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Holographic structured illumination combined with optogenetics enables patterned stimulation of neurons and glial cells in an intact living brain. Moreover, in vivo functional imaging of cellular activity with recent advanced microscope technologies allows for visualization of the cellular responses during learning, emotion and cognition. Integrating these techniques can be used to verify the link between cell function and behavior output. However, there are technical limitations to stimulate multiple cells with high spatial and temporal resolution with available techniques of optogenetic stimulation. Here, we summarized a two-photon microscope combined with holographic system to stimulate multiple cells with high spatial and temporal resolution for living mice and their biological application.

The challenge to understand the complex neuronal circuit functions in the mammalian brain has brought about a revolution in light-based neurotechnologies and optogenetic tools. However, while recent seminal works have shown excellent insights on the processing of basic functions such as sensory perception, memory, and navigation, understanding more complex brain functions is still unattainable with current technologies. We are just scratching the surface, both literally and figuratively. Yet, the path towards fully understanding the brain is not totally uncertain. Recent rapid technological advancements have allowed us to analyze the processing of signals within dendritic arborizations of single neurons and within neuronal circuits. Understanding the circuit dynamics in the brain requires a good appreciation of the spatial and temporal properties of neuronal activity. Here, we assess the spatio-temporal parameters of neuronal responses and match them with suitable light-based neurotechnologies as well as photochemical and optogenetic tools. We focus on the spatial range that includes dendrites and certain brain regions (e.g., cortex and hippocampus) that constitute neuronal circuits. We also review some temporal characteristics of some proteins and ion channels responsible for certain neuronal functions. With the aid of the photochemical and optogenetic markers, we can use light to visualize the circuit dynamics of a functioning brain. The challenge to understand how the brain works continue to excite scientists as research questions begin to link macroscopic and microscopic units of brain circuits.

The promise of artificial intelligence (AI) to process complex datasets has brought about innovative computing paradigms. While recent developments in quantum-photonic computing have reached significant feats, mimicking our brain’s ability to recognize images are poorly integrated in these ventures. Here, I incorporate orbital angular momentum (OAM) states in a classical Vander Lugt optical correlator to create the holographic photonic neuron (HoloPheuron). The HoloPheuron can memorize an array of matched filters in a single phase-hologram, which is derived by linking OAM states with elements in the array. Successful correlation is independent of intensity and yields photons with OAM states of lℏ, which can be used as a transmission protocol or qudits for quantum computing. The unique OAM identifier establishes the HoloPheuron as a fundamental AI device for pattern recognition that can be scaled and integrated with other computing platforms to build-up a robust neuromorphic quantum-photonic processor.

We demonstrate the first biological measurement with precision surpassing the quantum noise limit. Lipid particles within a living yeast cell are tracked with sub-shot noise sensitivity, thereby revealing the biological dynamics of the cellular cytoplasm., Comment: 10th Conference on Lasers and Electro-Optics Pacific Rim, CLEO-PR 2013, 2013

A dendritic spike is a vital computing mechanism in neurons and spikes occurring in complex dendritic arborizations are difficult to probe. Here, we use laser dendrotomy to study dendritic spikes in oblique branches of cortical neurons.

Metasurfaces exhibit unique optical properties that depend on the ratio of their refractive index and that of their surroundings. As such, they are effective for sensing global changes in refractive index based on the shifts of resonances in their reflectivity spectra. However, when used as a biosensor, the metasurface can be exposed to a spatial distribution of biomolecules that brings about gradients in refractive index along the plane of the metasurface. Such gradients produce complex global reflectivity spectrum but with distinct optical enhancements in localized areas along the metasurface. Here, we propose a unique sensing paradigm that images and maps out the optical enhancements that are correlated with the spatial distribution of the refractive index. Moreover, we designed a metasurface whose resonances can be tuned to detect a range of refractive indices. Our metasurface consists of silicon nanopillars with a cylindrical nanotrench at their centers and a metal plane at the base. To assess its feasibility, we performed numerical simulations to show that the design effectively produces the desired reflectivity spectrum with resonances in the near-infrared. Using an incident light tuned to one of its resonances, our simulations further show that the field enhancements are correlated with the spatial mapping of the gradients of refractive indices along the metasurface.

