Focal Depth Extension Techniques for Multiphoton Microscopy
The combination of two-photon laser scanning microscope and calcium indicator is the gold standard for in vivo neuronal signal detection. The neurons in neural networks are distributed in three-dimensional space, and monitoring their activity dynamics requires a way to quickly improve the volume imaging rate. However, using a grating scanning multiphoton microscope to image a large number of images, if a high numerical aperture (NA) objective is used to achieve higher lateral resolution, it will result in a smaller focal depth. In order to obtain volume imaging at a small focal depth,
It is necessary to perform Z-axis scanning through some means, imaging many planes by scanning each focal plane, which greatly limits the imaging speed. If axial image information can be sacrificed and volume scanning can be achieved in one lateral scan by extending the depth of focus, that is, the volume information is projected onto a single 2D image, the imaging speed can be greatly improved. This is called Extended Depth of Focus (EDF) imaging, which is particularly useful for imaging sparse population structures that require high temporal resolution, such as functional imaging of neuronal activity.
The axial and lateral resolutions of a microscope are determined by the numerical aperture (NA) of the objective lens. High NA can maximize axial and lateral resolution as well as the amount of light collected; Lower NA will result in lower axial resolution, i.e. longer depth of focus, but at the same time sacrifice lateral resolution and light collection efficiency. The method of extending the depth of focus that will be introduced next can achieve this while maintaining high lateral resolution and sufficient light flux.
The use of spatial light modulators to generate focal slender Bessel beams can achieve EDF imaging, but spatial light modulators are bulky and difficult to be compatible with narrow microscope spaces; In contrast, Bessel modules based on axial pyramids are cheap and compact, but they can only generate focal points of fixed depth and are not suitable for various experiments that require continuous changes in focal depth. To address this issue, in 2018, RONGWEN LU et al. demonstrated a Bessel module based on an axicon, in which only one lens needs to be translated along the optical axis to continuously adjust the axial length of the Bessel focal point.
