Using out-of-focus light to improve image acquisition time in confocal microscopy

Light that would typically be discarded at a confocal microscope's detector pinhole will be collected and processed to allow a reduced spatial sampling rate and thus an improved image acquisition time. It is shown that collecting and appropriately processing the out-of-focus light will allow an axial sampling rate below that specified by the Nyquist criterion. To achieve this, a central detector pinhole and a number of out-of-focus regions are collected concurrently. This corresponds to imaging through several different channels, with differing point spread functions, in parallel. Since the spatial sampling rate is below the Nyquist frequency, aliasing occurs in the data from each of the channels. However, since the point spread functions are different, the aliasing effects are different in each channel. This allows the ensemble of aliased images to be processed into a single dealiased and deconvolved image. This potential utility of out-of-focus light is demonstrated through simulated examples for differing collection schemes and scanning rates. Results are shown for under-sampling by up to a factor of four. Collecting the out-of-focus light also improves instrument collection efficiency.