Dendritic spikes in layer 5 pyramidal neurons (L5PNs) play a major role in cortical computation. While dendritic spikes have been studied extensively in apical and basal dendrites of L5PNs, whether oblique dendrites, which ramify in the input layers of the cortex, also generate dendritic spikes is unknown. Here we report the existence of dendritic spikes in apical oblique dendrites of L5PNs. In silico investigations indicate that oblique branch spikes are triggered by brief, low-frequency action potential (AP) trains (~40 Hz) and are characterized by a fast sodium spike followed by activation of voltage-gated calcium channels. In vitro experiments confirmed the existence of oblique branch spikes in L5PNs during brief AP trains at frequencies of around 60 Hz. Oblique branch spikes offer new insights into branch-specific computation in L5PNs and may be critical for sensory processing in the input layers of the cortex.

Significance: Wide-field measurement of cellular membrane dynamics with high spatiotemporal resolution can facilitate analysis of the computing properties of neuronal circuits. Quantum microscopy using a nitrogen-vacancy (NV) center is a promising technique to achieve this goal. Aim: We propose a proof-of-principle approach to NV-based neuron functional imaging. Approach: This goal is achieved by engineering NV quantum sensors in diamond nanopillar arrays and switching their sensing mode to detect the changes in the electric fields instead of the magnetic fields, which has the potential to greatly improve signal detection. Apart from containing the NV quantum sensors, nanopillars also function as waveguides, delivering the excitation/emission light to improve sensitivity. The nanopillars also improve the amplitude of the neuron electric field sensed by the NV by removing screening charges. When the nanopillar array is used as a cell niche, it acts as a cell scaffolds which makes the pillars function as biomechanical cues that facilitate the growth and formation of neuronal circuits. Based on these growth patterns, numerical modeling of the nanoelectromagnetics between the nanopillar and the neuron was also performed. Results: The growth study showed that nanopillars with a 2-μm pitch and a 200-nm diameter show ideal growth patterns for nanopillar sensing. The modeling showed an electric field amplitude as high as ≈1.02×1010 mV/m at an NV 100 nm from the membrane, a value almost 10 times the minimum field that the NV can detect. Conclusion: This proof-of-concept study demonstrated unprecedented NV sensing potential for the functional imaging of mammalian neuron signals.

A novel versatile photo-responsive nanocarrier that is able to load and release several functional molecules is obtained by one-step conjugation of scalable flame-made titania agglomerates. Highly crystalline anatase nano-crystals are synthesized by scalable flame spray pyrolysis of organometallic precursor solutions. Nanocarriers are self-assembled by adsorption of lysine molecules on the photocatalytic nanoparticles’ surface leading to a minimal flocculation and highly reactive amine terminations. Time-controlled photo-release of the ligand and end-loaded molecules is achieved by short exposure to UV light. The application of these flexible nanoplatforms to intracellular delivery is demonstrated by dye loading and two-photon microscopic in vitro imaging of their penetration in living neurons of Wistar rat brain tissue. These scalable photo-responsive nanocarriers are a flexible platform with potential for in vivo controlled release of amine-reactive dyes and amino-acid modified pro-drugs, as demonstrated by the successful loading and release of fluorescein isothiocyanate dye (FITC) and ketoprofen.

Commercial microscopy systems make use of tandem scanning i.e. either slow or fast scanning. We constructed, for the first time, an advanced control system capable of delivering a dynamic line scanning speed ranging from 2.7 kHz to 27 kHz and achieve variable frame rates from 5 Hz to 50 Hz (512 × 512). The dynamic scanning ability is digitally controlled by a new customized open-source software named PScan1.0. This permits manipulation of scanning rates either to gain higher fluorescence signal at slow frame rate without increasing laser power or increase frame rates to capture high speed events. By adjusting imaging speed from 40 Hz to 160 Hz, we capture a range of calcium waves and transient peaks from soma and dendrite of single fluorescence neuron (CAL-520AM). Motion artifacts arising from respiratory and cardiac motion in small animal imaging reduce quality of real-time images of single cells in-vivo. An image registration algorithm, integrated with PScan1.0, was shown to perform both real time and post-processed motion correction. The improvement is verified by quantification of blood flow rates. This work describes all the steps necessary to develop a high performance and flexible polygon-mirror based multiphoton microscope system for in-vivo biological imaging.

