Introduction to Aperture Correlation Microscopy
The primary advantage of confocal microscope imaging over traditional widefield fluorescence microscopy is the rejection of light from regions positioned away from the focal plane, leading to the ability to obtain thin optical sections with increased contrast that can be assembled into interpretable three-dimensional image stacks. Because optical sectioning is a prerequisite for a number of applications, including precise localization studies, co-localization experiments, and spectral unmixing, a large variety of different optical sectioning techniques have been developed and commercialized over the past few years. The laser scanning confocal microscope (LSCM) remains the gold standard for many applications, but single point scanning is severely limited in acquisition speed. Due to the fact that the image is assembled point-by-point (one pixel at a time), attempting to employ high frame rates will ultimately lead to very short pixel dwell times and, therefore, often insufficient signal-to-noise ratio (SNR) in the final images or optical sections. Alternative microscope designs attempt to overcome this unfortunate speed limitation by scanning multiple points in parallel, which can increase acquisition speed dramatically, but at the cost of spatial or axial resolution. It should be noted, however, that all of these instruments are based on laser illumination, which restricts their spectral flexibility and results in high system prices.
Microscope systems that do not rely on laser illumination but instead use a simple white light source, such as arc-discharge lamps or LEDs, present an attractive alternative to laser-based instruments for optical sectioning as they offer dramatically enhanced spectral flexibility for a relatively low price. The concept of structured illumination microscopy (SIM), which can be implemented on a standard widefield fluorescence microscope, offers such an option. In structured illumination systems, the excitation light is patterned in a defined geometrical pattern, and this additional information is used to mathematically extract an optical section from a series of raw data images. SIM systems all follow the same technical principle that consists of an illumination mask to structure the excitation light with a specific (usually based on a line grid) pattern. As the illumination mask is located in a conjugate image plane, the pattern will only appear in focus at the focal plane of the instrument. At positions residing away from the focal plane, the pattern will be blurred. Thus, the patterned structured illumination will generate fluorescence emission from the focal plane that is modulated in accordance to the grid pattern, whereas emission from other planes will not be modulated.
The key to calculation of an optical section image when using structured illumination is demodulation of the raw data image set. The software algorithm must identify the modulated part of the signal representing the in-focus information and separate that from the unmodulated signal arising from other focal planes. Image processing of SIM data is usually conducted post-acquisition by demodulating the image set (usually three or more images), which consists of images having the illumination pattern in different positions. Changes in pixel intensities in between the three or more images can be attributed to the different pattern positions, representing the modulated response from in-focus structures, while intensities that are not influenced by the position of the illumination pattern represent out-of-focus information. Such systems, as realized in the ZEISS ApoTome, have matured over the last years into viable low-cost alternatives for optical sectioning. However, the acquisition speed of such a system remains limited by the necessity to acquire several images in order to calculate one optical section.