Light sheet microscopy

Light sheet microscopy – also referred to as single plane illumination microscopy (SPIM) is a is a gentle way of imaging sensitive samples or fast biological processes /in vivo/. The specimen is illuminated only in a single plane at a time and detected from the perpendicular direction. Since there is no out-of-focus excitation, phototoxic effects are reduced to the focal plane. Light sheet imaging has intrinsic optical sectioning. By moving the sample through the light sheet, 3D images of a specimen can be recorded. With this technique large 3D volumes are captured at a much higher speed but slightly lower resolution compared to confocal imaging.

Leica SP8 DLS Photo: Erling Jirle

Leica SP8 DLS specifications

Our Leica SP8 DLS (inverted microsocpe) is equipped with:

Light source: five lasers with wavelenghts:

• 405 nm
• Solid state 488 nm
• Solid state 514 nm
• Solid state 552 nm
• Solid state 638 nm

Detectors = two PMT:s (photomultiplier tubes), one HyD GaAsP-spectral detector, one PMT for transmitted ligth
Objectives for confocal imaging: 10x/0.3, 20x/0.75 IMM, 25x/0.95W, 63x/1.4 Oil, 63x/1.3 GLYC
Objectives and mirrors for light sheet microscopy: imaging 25x/0.95W DLS, 10x/0.3W DLS, illumination 2,5x/0.07, 5x/0.15, DLS TwinFlect 2.5mm, DLS TwinFlect 5mm
Software: LAS X with HyVolution, Dye Finder, 3D Visualisation, Microlab (for FRAP, FLIP and FRET) and Huygens Base Package for Deconvolution

Confocal microscopy

Confocal microscopy – or to be more precise: confocal laser scanning microscopy (CLSM) – is a method that is particularly suited for microscopy of fluorescent or reflecting specimens. The confocal microscope eliminates out-of-focus light, and creates a clear view of structures in the focal plane – also in specimens that are to thick for conventional fluorescence microscopy.

If you acquire a series of confocal images at different depths in a thick specimen, the image series can be used for 3D analysis and 3D rendering and you can visualize structures that could not be distinguished in conventional light microscopy.

Our Zeiss LSM 510 META is equipped with:

Light source : four lasers, with the wavelengths:

• DPSS 405 nm (excitation of e.g. DAPI, Hoechst 34580)
• Ar ion laser: 458, 477, 488, 514 nm (488 nm for excitation of e.g. Cy2, Alexa488, GFP, FITC)
• DPSS 561 nm (excitation of e.g. Cy3, Alexa 546 – Alexa 592, Texas Red, TRITC)
• HeNe ion laser: 633 nm (excitation of e.g. Cy5, Alexa633 - Alexa 647)

Detectors: two PMT:s (photomultiplier tubes) and a META detector
Objectives: 5x/0.15, 10x/0.45W, 20x/0.8, 25x/0.8IMM, 40x/1.3Oil, 63x/1.4Oil
Software: ZEN 2009

Zeiss LSM 510. Photo: Peter Ekström.