Doing 3D eSRRF
The main message about 3D eSRRF is that it requires acquisition of fluorescence data at high resolution as in 2D, but this time, simultaneously across several Z planes. This is made possible typically with Multi-Focus Microscopy (MFM) and could also be done with image splitters combined with different defocus (this latter approach may however introduce spherical aberrations for certain planes).
Otherwise, all the restrictions in terms of frame rate and number of frames, high resolution from the 2D approach still apply. This means it will often require high NA objectives and fast acquisition rates.
Because the Z planes are recorded simultaneously, they are usually acquired either on the same camera but on different position of the chip or on different cameras altogether. Either way, the planes need to be registered spatially to take into account potential offsets and/or rotations. This therefore requires the acquisition of calibration data in imaging conditions as close as possible as that used for the real data of interests, but this time, of a sharp structure that can be found in all planes while performing Z scanning. Typically, this is done with fluorescent beads on a cover slip and a Z-stack acquisition to sequentially observe the beads in focus in each one of the planes.
The Get spatial registration from MFM data plugin will analyse that data and export a calibration table, containing information about the X and Y shift of each plane, the rotation angle and their respective axial offset. This is currently only implemented for 3x3 MFM dataset, corresponding to 9 planes.
The reconstruction is performed from the same plugin as for 2D eSRRF - Analysis, simply by enabling the 3D eSRRF parameter and making sure that the average Z Axial offset is also provided (in nm). The plugin will then ask the user to provide the calibration table that was created in (c).
The reconstruction then provides volumes time-course data of 3D eSRRF reconstruction. These can be viewed in other 3D viewer software such as ClearVolume or 3D Script.