oil | 1.3 | E | 0.17 |
| Water Dipping Lenses | |||||
| Magnification | Type | Immersion | NA | DIC | |
| 10x | HCX APO | Water Dipping | no | None | |
| 20x | HCX APO | Water Dipping | 0.5 | no | None |
| 40x | HCX APO | Water Dipping | 0.8 | no | None |
| 63x | HCX APO | Water Dipping | 0.9 | no | None |
| Letters in column 5 indicate the correct DIC prism to use with the lens | |||||
Lasers
| Laser | Typical Fluorophores | Dichroic Mirror Position | Notes | |
|---|---|---|---|---|
| 405 | DAPI, Hoechst | 405/532 | ||
| 488 | EGFP, FITC, Alexa 488 | 488/635 | ||
| 532 | Alexa 532, TRITC, Cy3 | 405/532 | This laser is not brilliant for exciting very red dyes like Alexa 594 and mCherry | |
| 635 | Cy5, Alexa 633, Alexa 647, Si Rhodamine dyes, DRAQ 5 | 488/635 |
Dichroic Mirror Position: There is a motorised dichroic mirror block that has to switch positions between 405/532 and 488/635 illumination paths, which will limit the speed with which green and red fluorophores (or blue and far red fluorophores) can be imaged. To optimise speed you should set up your multichannel imaging so the blue and red channels are acquired one after another and the green and far red channels are acquired one after another (e.g. blue/red/green/far red). That way the dichroic mirror only has to change position once between channels.
User Guides
- Quick Start Guide
- Setting up DIC on the SPE confocals
- Correcting misregistration of colour channels in images using Volocity
FAQ
- Can I use the SPEs to look at colocalisation in specimens?
- What steps should I take to avoid crashes and hangs - especially during time-lapse?