This study presents a system that combines a conventional refrigerator, microcontrollers and a smart phone to create an inventory monitoring that can monitor the stocks inside the refrigerator wirelessly by accessing an Android application. The developed refrigerator uses a sensor network system that is installed in a respective compartment inside the refrigerator. Each sensor will transmit data to the microcontrollers, such as Arduino Yun and Arduino Uno, which are interconnected by the I2C communications. All data and images will be processed to provide the user an Internet of Things application through the cloud-based website Temboo. Temboo will have access to send data to the Dropbox. A smartphone is connected to the Dropbox where all the data and images are stored. The user can monitor the stocks or contents of the refrigerator wirelessly using an Android Application.

The spectacular facets of light have made light ubiquitous in all fields of science. Light’s interaction with matter allows for accurate manipulation of atomic and molecular structures that enabled fundamental breakthroughs in physics, chemistry, and biomedical research. The transfer of light’s energy on molecules and genetically expressed proteins can be used to stimulate cells and emulate cellular processes such as synaptic inputs spatially distributed along the neuron’s dendritic tree. Here, we show basic neuronal functions derived via numerical modelling and describe how we can use light to emulate these functions in order to provide a systematic study of the neuron’s response. We focus on cortical pyramidal neurons and use the NEURON simulation environment to analyze how spatio-temporal stimulation patterns along various dendritic locations sets the neuron to fire an output. We then show an equivalent response from experiments via complex spatial light patterns for stimulating across different regions along the dendritic tree. Furthermore, we use the same spatial light patterns to simultaneously visualize neuronal responses via functional calcium imaging predicted via the same neuron model. Visualizing dendritic responses from back-propagating action potentials can provide new insights to some important features of dendritic computation.

Functional imaging provides efficient optical recording of Ca2+ activity from single neurons as well as networks of neurons forming a circuit. Sufficient spatial resolution needs to be achieved in order to resolve cell bodies as well as fine neuronal structures such as dendrites and spines. On the other hand, the temporal resolution should be sufficient to capture Ca2+ events from multiple sites across the sample. Here, we briefly describe various imaging modalities and focus on two-photon holographic multi-foci excitation to perform multi-site Ca2+ imaging. Holographically projected multi-foci can be used to excite two-photon fluorescence from multiple sites along the dendritic tree of a single neuron or multiple cell bodies in a neuronal network. By simultaneously projecting multiple foci onto the sample, the fluorescence emanating from the different foci can be collected simultaneously using a camera, thereby setting the temporal resolution to the frame-rate of the camera. We describe the generation of appropriate holograms as well as discuss the limitations of the approach. We also discuss a solution to improve the signal-to-noise ratio via the application of temporal gating. The multi-foci holographic technique is a promising approach to perform high-speed optical recording of neuronal activity.

To understand how fast neuronal signals propagate along the dendritic tree of neurons, we simultaneously capture Ca2+ activity along thin oblique dendrites via two-photon multi-foci excitation and simultaneous multi-channel recording of the fluorescence.

Identifying the specific role of physical guidance cues in the growth of neurons is crucial for understanding the fundamental biology of brain development and for designing scaffolds for tissue engineering. Here, we investigate the structural significance of nanoscale topographies as physical cues for neurite outgrowth and circuit formation by growing neurons on semiconductor nanowires. We monitored neurite growth using optical and scanning electron microscopy and evaluated the spontaneous neuronal network activity using functional calcium imaging. We show, for the first time, that an isotropic arrangement of indium phosphide (InP) nanowires can serve as physical cues for guiding neurite growth and aid in forming a network with neighboring neurons. Most importantly, we confirm that multiple neurons, with neurites guided by the topography of the InP nanowire scaffolds, exhibit synchronized calcium activity, implying intercellular communications via synaptic connections. Our study imparts new fundamental insights on the role of nanotopographical cues in the formation of functional neuronal circuits in the brain and will therefore advance the development of neuroprosthetic scaffolds.

