Automatic laser alignment for multifocal microscopy using a LCOS SLM and a 32×32 pixel CMOS SPAD array

Abstract

Alignment of a laser to a point source detector for confocal microscopy can be a time-consuming task. The problem is further exacerbated when multiple laser excitation spots are used in conjunction with a multiple pixel single photon detector; in addition to X, Y and Z positioning, pixels in a 2D array detector can also be misaligned in roll, pitch and yaw with respect to each other, causing magnification, rotation and focus variation across the array. We present a technique for automated multiple point laser alignment to overcome these issues using closed-loop feedback between a laser illuminated computer controlled Liquid Crystal on Silicon Spatial Light Modulator (LCOS-SLM) acting as the excitation source and a 32×32 pixel CMOS Single Photon Avalanche Diode (SPAD) array as the multiple pixel detection element. The alignment procedure is discussed and simulated to prove its feasibility before being implemented and tested in a practical optical system. We show that it is possible to align each independent laser point in a sub-second time scale, significantly simplifying and speeding up experimental set-up times. The approach provides a solution to the difficulties associated with multiple point confocal laser alignment to multiple point detector arrays, paving the way for further advances in applications such as Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Imaging Microscopy (FLIM).