Publications

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2016

Widefield fluorescence microscopy with sensor-based conjugate adaptive optics using oblique back illumination

Jiang Li, Thomas G. Bifano, Jerome Mertz,

Journal of Biomedical Optics

We describe a wavefront sensor strategy for the implementation of adaptive optics (AO) in microscope applications involving thick, scattering media. The strategy is based on the exploitation of multiple scattering to provide oblique back illumination of the wavefront-sensor focal plane, enabling a simple and direct measurement of the flux-density tilt angles caused by aberrations at this plane. Advantages of the sensor are that it provides a large measurement field of view (FOV) while requiring no guide star, making it particularly adapted to a type of AO called conjugate AO, which provides a large correction FOV in cases when sample-induced aberrations arise from a single dominant plane (e.g., the sample surface). We apply conjugate AO here to widefield (i.e., nonscanning) fluorescence microscopy for the first time and demonstrate dynamic wavefront correction in a closed-loop implementation.

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2015

High-resolution 3D phase imaging using a partitioned detection aperture: a wave-optic analysis

R. Barankov, J.-C. Baritaux, J. Mertz,

Journal of the Optical Society of America A

Quantitative phase imaging has become a topic of considerable interest in the microscopy community. We have recently described one such technique based on the use of a partitioned detection aperture, which can be operated in a single shot with an extended source [Opt. Lett. 37, 4062 (2012)]. We follow up on this work by providing a rigorous theory of our technique using paraxial wave optics, where we derive fully 3D spread functions for both phase and intensity. Using these functions, we discuss methods of phase reconstruction for in- and out-of-focus samples, insensitive to weak attenuations of light. Our approach provides a strategy for detection-limited lateral resolution with an extended depth of field and is applicable to imaging smooth and rough samples.

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Conjugate adaptive optics in widefield microscopy with an extended-source wavefront sensor

J. Li, D. R. Beaulieu, H. Paudel, R. Barankov, T. G. Bifano, and J. Mertz,

Optica

Subsurface microscopy is often limited by poor image quality due to sample-induced aberrations. Adaptive optics (AO) can counter such aberrations, though generally over limited fields of view. In most applications, AO is either slow or requires a “guide star” in the sample to serve as a localized reference target. We describe a fast closed-loop feedback implementation of AO that requires no guide stars, where the sample itself serves as the reference. Several features of our implementation are new. First, it is based on a high-resolution, single-shot wavefront sensor that is compatible with extended samples. Second, it is applied to widefield (i.e., nonscanning) microscopy in a conjugate AO configuration that increases field of view. Third, it makes use of a fast algorithm to identify sample-induced aberrations using illumination from an arbitrarily shaped source. We present the principle of our technique and proof-of-concept experimental demonstrations.

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Axial range of conjugate adaptive optics in two-photon microscopy

H. P. Paudel, J. Taranto, J. Mertz, and T. Bifano,

Optics Express

We describe an adaptive optics technique for two-photon microscopy in which the deformable mirror used for aberration compensation is positioned in a plane conjugate to the plane of the aberration. We demonstrate in a proof-of-principle experiment that this technique yields a large field of view advantage in comparison to standard pupil-conjugate adaptive optics. Further, we show that the extended field of view in conjugate AO is maintained over a relatively large axial translation of the deformable mirror with respect to the conjugate plane. We conclude with a discussion of limitations and prospects for the conjugate AO technique in two-photon biological microscopy.

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Field of view advantage of conjugate adaptive optics in microscopy applications

J. Mertz, H. Paudel, and T. G. Bifano,

Applied Optics

The imaging performance of an optical microscope can be degraded by sample-induced aberrations. A general strategy to undo the effect of these aberrations is to apply wavefront correction with a deformable mirror (DM). In most cases the DM is placed conjugate to the microscope pupil, called pupil adaptive optics (AO). When the aberrations are spatially variant an alternative configuration involves placing the DM conjugate to the main source of aberrations, called conjugate AO. We provide a theoretical and experimental comparison of both configurations for the simplified case where spatially variant aberrations are produced by a well-defined phase screen. We pay particular attention to the resulting correction field of view (FOV). Conjugate AO is found to provide a significant FOV advantage. While this result is well known in the astronomical community, our goal here is to recast it specifically for the optical microscopy community.

