9 – LSM 880 Training Guide
Light Path Configuration
The confocal portion of the LSM 880 is equipped with a 34-channel spectral detector plus transmitted light and has 5 lasers with
wavelengths 405nm, 458nm, 488nm, 514nm, 561nm, 594nm, and 633nm. The Light Path configuration allows you to program
precisely how your image acquisition will be executed.
The first step is to decide how you wish to collect each fluorophore. You have 3 choices:
1.
Simultaneous
– collect up to 4 fluorophores simultaneously.
Note: While this mode is the fastest approach – many fluorophore combinations will exhibit enough spectral overlap to be
able to detect unwanted signal from neighboring fluorophores using this method. This phenomenon is known as spectral
overlap or ‘bleed’.
2.
Sequential
– collect fluorophores sequentially.
Note: This mode tends to be the slowest mode – but provides accurate spectral separation of multiple fluorophores.
3.
Spectral
– lambda mode allows you to collect fluorophores simultaneously and unmix based on reference spectra.
Note: This mode is both the fastest AND the most accurate mode of spectral separation, however it requires single label
controls to differentiate between the unique spectral trace(s) from your fluorophore(s).