Zeiss LSM 510 Manual

Also see for LSM 510: Quick start

MULTIPHOTON LASER SCANNING MICROSCOPYCarl Zeiss Using the LSM 510 NLO direct coupled system LSM 510 META NLO9-16 B 45-0021 e 03/069.3.2.2 Advanced alignment protocolWhen the system is installed, the service engineer will establish alignment of the laser beam into the scanhead by verifying the overlay between the NIR laser and one of the VIS lasers. Although routine useusually does not usually require a complete realignment with the scan head, tuning the laser over largewavelength increments or changing or cleaning the optics in the laser can cause gross changes in beampointing. If this occurs, it is best to use the following protocol to re-establish proper alignment with thescan head. For this protocol, a partially reflective grid slide is needed (Part number 474028-0000-000,Test Grid Specimen for LSM). Care must be taken to use low amounts of laser power when using thisslide or the grid can be easily burned.Begin by confirming that the correct wavelength for the laser is set in the laser control panel in thesoftware. The wavelength can be changed by selecting the modify option. Refer to the Rees analyzer orthe BRF setting to determine the wavelength of the MIRA 900F.Next, place the partially reflective grid slide on the microscope stage. Bring the grid slide in focus usingthe 10x Plan-Neofluar objective and transmitted light. Configure the scan head as follows (Fig. 9-6):This configuration is designed to use the PMTs todetect the reflection of the NIR and VIS laser lightoff of the grid slide so an image of the grid ismade on the screen. For this protocol, the 543 nmline has been chosen. The HFT KP 700/488 beamsplitter is used to avoid sending too much 543 nmlaser light to the reflective slide. A KP 700/543beam splitter can be used, but care must be takennot to burn the slide. Alignment using thisprocedure will ensure that the NIR and VIS lasersoverlay on the combining mirror when they enterthe scan head.To perform the alignment, set the laser power forboth lasers at 3 % (1 % for a MIRA 900F with a10W pump laser) and adjust only the gain toimprove the intensity of the signal.WARNING: Too much laser powercan cause damage to the grid slide!Be sure attenuation is set beforescanning.Fig. 9-6 Configuration for testing theoverlay of the grid reflectionimages gained with the vis (543nm) laser and the NIR laser