This work was supported by the Australian Research Council (ARC) Discovery Projects (DP130101364, DP170100908 and DP140101555), the National Health and Medical Research Council (NHMRC) Project Grants (PG1124411 and PG1105944) and the ARC Centre of Excellence for Integrative Brain Function (CE140100007).

Quantum metrology allows high sensitivity measurements to proceed with a lower light intensity than classically possible [1]. An important frontier for this technology is in biological measurements, where photochemical interactions often disturb biological processes and can damage the specimen [2]. Here we report the first demonstration of biological measurement with precision surpassing the quantum noise limit [3]. This was enabled through the development of a new microscopy system which extended previous methods used to track the motion of highly reflective mirrors with non-classical light to measurements of microscopic particles with non-paraxial fields (see Fig. 1). Biological dynamics in the critical Hz-kHz frequency range were made accessible by applying a quantum optical lock-in technique for the first time. This straightforward technique allowed quantum enhancement over a frequency range which reached as low as the range reported for squeezed light sources developed for gravity wave interferometers [4].

Neurons are classified according to action potential firing in response to current injection. While such firing patterns are shaped by the composition and distribution of ion channels, modelling studies suggest that the geometry of dendritic branches also influences temporal firing patterns. Verifying this link is crucial to understanding how neurons transform their inputs to output but has so far been technically challenging. Here, we investigate branching-dependent firing by pruning the dendritic tree of pyramidal neurons. We use a focused ultrafast laser to achieve highly localized and minimally invasive cutting of dendrites, thus keeping the rest of the dendritic tree intact and the neuron functional. We verify successful dendrotomy via two-photon uncaging of neurotransmitters before and after dendrotomy at sites around the cut region and via biocytin staining. Our results show that significantly altering the dendritic arborisation, such as by severing the apical trunk, enhances excitability in layer V cortical pyramidal neurons as predicted by simulations. This method may be applied to the analysis of specific relationships between dendritic structure and neuronal function. The capacity to dynamically manipulate dendritic topology or isolate inputs from various dendritic domains can provide a fresh perspective on the roles they play in shaping neuronal output.

We present a technique to improve the two-photon fluorescence yield from holographically projected multiple foci with less photo-damage on living cells. By positioning multiple foci to neurons in a circuit, we observed correlated calcium activity

The flow of information in the brain theorizes that each neuron in a network receives synaptic inputs and sends off its processed signals to neighboring neurons. Here, we model these synaptic inputs to understand how each neuron processes these inputs and transmits neurotransmitters to neighboring neurons. We use the NEURON simulation package to stimulate a neuron at multiple synaptic locations along its dendritic tree. Accumulation of multiple synaptic inputs causes changes in the neuron's membrane potential leading to firing of an action potential. Our simulations show that simultaneous synaptic stimulation approaches firing of an action potential at lesser inputs compared to sequential stimulation at multiple sites distributed along several dendritic branches.

We report a laser-scanning confocal reflectance microscope with a wavelet-profiled point spread function (PSF) for rapid multi-resolution extraction and analysis of microscopic object features. The PSF is generated via holography by encoding a π-phase shifting disk unto a collimated laser beam via a phase-only spatial light modulator (SLM) that is positioned at the pupil plane of the focusing objective lens. Scaling of the transverse PSF distribution is achieved by selecting a suitable ratio of the π-phase shifting disk radius and the pupil aperture radius. With one and the same objective lens and one SLM to control the phase profile of the pupil function, we produce amplitude PSF distributions that are accurate scaled representations of the circularly-symmetric Mexican hat mother wavelet.