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2014

High-throughput imaging of self-luminous objects through a single optical fibre

R. Barankov, J. Mertz,

Nature Communications

Imaging through a single optical fibre offers attractive possibilities in many applications such as micro-endoscopy or remote sensing. However, the direct transmission of an image through an optical fibre is difficult because spatial information is scrambled upon propagation. We demonstrate an image transmission strategy where spatial information is first converted to spectral information. Our strategy is based on a principle of spread-spectrum encoding, borrowed from wireless communications, wherein object pixels are converted into distinct spectral codes that span the full bandwidth of the object spectrum. Image recovery is performed by numerical inversion of the detected spectrum at the fibre output. We provide a simple demonstration of spread-spectrum encoding using Fabry–Perot etalons. Our technique enables the two-dimensional imaging of self-luminous (that is, incoherent) objects with high throughput in principle independent of pixel number. Moreover, it is insensitive to fibre bending, contains no moving parts and opens the possibility of extreme miniaturization.

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Nonlinear Midinfrared Photothermal Spectroscopy Using Zharov Splitting and Quantum Cascade Lasers

A. Mertiri, H. Altug, M. K. Hong, P. Mehta, J. Mertz, L. D. Ziegler, S. Erramilli,

ACS Photonics

We report on the mid-infrared nonlinear photothermal spectrum of the neat liquid crystal 4-octyl-4′-cyanobiphenyl (8CB) using a tunable Quantum Cascade Laser (QCL). The nonequilibrium steady state characterized by the nonlinear photothermal infrared response undergoes a supercritical bifurcation. The bifurcation, observed in heterodyne two-color pump–probe detection, leads to ultrasharp nonlinear infrared spectra similar to those reported in the visible region. A systematic study of the peak splitting as a function of absorbed infrared power shows the bifurcation has a critical exponent of 0.5. The observation of an apparently universal critical exponent in a nonequilibrium state is explained using an analytical model analogous of mean field theory. Apart from the intrinsic interest for nonequilibrium studies, nonlinear photothermal methods lead to a dramatic narrowing of spectral lines, giving rise to a potential new contrast mechanism for the rapidly emerging new field of mid-infrared microspectroscopy using QCLs.

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View synthesis with a partitioned-aperture microscope

J.-C. Baritaux, C. R. Chan, J. Li, J. Mertz,

Optics Letters

We present a simple and fast algorithm for view synthesis based on the acquisition of four high-resolution oblique images with a conventional widefield microscope. The images are acquired simultaneously using a partitioned aperture add-on. The technique provides physically valid views of thin samples that are transmitting or fluorescent, as demonstrated with biopsied tissue or green fluorescent protein-labeled brain slices. The goal of this technique is to facilitate image interpretation by conferring impressions of depth that are otherwise absent in standard microscope images.

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Fast volumetric phase-gradient imaging in thick samples

J. D. Giese, T. N. Ford, J. Mertz,

Optics Express

Oblique back-illumination microscopy (OBM) provides high resolution, sub-surface phase-gradient images from arbitrarily thick samples. We present an image formation theory for OBM and demonstrate that OBM lends itself to volumetric imaging because of its capacity for optical sectioning. In particular, OBM can provide extended depth of field (EDOF) images from single exposures, by rapidly scanning the focal plane with an electrically tunable lens. These EDOF images can be further enhanced by deconvolution. We corroborate our theory with experimental volumetric images obtained from transparent bead samples and mouse cortical brain slices.

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Phase-gradient contrast in thick tissue with a scanning microscope

J. Mertz, A. Gasecka, A. Daradich, I. Davison, D. Cote ,

Biomedical Optics Express

It is well known that the principle of reciprocity is valid for light traveling even through scattering or absorptive media. This principle has been used to establish an equivalence between conventional widefield microscopes and scanning microscopes. We make use of this principle to introduce a scanning version of oblique back-illumination microscopy, or sOBM. This technique provides sub-surface phase-gradient and amplitude images from unlabeled tissue, in an epi-detection geometry. That is, it may be applied to arbitrarily thick tissue. sOBM may be implemented as a simple, cost-effective add-on with any scanning microscope, requiring only the availability of an extra input channel in the microscope electronics. We demonstrate here its implementation in combination with two-photon excited fluorescence (TPEF) microscopy and with coherent anti-Stokes Raman scattering (CARS) microscopy, applied to brain or spinal cord tissue imaging. In both cases, sOBM provides information on tissue morphology complementary to TPEF or CARS contrast. This information is obtained simultaneously and is automatically co-registered. Finally, we show that sOBM can be operated at video rate.

