Correlation of chromatic shifts and focal depth in spectral precision distance microscopy measured by microaxial tomography

Confocal laser scanning fluorescence microscopy is presently being used widely in biomedical research. A severe limitation for its use is its often still insufficient resolution. In situ measurements in 3D conserved human cell nuclei showed that distance measurements between fluorescent targets located in the interior of such objects are limited a resolution regime of appr. greater than or equal to 0.3 micrometer in lateral and appr. greater than or equal to 0.7 micrometer in axial direction. A technique to overcome these restrictions is the recently developed Spectral Precision Distance Microscopy (SPM). This approach allows the determination of distances between targets which carry different spectral signatures with high precision. In situ measurements revealed that the SPM approach allows the determination of distances in 3D intact cell nuclei with a 'Resolution Equivalent' better than 50 nm. Here we present an improved chromatic shift calibration method for Spectral Precision Distance Microscopy. Furthermore, micro axial tomography allows the tilting of objects perpendicular to the optical axis; thus two objects can always be tilted in such a way that they can be recorded in the same focal plane. Therefore the error in distance determination is minimized. Here we present some preliminary data for the applicability of spectral precision distance microscopy (SPM) to micro axial microscopy.