BACKGROUND: Although recent developments in methodologies for light microscopy have enabled imaging of fine biological structures, such imaging is often accompanied by two types of problems. One is a tilting of the specimen with respect to the x-y plane (i.e. rotation around the x- or y-axis) such that the sample is not perpendicular to the optical z-axis, and the other is rotation around the z-axis that precludes optimal orientations for imaging and experimentation. These rotation problems can cause optical aberrations and hamper imaging experiments, even when the angular difference from the ideal position is small. NEW METHOD: In order to correct for these practical issues, we have developed a specimen holder with 3-axis (x-y-z) rotation for an inverted light microscope. This allows for full-range rotations of 2-4° for x-, y-axes, ~24° for z-axis, and a small-angle control of <0.1° for either axis. RESULTS: Using this device, we observed the cultured hippocampal neurons stained by immunofluorescence for a dendritic marker, or the sub-resolution fluorescent beads plated on a glass coverslip. The rotations and associated problems could be manipulated, while viewing the specimens by laser-scanning confocal microscopy. COMPARISON WITH EXISTING METHODS: This tilting/rotation device is easily manufactured and installed on a conventional microscope stage without requiring changes to the existing optical components. Similar devices with full capability have not been available. CONCLUSIONS: It will be useful for imaging experiments with biomedical applications.
BACKGROUND: Although recent developments in methodologies for light microscopy have enabled imaging of fine biological structures, such imaging is often accompanied by two types of problems. One is a tilting of the specimen with respect to the x-y plane (i.e. rotation around the x- or y-axis) such that the sample is not perpendicular to the optical z-axis, and the other is rotation around the z-axis that precludes optimal orientations for imaging and experimentation. These rotation problems can cause optical aberrations and hamper imaging experiments, even when the angular difference from the ideal position is small. NEW METHOD: In order to correct for these practical issues, we have developed a specimen holder with 3-axis (x-y-z) rotation for an inverted light microscope. This allows for full-range rotations of 2-4° for x-, y-axes, ~24° for z-axis, and a small-angle control of <0.1° for either axis. RESULTS: Using this device, we observed the cultured hippocampal neurons stained by immunofluorescence for a dendritic marker, or the sub-resolution fluorescent beads plated on a glass coverslip. The rotations and associated problems could be manipulated, while viewing the specimens by laser-scanning confocal microscopy. COMPARISON WITH EXISTING METHODS: This tilting/rotation device is easily manufactured and installed on a conventional microscope stage without requiring changes to the existing optical components. Similar devices with full capability have not been available. CONCLUSIONS: It will be useful for imaging experiments with biomedical applications.