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2013

Single-exposure surface profilometry using partitioned aperture wavefront imaging

R. Barankov, J. Mertz,

Optics Letters

We demonstrate a technique for instantaneous measurements of surface topography based on the combination of a partitioned aperture wavefront imager with a lamp-based reflection microscope using standard objectives. The technique can operate at video rate over large fields of view, and provides nanometer axial resolution and submicrometer lateral resolution. We discuss performance characteristics of this technique, which we experimentally compare with scanning white light interferometry.

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Focusing polychromatic light through strongly scattering media

H. P. Paudel, C. Stockbridge, J. Mertz, T. Bifano,

Optics Express

We demonstrate feedback-optimized focusing of spatially coherent polychromatic light after transmission through strongly scattering media, and describe the relationship between optimized focus intensity and initial far-field speckle contrast. Optimization is performed using a MEMS spatial light modulator with camera-based or spectrometer-based feedback. We observe that the spectral bandwidth of the optimized focus depends on characteristics of the feedback signal. We interpret this dependence as a modification in the number of independent frequency components, or spectral correlations, transmitted by the sample, and introduce a simple model for polychromatic focus enhancement that is corroborated by experiment with calibrated samples.

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Video-rate imaging of microcirculation with single-exposure oblique back-illumination microscopy

T. N. Ford, J. Mertz,

Journal of Biomedical Optics

Oblique back-illumination microscopy (OBM) is a new technique for simultaneous, independent measurements of phase gradients and absorption in thick scattering tissues based on widefield imaging. To date, OBM has been used with sequential camera exposures, which reduces temporal resolution, and can produce motion artifacts in dynamic samples. Here, a variation of OBM that allows single-exposure operation with wavelength multiplexing and image splitting with a Wollaston prism is introduced. Asymmetric anamorphic distortion induced by the prism is characterized and corrected in real time using a graphics-processing unit. To demonstrate the capacity of single-exposure OBM to perform artifact-free imaging of blood flow, video-rate movies of microcirculation in ovo in the chorioallantoic membrane of the developing chick are presented. Imaging is performed with a highresolution rigid Hopkins lens suitable for endoscopy.

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2012

Phase-gradient microscopy in thick tissue with oblique back-illumination

Tim N. Ford, Kengyeh K. Chu, Jerome Mertz,

Nature Methods

Phase-contrast techniques, such as differential interference contrast microscopy, are widely used to obtain morphological images of unstained biological samples. The transillumination geometry required for these techniques restricts their application to thin samples. We introduce oblique back-illumination microscopy, a method of collecting en face phase-gradient images of thick scattering samples, enabling near-video-rate in vivo phase imaging with a miniaturized probe suitable for endoscopy.

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Quantitative phase imaging using a partitioned detection aperture

Ashwin B. Parthasarathy, Kengyeh K. Chu, Tim N. Ford, and Jerome Mertz,

Optics Letters

We present a technique to quantitatively image the phase of thin quasi-transparent samples using extended source incoherent illumination and off-axis detection apertures. Our technique is achromatic and polarization independent, requires no active elements, and can be readily adapted to standard bright-field microscopes. We demonstrate our technique by quantitatively reconstructing the phase of cheek cells and a microlens. The light efficient, single-shot nature of our technique enables phase imaging at frame rates that are camera limited.

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Single exposure complementary aperture phase microscopy with polarization encoding

K. K. Chu and J. Mertz,

Optics Letters

We introduce a single-exposure widefield system of producing phase gradient images by dividing the illumination and detection apertures into two oblique complementary components and encoding them using polarization. A Wollaston prism splits the images formed by the two respective aperture halves to allow both components to be simultaneously imaged by a single camera. By producing images characteristically similar to differential interference contrast while using a darkfield illumination scheme, sensitivity to weak phase gradients is improved.

