Jordi Andilla

PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking

We present a novel approach for three-dimensional localization of single molecules using adaptive optics. A 52-actuator deformable mirror is used to both correct aberrations and induce two-dimensional astigmatism in the point-spread-function. The dependence of the z-localization precision on the degree of astigmatism is discussed. We achieve a z-localization precision of 40 nm for fluorescent proteins and 20 nm for fluorescent dyes, over an axial depth of ~800 nm. We illustrate the capabilities of our approach for three-dimensional high-resolution microscopy with super-resolution images of actin filaments in fixed cells and single-molecule tracking of quantum-dot labeled transmembrane proteins in live HeLa cells.

Fast generation of holographic optical tweezers by random mask encoding of Fourier components

The random mask encoding technique of multiplexing phase-only filters can be easily adapted to the generation of holographic optical tweezers. The result is a direct, non-iterative and extremely fast algorithm that can be used for computing arbitrary arrays of optical traps. Additional benefits include the possibility of modifying any existing hologram to quickly add more trapping sites and the inexistence of ghost traps or replicas.

Design strategies for optimizing holographic optical tweezers set-ups

We provide a detailed account of the construction of a system of holographic optical tweezers. While a lot of information is available on the design, alignment and calibration of other optical trapping configurations, those based on holography are relatively poorly described. Inclusion of a spatial light modulator in the set-up gives rise to particular design trade-offs and constraints, and the system benefits from specific optimization strategies, which we discuss.

Correction of aberration in holographic optical tweezers using a Shack-Hartmann sensor

Optical aberration due to the nonflatness of spatial light modulators used in holographic optical tweezers significantly deteriorates the quality of the trap and may easily prevent stable trapping of particles. We use a Shack-Hartmann sensor to measure the distorted wavefront at the modulator plane; the conjugate of this wavefront is then added to the holograms written into the display to counteract its own curvature and thus compensate the optical aberration of the system. For a Holoeye LC-R 2500 reflective device, flatness is improved from 0.8λ to λ/16 (λ=532 nm), leading to a diffraction-limited spot at the focal plane of the microscope objective, which makes stable trapping possible. This process could be fully automated in a closed-loop configuration and would eventually allow other sources of aberration in the optical setup to be corrected for.

Decoupled illumination detection in light sheet microscopy for fast volumetric imaging

Current microscopy demands the visualization of large three-dimensional samples with increased sensitivity, higher resolution, and faster speed. Several imaging techniques based on widefield, point-scanning, and light-sheet strategies have been designed to tackle some of these demands. Although successful, all these require the illuminated volumes to be tightly coupled with the detection optics to accomplish efficient optical sectioning. Here, we break this paradigm and produce optical sections from out-of-focus planes. This is done by extending the depth of field of the detection optics in a light-sheet microscope using wavefront-coding techniques. This passive technique allows accommodation of the light sheet at any place within the extended axial range. We show that this enables quick scanning of the light sheet across a volumetric sample. As a consequence, imaging speeds faster than twice the volumetric video rate (>70  volumes/s) can be achieved without needing to move the sample. These capabilities are demonstrated for volumetric imaging of fast dynamics in vivo as well as for fast, three-dimensional particle tracking.

HoloTrap: Interactive hologram design for multiple dynamic optical trapping

Abstract This work presents an application that generates real-time holograms to be displayed on a holographic optical tweezers setup; a technique that allows the manipulation of particles in the range from micrometres to nanometres. The software is written in Java, and uses random binary masks to generate the holograms. It allows customization of several parameters that are dependent on the experimental setup, such as the specific characteristics of the device displaying the hologram, or the presence of aberrations. We evaluate the softwares performance and conclude that real-time interaction is achieved. We give our experimental results from manipulating 5 μm microspheres using the program. Program summary Title of program: HoloTrap Catalogue identifier:ADZB_v1_0 Program summary URL: http://cpc.cs.qub.ac.uk/summaries/ADZB_v1_0 Program obtainable from: CPC Program Library, Queens University of Belfast, N. Ireland Computer for which the program is designed and others on which it has been tested: General computer Operating systems or monitors under which the program has been tested: Windows, Linux Programming language used: Java Memory required to execute with typical data: up to 34 MB including the Java Virtual Machine No. of bits in a word: 8 bits No. of processors used: 1 Has the code been vectorized or parallelized?: No No. of lines in distributed program, including test data, etc.: 471 145 No. of bytes in distributed program, including test data, etc.: 1 141 457 Distribution format: tar.gz Nature of physical problem: To calculate and display holograms for generating multiple and dynamic optical tweezers to be reconfigured interactively. Method of solution: Fast random binary mask for the simultaneous codification of multiple phase functions into a phase modulation device. Typical running time: Up to 10 frames per second Unusual features of the program: None References: The method for calculating holograms can be found in [M. Montes-Usategui, E. Pleguezuelos, J. Andilla, E. Martin-Badosa, Fast generation of holographic optical tweezers by random mask encoding of Fourier components, Opt. Express 14 (2006) 2101–2107].

Imaging tissue-mimic with light sheet microscopy: A comparative guideline

Tissue mimics (TMs) on the scale of several hundred microns provide a beneficial cell culture configuration for in vitro engineered tissue and are currently under the spotlight in tissue engineering and regenerative medicine. Due to the cell density and size, TMs are fairly inaccessible to optical observation and imaging within these samples remains challenging. Light Sheet Fluorescence Microscopy (LSFM)- an emerging and attractive technique for 3D optical sectioning of large samples- appears to be a particularly well-suited approach to deal with them. In this work, we compared the effectiveness of different light sheet illumination modalities reported in the literature to improve resolution and/or light exposure for complex 3D samples. In order to provide an acute and fair comparative assessment, we also developed a systematic, computerized benchmarking method. The outcomes of our experiment provide meaningful information for valid comparisons and arises the main differences between the modalities when imaging different types of TMs.

Light-sheet microscopy: a tutorial

This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.

Holographic optical manipulation of motor-driven membranous structures in living NG-108 cells

Optical tweezer experiments have partially unveiled the me- chanical properties of processive motor proteins while driving polysty- rene or silica microbeads in vitro. However, the set of forces underlying the more complex transport mechanisms in living samples remains poorly understood. Several studies have shown that optical tweezers are capable of trapping vesicles and organelles in the cytoplasm of living cells, which can be used as handles to mechanically interact with en- gaged active motors, or other components regulating transport. This may ultimately enable the exploration of the mechanics of this trafficking mechanism in vivo. These cell manipulation experiments have been car- ried out using different strategies to achieve dynamic beam steering ca- pable of trapping these subcellular structures. We report here the first trapping and manipulation, to our knowledge, of such small motor- propelled cargos in living cells using holographic technology.

Design of a low-cost interactive holographic optical tweezers system

The paper describes the design of an inexpensive holographic optical tweezers setup. The setup is accompanied by software that allows real-time manipulation of the sample and takes into account the experimental features of the setup, such as aberration correction and LCD modulation. The LCD, a HoloEye LCR-2500, is the physical support of the holograms, which are calculated using the fast random binary mask algorithm. The real-time software achieves 12 fps at full LCD resolution (including aberration correction and modulation) when run on a Pentium IV HT, 3.2 GHz computer.