New white paper details the technology behind ZEISS Apotome.2
In conventional widefield fluorescence microscopy an image contains light collected from the structures in the plane of focus as well as light from structures above and below this plane. The light from structures above and below becomes visible in the focal plane because of diffraction and interference and creates a blur in the image. Diffraction and interference are physical phenomena where light waves interact with each other causing a blurring of the object of interest. Consequently a point object which is smaller than the resolution limit of the objective when imaged in 3D is not imaged as a point but as a hour glass shape known as Point Spread Function (PSF).
When studying thick specimens, this out-of-focus light degrades the image quality by increasing the background fluorescence in the image and therefore decrease the signal-to-noise ratio (SNR) and the image contrast. Such images are often not suitable for reasonable analysis and interpretation of the 3-dimensional structure of the objects. Therefore, the motivation is to remove the out-of-focus light and obtain images of the objects of interest with highest contrast and highest resolution. This is the principle in optical sectioning. Besides the predominant confocal method using a pinhole in an optically conjugate plane, other approaches have been developed to obtain optical sectioning, including Structured Illumination.
As mentioned above, the main motivation of optical sectioning is improving the contrast by removing the out-of-focus light. This usually comes at the expense of decreasing the overall signal. When imaging weakly fluorescing samples this can be overcome by increasing the thickness of the optical section or by increasing the acquisition time. The latter has the negative side effect of putting higher burden on the sample leading to bleaching and increased phototoxicity, whereas the former increase the SNR. So the major trade-off when doing optical imaging is the decrease in signal, and the skill of the operator to define the compromise between signal, resolution (i.e. thickness), photodamage and acquisition time. The thickness of the optical section is determined differently dependent on the sectioning method applied.
For instance, in confocal laser scanning microscopy, the size of the pinhole determines the thickness of the optical section. Increasing the pinhole will increase the thickness of the optical sample and increase the overall signal. The optimal thickness of the optical section is also determined by the numerical aperture (NA) of the objective lens, where high NA allows for thinner optical sectioning. In structured illumination, the thickness of the optical section can be altered by changing the frequency of the grid pattern. This also means that different objective lenses with various numerical apertures require different grid pattern frequencies.
The principle in structured illumination used in Apotome.2 is based on projection of a defined grid into the focal plane of a widefield fluorescence microscope. As the grid is moving in a lateral manner, a defined number of images, termed phases, are captured by a camera. The light emitted by structures, which are in the focal plane, will vary strongly in intensities when the illuminating grid lines move across them. If structures are not in focus, the wandering grid lines will not cause the emitted light to fluctuate as strongly. By calculating the difference between the different phase images it is possible to discriminate between in-focus and out-of-focus light. The differences in intensities are calculated pixel-by-pixel and the final image contains only the in-focus information which is an optical section.