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Pyramidal cells accumulate chloride at seizure onset

K. P. Lillis, M. A. Kramer, J. Mertz, K. J. Staley and J. A. White,

Neurobiology of Disease

Seizures are thought to originate from a failure of inhibition to quell hyperactive neural circuits, but the nature of this failure remains unknown. Here we combine high-speed two-photon imaging with electrophysiological recordings to directly evaluate the interaction between populations of interneurons and principal cells during the onset of seizure-like activity in mouse hippocampal slices. Both calcium imaging and dual patch clamp recordings reveal that in vitro seizure-like events (SLEs) are preceded by pre-ictal bursts of activity in which interneurons predominate. Corresponding changes in intracellular chloride concentration were observed in pyramidal cells using the chloride indicator Clomeleon. These changes were measurable at SLE onset and became very large during the SLE. Pharmacological manipulation of GABAergic transmission, either by blocking GABAA receptors or by hyperpolarizing the GABAA reversal potential, converted SLEs to short interictal-like bursts. Together, our results support a model in which pre-ictal GABAA receptor-mediated chloride influx shifts EGABA to produce a positive feedback loop that contributes to the initiation of seizure activity.

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Mid-infrared photothermal heterodyne spectroscopy in a liquid crystal using a quantum cascade laser

A. Mertiri, T. Jeys, V. Liberman, M. K. Hong, J. Mertz, H. Altug and S. Erramilli,

Applied Physics Letters

We report a technique to measure the mid-infrared photothermal response induced by a tunable quantum cascade laser in the neat liquid crystal 4-octyl-4′-cyanobiphenyl (8CB), without any intercalated dye. Heterodyne detection using a Ti:sapphire laser of the response in the solid, smectic, nematic and isotropic liquid crystal phases allows direct detection of a weak mid-infrared normal mode absorption using an inexpensive photodetector. At high pump power in the nematic phase, we observe an interesting peak splitting in the photothermal response. Tunable lasers that can access still stronger modes will facilitate photothermal heterodyne mid-infrared vibrational spectroscopy.

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Fast optically sectioned fluorescence HiLo endomicroscopy

Tim N. Ford, Daryl Lim, Jerome Mertz,

Journal of Biomedical Optics

We describe a nonscanning, fiber bundle endomicroscope that performs optically sectioned fluorescence imaging with fast frame rates and real-time processing. Our sectioning technique is based on HiLo imaging, wherein two widefield images are acquired under uniform and structured illumination and numerically processed to reject out-of-focus background. This work is an improvement upon an earlier demonstration of widefield optical sectioning through a flexible fiber bundle. The improved device features lateral and axial resolutions of 2.6 and 17 μm, respectively, a net frame rate of 9.5 Hz obtained by real-time image processing with a graphics processing unit (GPU) and significantly reduced motion artifacts obtained by the use of a double-shutter camera. We demonstrate the performance of our system with optically sectioned images and videos of a fluorescently labeled chorioallantoic membrane (CAM) in the developing G. gallus embryo. HiLo endomicroscopy is a candidate technique for low-cost, high-speed clinical optical biopsies.

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2011

Control of local intracellular calcium concentration with dynamic-clamp controlled 2-photon uncaging

E. Idoux and J. Mertz,

PLoS ONE

The variations of the intracellular concentration of calcium ion ([Ca2+]i) are at the heart of intracellular signaling, and their imaging is therefore of enormous interest. However, passive [Ca2+]i imaging provides no control over these variations, meaning that a full exploration of the functional consequences of [Ca2+]i changes is difficult to attain. The tools designed so far to modify [Ca2+]i, even qualitatively, suffer drawbacks that undermine their widespread use. Here, we describe an electrooptical technique to quantitatively set [Ca2+]i, in real time and with sub-cellular resolution, using two-photon Ca2+ uncaging and dynamic-clamp. We experimentally demonstrate, on neurons from acute olfactory bulb slices of Long Evans rats, various capabilities of this technique previously difficult to achieve, such as the independent control of the membrane potential and [Ca2+]i variations, the functional knocking-in of user-defined virtual voltage-dependent Ca2+ channels, and the standardization of [Ca2+]i patterns across different cells. Our goal is to lay the groundwork for this technique and establish it as a new and versatile tool for the study of cell signaling.

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Optical sectioning microscopy with planar or structured illumination

Jerome Mertz,

Nature Methods

A key requirement for performing three-dimensional (3D) imaging using optical microscopes is that they be capable of optical sectioning by distinguishing in-focus signal from out-of-focus background. Common techniques for fluorescence optical sectioning are confocal laser scanning microscopy and two-photon microscopy. But there is increasing interest in alternative optical sectioning techniques, particularly for applications involving high speeds, large fields of view or long-term imaging. In this Review, I examine two such techniques, based on planar illumination or structured illumination. The goal is to describe the advantages and disadvantages of these techniques.