We report parallel two-photon photopolymerization of microgear patterns by exposing a photoresist to holographically generated optical vortices. The optical vortices are created by imparting a helical pitch onto the incident light using a programmable lithographic phase mask realized with a computer addressable phase-only spatial light modulator. By varying the phase levels of the spatial light modulator, the truncated helical phase of an optical vortex results in output intensity patterns that typifies that of microgears instead of perfect doughnut beams. Our experiments and simulations are in good agreement implying a more efficient and highly parallel two-photon photopolymerization scheme that can be subsequently used for non-scanning fabrication of microgears.

We propose a modified design of the phase contrast filter to optimize the synthetic reference wave (SRW) in the generalized phase contrast method. The modified filter consists of a π-phase shifting disk surrounded by an annular ring to modulate the curvature of the intensity distribution generally associated with the SRW. We identify optimal filter parameters to yield a uniform intensity distribution within the working area. Performance tests where done for both periodic and arbitrary input phase patterns using the optimal filter parameters. Assessment of the quality of the projected intensity patterns suggests a more uniform intensity distribution at the output when using the modified filter.

In this talk, I will describe our efforts to use light to understand information processing in the brain. I will start with an established tool of using light for 3D brain imaging via multi-photon microscopy, where within fundamental optical limits, provides a good spatial range for studying single neurons up to interconnected neurons in a circuit. Within this spatial range, I will then talk about using patterned light to photo-induce synaptic inputs and analyze how the spatio-temporal organization of these inputs cause the neuron to fire an output. We use a dynamically programmable hologram to produce 3D light patterns that can induce targeted and highly localized synaptic inputs along the dendritic tree of a neuron. Such technique can also be used for light-based recording of responses from multiple neurons – leading to an all-optical method for stimulating and recording neuronal activity. To target neurons deep within the brain tissue, the hologram can be added with an adaptive phase-pattern to correct for optical aberrations caused by the brain tissue. Towards the end, I will discuss preliminary results on using non-linear light-tissue interactions to dynamically prune the neuronʼs dendritic tree. The neuronʼs output and overall function in a neuronal circuit are said to be dependent on the spatial extent of its dendritic tree. Hence, laser dendrotomy allows us to study morphology-dependent neuronal function. These experiments are among many light-neuron interactions that facilitate our understanding of how the brain works.

We demonstrate a novel technique for generating tuneable complex field distributions for controllable coupling to high-order guided modes of micro-structured fibres. The optical Fourier transform of grating-based phase patterns, which are encoded on a computer-controlled spatial light modulator, generates complex field distributions for selective launching of a desired mode. Both the amplitude and the phase of the programmable fields are modulated by straightforward and fast adjustments of simple pre-defined binary phase-only diffractive patterns. Experiments demonstrate tuneable coupling to the second-order guided modes of a commercially available index-guiding silica fibre with a triangular lattice air-hole micro-structure.

An all-optical manipulation method has been used for transferring both linear and angular momentum resulting in translation, rotation and alignment of multiple dielectric polystyrene particles and cell structures. The trapping-beams are generated using the generalised phase contrast method where a direct correspondence exists between the input phase modulation and the output intensity distribution. A computer-controlled spatial light modulator is used to produce a dynamically programmable input phase pattern that is transformed into parallel optical traps without the need of mechanical movement of the optical components. This is advantageous for future miniaturised systems, e.g. lab-on-a-chip and micro-fluidic devices. Our method provides for computer-controlled laser trapping-beams with real-time orientation of the particles in a micro-fluidic system.

TRPA1 is a non-selective cation channel involved in pain sensation and neurogenic inflammation. Although TRPA1 is well established in a number of organs including the nervous system, its presence and function in the mammalian cortex remains unclear. Here, we demonstrate the expression of TRPA1 in rodent somatosensory cortex through immunostaining and investigate its functional activation by whole-cell electrophysiology, Ca 2+ imaging and two-photon photoswitching. Application of TRPA1 agonist (AITC) and antagonist (HC-030031) produced significant modulation of activity in layer 5 (L5) pyramidal neurons in both rats and mice; AITC increased intracellular Ca 2+ concentrations and depolarized neurons, and both effects were blocked by HC-030031. These modulations were absent in the TRPA1 knockout mice. Next, we used optovin, a reversible photoactive molecule, to activate TRPA1 in individual L5 neurons of rat cortex. Optical control of activity was established by applying a tightly focused femtosecond-pulsed laser to optovin-loaded neurons. Light application depolarized neurons ( n = 17) with the maximal effect observed at λ = 720 nm. Involvement of TRPA1 was further confirmed by repeating the experiment in the presence of HC-030031, which diminished the light modulation. These results demonstrate the presence of TRPA1 in L5 pyramidal neurons and introduce a highly specific approach to further understand its functional significance.

