Computer Generated Holography with Intensity-Graded Patterns

Abstract

Computer Generated Holography achieves patterned illumination at the sample plane through phase modulation of the laser beam at the objective back aperture. This is obtained by using liquid crystal-based spatial light modulators (LC-SLMs), which modulate the spatial phase of the incident laser beam. A variety of algorithms is employed to calculate the phase modulation masks addressed to the LC-SLM. These algorithms range from simple gratings-and-lenses to generate multiple diffraction-limited spots, to iterative Fourier-transform algorithms capable of generating arbitrary illumination shapes perfectly tailored on the base of the target contour. Applications for holographic light patterning include multi-trap optical tweezers, patterned voltage imaging and optical control of neuronal excitation using uncaging or optogenetics. These past implementations of computer generated holography used binary input profile to generate binary light distribution at the sample plane. Here we demonstrate that using graded input sources, enables generating intensity graded light patterns and extend the range of application of holographic light illumination. At first, we use intensity-graded holograms to compensate for LC-SLM position dependent diffraction efficiency or sample fluorescence inhomogeneity. Finally we show that intensity-graded holography can be used to equalize photo evoked currents from cells expressing different levels of chanelrhodopsin2 (ChR2), one of the most commonly used optogenetics light gated channels, taking into account the non-linear dependence of channel opening on incident light.

Multiplexed temporally focused light shaping for high-resolution multi-cell targeting

“…One can notice the speckled intensity profile, typical of holographic spots 53 , as well as the similarity of the speckle distribution for spots lying on different planes, confirming that each spot was indeed a replica of the original 2D shape. 30 As previously described 13,54,55 , by calculating the phase that we sent to SLM2 with a weighted 15…”

Imaging membrane potential changes from dendritic spines using computer-generated holography

“…In addition to spatial shaping of illumination, CGH also enables modulation of the relative intensity of individual excitation spots to achieve optimal recording conditions. 53 In the three examples shown in Fig. 3, dendritic regions at the base of the spine were not illuminated while the locations away from dendritic spines were illuminated with lower intensities.…”

“…47,52 The relative intensity between spots was controlled by adjusting their weight in the input pattern of the algorithm. 53,54 The pattern and the corresponding wGSgenerated phase profiles were defined, computed, and addressed to the SLM using "Wavefront Designer IV," in house software written in C++ with Qt 4.4.0 and fftw 3.1.2. Calibration and characterization of the generated patterns were performed by detecting induced fluorescence on a thin film of rhodamine 6G spin-coated on a coverslip.…”

Vortex-free phase profiles for uniform patterning with computer-generated holography

“…Non-homogeneous intensities are all the more critical in experiments involving non-linear light-matter interactions. For such systems exhibiting optical non-linearities (like two-photon absorption [8]), saturated optical responses (lasers [17], light-sentitive membrane channels [18]) or optical processes sensitive to temperature (photo-polymerization [19]), speckled patterns generated by regular CGH with phase-only modulation are thus detrimental and may result in experimental artifacts. Moreover, in applications using linear photo-excitation processes, the speckle pattern may also be problematic.…”