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Chromatic and spherical aberration correction for silicon aplanatic solid immersion lens for fault isolation and photon emission microscopy of integrated circuits

B. B. Goldberg, A. Yurt, Y. Lu, E. Ramsay, F. H. Koklu, J. Mertz, T. G. Bifano and M. S. Unlu,

Microelectronics Reliability

Current state-of-the-art in backside fault isolation and logic analysis utilizes solid immersion lens (SIL) imaging in the central configuration. An attractive advancement is the development and integration of an aplanatic SIL, which allows significant improvement in resolution, signal acquisition and isolation capabilities, especially for the 22 nm node and beyond. However, aplanatic SIL configurations introduce both chromatic and spherical aberrations. We have developed backing objective designs capable of correcting for chromatic aberrations allowing application in photon emission microscopy, as well as deformable mirror designs and experiments that eliminate spherical aberrations of aplanatic SILs to account for variations in substrate thickness and off-axis imaging.

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Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

Daryl Lim, Tim N. Ford, Kengyeh K. Chu, Jerome Mertz,

Journal of Biomedical Optics

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

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2010

Practical implementation of log-scale active illumination microscopy

K. K. Chu, D. Lim and J. Mertz,

Biomedical Optics Express

Active illumination microscopy (AIM) is a method of redistributing dynamic range in a scanning microscope using real-time feedback to control illumination power on a sub-pixel time scale. We describe and demonstrate a fully integrated instrument that performs both feedback and image reconstruction. The image is reconstructed on a logarithmic scale to accommodate the dynamic range benefits of AIM in a single output channel. A theoretical and computational analysis of the influence of noise on active illumination feedback is presented, along with imaging examples illustrating the benefits of AIM. While AIM is applicable to any type of scanning microscope, we apply it here specifically to two-photon microscopy.

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Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection

J. Mertz and J. Kim,

Journal of Biomedical Optics

t is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

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2009

Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle

Silvia Santos, Kengyeh K. Chu, Daryl Lim, Nenad Bozinovic, Tim N. Ford, Claire Hourtoule, Aaron C. Bartoo, Satish K. Singh, Jerome Mertz,

Journal of Biomedical Optics

We present an endomicroscope apparatus that exhibits out-of-focus background rejection based on wide-field illumination through a flexible imaging fiber bundle. Our technique, called HiLo microscopy, involves acquiring two images, one with grid-pattern illumination and another with standard uniform illumination. An evaluation of the image contrast with grid-pattern illumination provides an optically sectioned image with low resolution. This is complemented with high-resolution information from the uniform illumination image, leading to a full-resolution image that is optically sectioned. HiLo endomicroscope movies are presented of fluorescently labeled rat colonic mucosa.

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Two-photon fluorescence microscopy with differential aberration imaging

K. K. Chu, A. Leray, T. G. Bifano and J. Mertz,

Proceedings of the SPIE

We report our progress in the development of Differential Aberration Imaging (DAI), a technique that enhances two-photon fluorescence (TPEF) microscopy by improving rejection of out-of-focus background by means of a deformable mirror (DM). The DM is used to intentionally add aberrations to the imaging system, which causes dramatic losses to in-focus signal while preserving the bulk of the out-of-focus background. By taking the difference between TPEF images with and without added aberrations, the out-of-focus portion of the signal is further rejected. We now introduce an implementation of DAI using a new type of DM that can be produced at much lower cost.

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2008

Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy

D. Lim, K. K. Chu and J. Mertz,

Optics Letters

We describe a method of obtaining optical sectioning with a standard wide-field fluorescence microscope. The method involves acquiring two images, one with nonuniform illumination (in our case, speckle) and another with uniform illumination (in our case, randomized speckle). An evaluation of the local contrast in the speckle-illumination image provides an optically sectioned image with low resolution. This is complemented with high-resolution information obtained from the uniform-illumination image. A fusion of both images leads to a full resolution image that is optically sectioned across all spatial frequencies. This hybrid illumination method is fast, robust, and generalizable to a variety of illumination and imaging configurations.

