Frequently asked Questions

Multiphoton microscopy: bad performance

Q: Multiphoton microscopy - bad imaging performance!

A:here is a a short list of the most important issues regarding multiphoton performance:

• Check lens performance by evaluating the epifluorescence NDD signal from 200 micromolar fluorescein solution in PBS pH 8.9 excited by 890 nm IR pulse laser (or any wavelength between 720 and 930 nm). You can prepare a fluorescein solution and place it under a cover slip mounted onto a slide with spacers made from cover slip glass. Check at different imaging depth / imaging distance from dish (cover slip) surface.
• Use the appropriate lens for your sample, especially for in depth imaging >20 micrometers below cover slip (otherwise spherical aberration will cause bad in depth performance). Very important: for samples in buffer, use a water immersion lens designed for use with a standard (0.17 mm thick) cover slip. Our ZEISS 40x/1.2 W lens (for use with cover slip) performs very well. Check if the bottom of your sample dish meets this specification (0.17 mm +- 0.01 mm thickness, i.e cover slip # 1.5). Check specification using a micrometer measuring device from your local mechanical workshop. Do not use plastic bottom dishes, except you know of good multiphoton performance!
• Use high N.A. condenser for transmission NDD recording; focus condenser for maximum signal. Open all apertures (field stop and aperture stop) of the condenser, remove filters except multiphoton IR beam blocking filter.
• AOM or Pockels cell: check possible beam drift, align beam under measurement conditions, e.g. 100% AOM, bidirectional scan. For measurement, you can start scaning 30 sec. under identical conditions (until beam position is stable) before first significant recording. If you notice beam drift, ask your representative for AOM or Pockels cell.

Huygens2

Q: I cannot load files into Huygens!

Check file path / file name for special characters: Unix and the Tcl script language are very sensitive to non-alphabetical characters, e.g. =, %, #, +, -, whitespace etc (except underscore). Mac-users seem to be very happy in using such characters. While some non-alphabetical characters may work, it is strongly recommended to use only alphabetical characters and underscore for use with Huygens / Unix.

The Unix file system is case-sensitive!

Some file types have to be loaded into Imaris3 first and then saved as Imaris3 files (check parameters in Huygens!)

Scanning problems: bad images

Q: My confocal fluorescence scans show some blur and are not sharp at all!

Check DIC prism settings. Since DIC imaging is sometimes performed with the confocal (and widefield) microscopes, it is essential for everybody to check the correct positions of the polarizer and the DIC prism wheels.

People sometimes forget to set the DIC turret positions back to the brightfield position (generally labeled BF or HF). Colleagues using the microscope afterwards may not be aware of the change in settings and perform standard fluorescence scans with the prisms inserted. Especially at high magnifications/zoom factors, however, artifacts may be produced by the prisms (visible as blur, or as doubling of fine structures). Therefore, please:

1. Check the prism and polarizer settings at the beginning and the end of your session
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2. At the end of your session: set prism turrets and polarizers to their default (non-DIC) position. This will help to avoid both waste of time and bad scans.