Resolution in the ApoTome and the confocal laser scanning microscope: comparison

Area of Science:

• Microscopy
• Optical Imaging
• Biotechnology

Background:

• Confocal laser scanning microscopy (cLSM) generates optical sections by eliminating out-of-focus light.
• Structured illumination microscopy (SIM) emerged as an alternative for optical sectioning in biological specimens.

Purpose of the Study:

• To compare the resolution performance of ApoTome (a commercial SIM system) against a cLSM (Zeiss LSM 510).
• To evaluate axial and lateral resolution differences using various test objects.

Main Methods:

• Comparative analysis of resolution using fluorescent beads as test objects.
• Evaluation using subresolution homogeneous fluorescent layers as test objects.
• Assessment of axial resolution homogeneity and anisotropy across the field-of-view.

Main Results:

• ApoTome (SIM) demonstrated superior lateral resolution compared to cLSM, but lower axial resolution with fluorescent beads.
• ApoTome achieved higher axial resolution with homogeneous fluorescent layers, exhibiting uniform performance across the field-of-view.
• cLSM showed marked variations in axial resolution, while ApoTome's resolution anisotropy was negligible for standard applications.

Conclusions:

• ApoTome (SIM) offers distinct advantages in lateral resolution and axial resolution homogeneity over cLSM.
• The effective depth for resolving fine structures in stained tissues is limited in ApoTome compared to cLSM.
• The choice between SIM (ApoTome) and cLSM depends on specific application requirements for resolution and imaging depth.