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Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution

K. P. Lillis, A. Eng, J. A. White and J. Mertz,

Journal of Neuroscience Methods

We describe a simple two-photon fluorescence imaging strategy, called targeted path scanning (TPS), to monitor the dynamics of spatially extended neuronal networks with high spatiotemporal resolution. Our strategy combines the advantages of mirror-based scanning, minimized dead time, ease of implementation, and compatibility with high-resolution low-magnification objectives. To demonstrate the performance of TPS, we monitor the calcium dynamics distributed across an entire juvenile rat hippocampus (>1.5 mm), at scan rates of 100 Hz, with single cell resolution and single action potential sensitivity. Our strategy for fast, efficient two-photon microscopy over spatially extended regions provides a particularly attractive solution for monitoring neuronal population activity in thick tissue, without sacrificing the signal-to-noise ratio or high spatial resolution associated with standard two-photon microscopy. Finally, we provide the code to make our technique generally available.

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Autoconfocal microscopy with a cw laser and thermionic detection

Daryl Lim, Kengyeh K. Chu, and Jerome Mertz,

Optics Letters

We introduce an application of thermionic emission in a PMT photocathode. Because of the nonlinear dependence of thermionic emission on absorbed laser power, a conventional PMT is found to produce a virtual pinhole effect that rejects unfocused light at least as strongly as a physical pinhole. This virtual pinhole effect is exploited in a scanning transmission confocal microscope equipped with a cw laser source. Because the area of the PMT photocathode is large, signal descanning is not required and thermionic detection acts as a self-aligned pinhole. Our technique of thermionic-detection autoconfocal microscopy is further implemented with graded-field contrast to obtain enhanced phase-gradient sensitivity in unlabeled samples, such as rat hippocampal brain slices.

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Bonding of macromolecular hydrogels using perturbants

G. M. Price, K. K. Chu, J. G. Truslow, M. D. Tang-Schomer, A. P. Golden, J. Mertz and J. Tien,

Journal of the American Chemical Society

This work describes a method to bond patterned macromolecular gels into monolithic structures using perturbants. Bonding strengths for a variety of solutes follow a Hofmeister ordering; this result and optical measurements indicate that bonding occurs by reversible perturbation of contacting gels. The resulting microfluidic gels are mechanically robust and can serve as scaffolds for cell culture.

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Fluorescence endomicroscopy with structured illumination

Nenad Bozinovic, Cathie Ventalon, Tim Ford, Jerome Mertz,

Optics Express

We present an endomicroscope apparatus that utilizes structured illumination to produce high resolution (~2.6µm) optically sectioned fluorescence images over a field of view of about 240µm. The endomicroscope is based on the use of a flexible imaging fiber bundle with a miniaturized objective. We also present a strategy to largely suppress structured illumination artifacts that arise when imaging in thick tissue that exhibits significant out-of-focus background. To establish the potential of our endomicroscope for preclinical or clinical applications, we provide images of BCECF-AM labeled rat colonic mucosa.

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Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy

A. Leray, K. Lillis and J. Mertz,

Biophysical Journal

When a two-photon excited fluorescence (TPEF) microscope is used to image deep inside tissue, out-of-focus background can arise from both ballistic and nonballistic excitation. We propose a solution to largely reject TPEF background in thick tissue. Our technique is based on differential-aberration imaging with a deformable mirror. By introducing extraneous aberrations in the excitation beam path, we preferentially quench in-focus TPEF signal while leaving out-of-focus TPEF background largely unchanged. A simple subtraction of an aberrated, from an unaberrated, TPEF image then removes background while preserving signal. Our differential aberration (DA) technique is simple, robust, and can readily be implemented with standard TPEF microscopes with essentially no loss in temporal resolution when using a line-by-line DA protocol. We analyze the performance of various induced aberration patterns, and demonstrate the effectiveness of DA-TPEF by imaging GFP-labeled sensory neurons in a mouse olfactory bulb and CA1 pyramidal cells in a hippocampus slice.

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2007

Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination

K. K. Chu, D. Lim and J. Mertz,

Optics Letters

We describe a technique to enhance both the weak-signal relative sensitivity and the dynamic range of a laser scanning optical microscope. The technique is based on maintaining a fixed detection power by fast feedback control of the illumination power, thereby transferring high measurement resolution to weak signals while virtually eliminating the possibility of image saturation. We analyze and demonstrate the benefits of adaptive illumination in two-photon fluorescence microscopy.