This work was supported by the Australian Research Council Discovery Project (contract number DP140101555).

Crystalline TiO2 nanoparticles were produced by scalable flame spray pyrolysis of organometallic solutions. A protocol is presented for the optimized functionalization of these particles with fluorescein isothiocyanate (FITC), an important biomedical dye via a lysine linker. The pH, stoichiometry and time for lysine reaction were determined for highest dye loading and minimized degree of polylysine formation. Acidic reaction conditions, low lysine concentration and short reaction times were found to meet this aim. The resulting particles were used for imaging single neurons, showing high fluorescence emission and ability for the particles to diffuse into small neuron structures such as dendrites.

Neurons receive thousands of synaptic inputs that are distributed in space and time. The systematic study of how neurons process these inputs requires a technique to stimulate multiple yet highly targeted points of interest along the neuron's dendritic tree. Three-dimensional multi-focal patterns produced via holographic projection combined with two-photon photolysis of caged compounds can provide for highly localized release of neurotransmitters within each diffraction-limited focus, and in this way emulate simultaneous synaptic inputs to the neuron. However, this technique so far cannot achieve time-dependent stimulation patterns due to fundamental limitations of the hologram-encoding device and other factors that affect the consistency of controlled synaptic stimulation. Here, we report an advanced technique that enables the design and application of arbitrary spatio-temporal photostimulation patterns that resemble physiological synaptic inputs. By combining holographic projection with a programmable high-speed light-switching array, we have overcome temporal limitations with holographic projection, allowing us to mimic distributed activation of synaptic inputs leading to action potential generation. Our experiments uniquely demonstrate multi-site two-photon glutamate uncaging in three dimensions with submillisecond temporal resolution. Implementing this approach opens up new prospects for studying neuronal synaptic integration in four dimensions.

We report both subdiffraction-limited quantum metrology and quantum-enhanced spatial resolution for the first time in a biological context. Nanoparticles are tracked with quantum-correlated light as they diffuse through an extended region of a living cell in a quantum-enhanced photonic-force microscope. This allows spatial structure within the cell to be mapped at length scales down to 10 nm. Control experiments in water show a 14% resolution enhancement compared to experiments with coherent light. Our results confirm the long-standing prediction that quantum-correlated light can enhance spatial resolution at the nanoscale and in biology. Combined with state-of-the-art quantum light sources, this technique provides a path towards an order of magnitude improvement in resolution over similar classical imaging techniques.

Two-photon imaging using high-speed multi-channel detectors is a promising approach for optical recording of cellular membrane dynamics at multiple sites. A main bottleneck of this technique is the limited number of photons captured within a short exposure time (~1ms). Here, we implement temporal gating to improve the two-photon fluorescence yield from holographically projected multiple foci whilst maintaining a biologically safe incident average power. We observed up to 6x improvement in the signal-to-noise ratio (SNR) in Fluorescein and cultured hippocampal neurons showing evoked calcium transients. With improved SNR, we could pave the way to achieving multi-site optical recording of fluorogenic probes with response times in the order of ~1ms.

We use patterned 3D multi-spot illumination to perform neuronal multi-site stimulation in rat brain tissue. Using a spatial light modulator, we holograpically project 3D light fields for multi-site two-photon photolysis of caged neurotransmitters to generate synaptic inputs to a neuron. Controlled photostimulation of multiple synapses from various locations in the dendritic tree provides a way to analyze how neurons integrate multiple inputs. Our holographic projection setup is incorporated into a two-photon 3D imaging microscope for visualization and for accurate positioning of specific uncaging sites along the neuron's dendritic tree. We show two-photon images and the neuron's response to holographic photostimulation of synapses along dendrites.

