aFLOT

Confocal theta microscopy suggests that angled illumination and detection reduce the observation volume, thereby increasing resolution. The best angle between illumination and detection axes is 90 degree. Confocal theta microscopy mainly focused its interest in 2D imaging and has been extended its capability in 3D imaging for transparent small samples recently. For example, plane sheet illumination is a new approach that adopts the 90 degree advantage. However, as PSF is determined by not only the imaging system but also the imaged subject, if the imaged subject is scattering, the conclusion of 90 degree may not be valid. We find out that in scattering samples, the best angle between illumination and detection axes is 60 degree instead. This is because of photons being scattered toward deeper region. In other words, along illumination axis, photons distribute tightly close to and loosely away from the illuminating source. As a result, the photon distribution can not maintain symmetric axially as in transparent subjects. (In coherent detection, PSF along axis is sinc shape.) To implement optics to have this 60 degree intersection, configuring both illumination and detection arms at approximately 30/-30 degree in the subject (assuming n=1.4) is possible, but this configuration needs to solve the additionally raised off-focused detection. This off-focused detection is not negligible because fluorescence escaping from the surface of subject is typically widely distributed about several mm, especially when the subject is highly scattering or the fluorescence source/object is deep within. Since CCD detector is placed with an angle, only a minor portion of detected fluorescence is in focus, leaving major portion out of focus. This engineering problem can be solved independently such as replacing the single focusing lens with micro lens array. Nevertheless, given the current available resource, placing the EMCCD normal to the subject surface seems wise to eliminate the off-focus detection issue. This leaves adjusting the illumination angle the only other degree of freedom. However, directing illumination to have 30 degree angle below the surface of subject (60 degree transmission angle) may not be practically implementable in remission/epitaxial imaging. it is because 90 degree incidence is simply no incidence in remission mode imaging. It is possible for plane sheet illumination because the subject is small. As a result, our conclusion is that while the 60 degree is not obtainable, practically large angled incidence however would still be better than simply normal incidence/detection. Another consideration is the penetration depth. Plane sheet illumination usually does not have such issue because it focuses on small objects.