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Dynamic speckle illumination microscopy with wavelet prefiltering

C. Ventalon, R. Heintzmann and J. Mertz,

Optics Letters

Dynamic speckle illumination (DSI) provides a simple and robust technique to obtain fluorescence depth sectioning with a widefield microscope. We report a significant improvement to DSI microscopy based on a statistical image-processing algorithm that incorporates spatial wavelet prefiltering. The resultant gain in sectioning strength leads to a fundamentally improved scaling law for the out-of-focus background rejection.

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Graded-field autoconfocal microscopy

Kengyeh K. Chu, Ran Yi, and Jerome Mertz,

Optics Express

Autoconfocal microscopy (ACM) is a simple implementation of a transmitted-light confocal microscopy where a nonlinear detector plays the role of a virtual self-aligned pinhole. We report here a significant improvement of ACM based on the use of graded-field (GF) imaging. The technique of GF imaging involves introducing partial beam blocks in the illumination and detection apertures of an imaging system. These partial beam blocks confer phase-gradient sensitivity to the imaging system and allow control over its background level. We present the theory of the GF contrast in the context of ACM, comparing it to GF contrast in a non-scanning widefield microscope, and discuss various performance characteristics of GF-ACM in terms of resolution, sectioning strength, and an “under-detection” light collection geometry. An advantage of ACM is that it can be readily combined with two-photon excited fluorescence (TPEF) microscopy. We present images of rat brain hippocampus using simultaneous GF-ACM and TPEF microscopy. These images are inherently co-registered.

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2006

Rejection of two-photon fluorescence background in thick tissue by differential aberration imaging

A. Leray and J. Mertz,

Optics Express

We present a simple and robust way to reject out-of-focus background when performing deep two-photon excited fluorescence (TPEF) imaging in thick tissue. The technique is based on the use of a deformable mirror (DM) to introduce illumination aberrations that preferentially degrade TPEF signal while leaving TPEF background relatively unchanged. A subtraction of aberrated from unaberrated images leads to background rejection. We present a heuristic description of our technique, which we corroborate with experiment. An added benefit of our technique is that it leads to somewhat improved image resolution.

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Dynamic speckle illumination microscopy with translated versus randomized speckle patterns

C. Ventalon and J. Mertz,

Optics Express

Dynamic speckle illumination (DSI) microscopy is a widefield fluorescence imaging technique that provides depth discrimination. The technique relies on the illumination of a sample with a sequence of speckle patterns. We consider an image processing algorithm based on a differential intensity variance between consecutive images, and demonstrate that DSI sectioning strength depends on the dynamics of the speckle pattern. Translated speckle patterns confer greater sectioning strength than randomized speckle patterns because they retain out-of-focus correlations that lead to better background rejection. We present a theory valid for arbitrary point-spread-functions, which we corroborate with experimental results.

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Graded-field microscopy with white light

Ran Yi, Kengyeh K. Chu, and Jerome Mertz,

Optics Express

We present a general imaging technique called graded-field microscopy for obtaining phase-gradient contrast in biological tissue slices. The technique is based on introducing partial beam blocks in the illumination and detection apertures of a standard white-light widefield transillumination microscope. Depending on the relative aperture sizes, one block produces phase-gradient contrast while the other reduces brightfield background, allowing a full operating range between brightfield and darkfield contrast. We demonstrate graded-field imaging of neurons in a rat brain slice.

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Membrane potential detection with second-harmonic generation and two-photon excited fluorescence: a theoretical comparison

T. Pons and J. Mertz,

Optics Communications

We theoretically compare the performance of TPEF and SHG microscopy for membrane potential imaging. We argue that electrochromic TPEF and SHG membrane potential responses are reflections of the same phenomenon, and can be described in a unified manner as resulting from the linear Stark effect. We also show that TPEF and SHG exhibit similar sensitivities in the case of both electrochromic and orientational response mechanisms. Despite their similar sensitivities, SHG nevertheless presents advantages over TPEF for membrane potential imaging because of its remarkable spatial and spectral contrast, and because of its insensitivity to non-radiative excited-state damping mechanisms.