We demonstrate simultaneous multi-site two-photon photolysis of caged neurotransmitters with close to diffraction-limited resolution in all three dimensions (3D). We use holographic projection of multiple focal spots, which allows full control over the 3D positions of uncaging sites with a high degree of localized excitation. Our system incorporates a two-photon imaging setup to visualize the 3D morphology of the neurons in order to accurately determine the photostimulation sites. We show its application to studies of synaptic integration by performing simultaneous and controlled glutamate delivery at multiple locations on dendritic trees. (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Light beams with helical phase profile correspond to photons having orbital angular momentum (OAM). A Laguerre-Gaussian (LG) beam is an example where its helical phase sets a phase-singularity at the optical axis and forms a ring-shaped transverse amplitude profile. Here, we describe a unique beam where both phase and amplitude express a helical profile as the beam propagates in free space. Such a beam can be accurately referred to as an optical twister. We characterize optical twisters and demonstrate their capacity to induce spiral motion on particles trapped along the twisters’ path. Unlike LG beams, the far field projection of the twisted optical beam maintains a high photon concentration even at higher values of topological charge. Optical twisters have therefore profound applications to fundamental studies of light and atoms such as in quantum entanglement of the OAM, toroidal traps for cold atoms and for optical manipulation of microscopic particles.

By the use of analytic continuation, the correct spectrum of an undersampled analog input signal fa(t) of a true bandwidth B is recovered from an aliased Fourier spectrum that is computed directly from a data set consisting of sinusoid-crossing locations {ti}, where the signal fa(t) intersects with a reference sinusoid r(t) with a frequency of W < B/2 and an amplitude of A. If A ≥ |fa(t)| within the sampling period T, then a crossing exists within each time interval Δ = 1/2W, and a total of 2WT = 2M sinusoid crossings are detected, where M is a positive integer. The cut-off frequency for sampling is W = ±M/T. In a crossing detector, a trade-off exists between the size of Δ and the accuracy with which a crossing can be located within it because the detector has a finite response time. Low-accuracy detection of the crossing positions degrades the detection limit of the detector and results in a computed Fourier spectrum that contains spurious wideband frequencies. We show however that, if fa(t) has a known compact support within T, then sampling at a frequency of W < B/2 may still be possible because the correct fa(t) spectrum can be recovered from the aliased spectrum by means of analytic continuation. The technique is demonstrated for an interferogram test signal in both the absence and presence of additive Gaussian noise.

We demonstrate a multi-functional system capable of multiple-site two-photon excitation of photo-sensitive compounds as well as transfer of optical mechanical properties on an array of mesoscopic particles. We use holographic projection of a single Ti:Sapphire laser operating in femtosecond pulse mode to show that the projected three-dimensional light patterns have sufficient spatiotemporal photon density for multi-site two-photon excitation of biological fluorescent markers and caged neurotransmitters. Using the same laser operating in continuous-wave mode, we can use the same light patterns for non-invasive transfer of both linear and orbital angular momentum on a variety of mesoscopic particles. The system also incorporates high-speed scanning using acousto-optic modulators to rapidly render 3D images of neuron samples via two-photon microscopy.

We use the holographic method to project an arbitrary array of diffraction-limited focal spots suitable for multi-site twophoton excitation. The spot array can be projected arbitrarily within a three-dimensional (3D) volume, while the fourth dimension in time is attributed to high temporal resolution via high-speed non-iterative calculation of the hologram using a video graphics accelerator board. We show that the spots have sufficient energy and spatiotemporal photon density for localized two-photon excitation at individual spots in the array. The significance of this work points to 3D microscopy, non-linear micro-fabrication, volume holographic optical storage and biomedical instrumentation. In neuroscience, timecritical release of neurotransmitters at multiple sites within complex dendritic trees of neurons can lead to insights on the mechanisms of information processing in the brain.

The performance of a zero-crossing-based optical spectrum analyzer is presented. With the use of only a single comparator, the analyzer circuit detects one zero crossing per Nyquist interval with a location accuracy of 1 part in 256. The dynamic range of sampling is limited only by the supply voltages of the comparator and the noise arising from truncation errors associated with computation. The 512-component spectra of various interferogram inputs are directly computed by Newton's formula from the measured crossings. The single-channel spectrum analyzer with a preselectable Nyquist sampling interval setting is constructed with discrete transistor–transistor logic and analog components. The ultimate performance characteristics of the analyzer are set by the response capabilities of the electronic components used.

We demonstrate dynamic and arbitrary multisite two-photon excitation in three dimensions using the holographic projection method. Rapid response (fourth dimension) is achieved through high-speed noniterative calculation of the hologram using a video graphics accelerator board. We verify that the projected asymmetric spot configurations have sufficient spatiotemporal photon density for localized two-photon excitation. This system is a significant advance and can be applied to time-resolved photolysis of caged compounds in biological cells and complex neuronal networks, nonlinear microfabrication and volume holographic optical storage., 10 pages including 4 figures

To gain a better understanding of the spatiotemporal problems that are encountered in two-photon excitation fluorescence imaging through highly scattering media, we investigate how diffraction affects the three-dimensional intensity distribution of a focused, pulsed optical beam propagating inside a scattering medium. In practice, the full potential of the two-photon excitation fluorescence imaging is unrealized at long scattering depths, owing to the unwanted temporal and spatial broadening of the femtosecond excitation light pulse that reduces the energy density at the geometric focus while it increases the excitation energy density in the out-of-focus regions. To analyze the excitation intensity distribution, we modify the Monte Carlo-based photon-transport model to a semi-quantum-mechanical representation that combines the wave properties of light with the particle behavior of the propagating photons. In our model the propagating photon is represented by a plane wave with its propagation direction in the scattering medium determined by the Monte Carlo technique. The intensity distribution in the focal region is given by the square of the linear superposition of the various plane waves that arrive at different incident angles and optical path lengths. In the absence of scattering, the propagation model yields the intensity distribution that is predicted by the Huygens-Fresnel principle. We quantify the decrease of the energy density delivered at the geometric focus as a function of the optical depth to the mean-free-path ratio that yields the average number of scattering events that a photon encounters as it propagates toward the focus. Both isotropic and anisotropic scattering media are considered. Three values for the numerical aperture (NA) of the focusing lens are considered: NA = 0.25, 0.5, 0.75.

We perform single cell analysis of cell growth in a mixed cell culture. Two species of yeast cells: Saccharomyces cerevisiae and Candida albicans, are optically trapped using focused continuous‐wave near infrared laser. Cell growth for both cells is inhibited only when the two species of cells are in contact with each other. This indicates cell‐cell interaction mediated cell growth inhibition mechanism. Single cell level analysis of cell growth studied here contributes to the further understanding of yeast growth arrest in a mixed yeast culture.

We demonstrate the feasibility of implementing the generalized phase-contrast (GPC) method in a planar-integrated micro-optics platform. An entire 4-f diffractive lens system with a Fourier plane phase-only filter has been designed and manufactured in a 50-mm-diameter fused-silica substrate by use of a four-level multimask lithography. Experimental results show successful conversion of an input phase distribution into a high-contrast intensity distribution. The diffraction efficiency of the optical components is found to limit the operating range of the GPC system.

We present what is to our knowledge a novel technique for efficient suppression of the zero-order beam inherent in light patterns projected via phase-only computer-generated holograms (CGHs). Encoding a CGH on a spatial light modulator (SLM) with a limited fill factor produces a disturbing zero-order beam at the optical axis. Here, we propose to derive a CGH, which includes holographic information to project a corrective beam that destructively interferes with the zero-order beam. The CGH for projecting arbitrary light patterns plus a corrective beam are derived using the Gerchberg-Saxton algorithm where the iterations impose both amplitude and phase constraints for the target field pattern at the Fourier plane. As proof of principle, we analyze the viability of the technique by simulating the performance when applied on a practical SLM with a limited fill factor, fixed number of phase-shifting pixels, and wavefront distortion associated with the surface roughness of the SLM.