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2005

Quasi-confocal fluorescence sectioning with dynamic speckle illumination

C. Ventalon and J. Mertz,

Optics Letters

We present a simple modification to a conventional wide-field fluorescence microscope that provides depth discrimination in thick tissues. The technique consists of illuminating a sample with a sequence of independent speckle patterns and displaying the rms of the resultant sequence of fluorescence images. The advantage of speckle illumination is that it provides diffraction-limited illumination granularity that is highly contrasted even in scattering media. We demonstrate quasi-confocal imaging in a mouse olfactory bulb labeled with green fluorescent protein.

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Effects of (multi)branching of dipolar chromophores on photophysical properties and two-photon absorption

C. Katan, F. Terenziani, O. Mongin, M. H. V. Werts, L. Porrès, T. Pons, J. Mertz, S. Tretiak and M. Blanchard-Desce,

Journal of Physical Chemistry A

To investigate the effect of branching on linear and nonlinear optical properties, a specific series of chromophores, epitome of (multi)branched dipoles, has been thoroughly explored by a combined theoretical and experimental approach. Excited-state structure calculations based on quantum-chemical techniques (time-dependent density functional theory) as well as a Frenkel exciton model nicely complement experimental photoluminescence and one- and two-photon absorption findings and contribute to their interpretation. This allowed us to get a deep insight into the nature of fundamental excited-state dynamics and the nonlinear optical (NLO) response involved. Both experiment and theory reveal that a multidimensional intramolecular charge transfer takes place from the donating moiety to the periphery of the branched molecules upon excitation, while fluorescence stems from an excited state localized on one of the dipolar branches. Branching is also observed to lead to cooperative enhancement of two-photon absorption (TPA) while maintaining high fluorescence quantum yield, thanks to localization of the emitting state. The comparison between results obtained in the Frenkel exciton scheme and ab initio results suggests the coherent coupling between branches as one of the possible mechanisms for the observed enhancement. New strategies for the rational design of NLO molecular assemblies are thus inferred on the basis of the acquired insights.

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2004

Two-photon absorption and fluorescence with quadrupolar and branched chromophores – effect of structure and branching

L. Porrès, O. Mongin, C. Katan, M. Charlot, B. K. G. Batthula, V. Jouikov, T. Pons, J. Mertz and M. Blanchard-Desce,

Journal of Nonlinear Optical Physics & Materials

The photophysical and two-photon absorption (TPA) properties of three homologous quadrupolar and one related three-branched chromophores were investigated. Design of the quadrupoles is based on the symmetrical functionalization of a biphenyl core. Modulation of the nonlinear absorptivity/transparency/photostability trade-off can be achieved by playing with the twist angle of the core and on the spacers (phenylene-vinylene versus phenylene-ethynylene). The quadrupolar chromophores combine high TPA cross-sections, high fluorescence quantum yield and solvent sensitive photoluminescence properties. The branched structure exhibits spectrally broadened TPA in the NIR region (up to 3660 GM at 740 nm measured in the femtosecond regime) but reduced sensitivity to the environment.

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Two-photon absorption and fluorescence in nanoscale multipolar chromophores: effect of dimensionality and charge-symmetry

L. Porrès, C. Katan, O. Mongin, T. Pons, J. Mertz and M. Blanchard-Desce,

Journal of Molecular Structure

A series of structurally related chromophores of different symmetry (quadrupolar, C2v, octupolar,…) and shape (rod-like, propeller-shaped, Y-shaped, dendritic,…) were investigated and compared for optimization of molecular two-photon absorption (TPA). Their design is based on the functionalization of linear or branched conjugated backbones with electron-releasing and/or electron-withdrawing peripheral groups. Their TPA spectra were determined by investigating their two-photon-excited fluorescence properties in the NIR region using pulsed excitation in the femtosecond regime. These studies provide evidence that the charge symmetry plays an important role in determining the TPA magnitude, the quadrupolar chromophore leading to the highest TPA cross-section. However, higher-order charge symmetries and branched structures provide an interesting route for improvement of the non-linear absorptivity/transparency range trade-off as well as for TPA spectral broadening in the NIR region.

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Nonlinear microscopy: new techniques and applications

Jerome Mertz,

Current Opinion in Neurobiology

Nonlinear microscopy, a general term that embraces any microscopy technique based on nonlinear optics, is further establishing itself as an important tool in neurobiology. Recent advances in labels, labeling techniques, and the use of native or genetically encoded contrast agents have bolstered the capacity of nonlinear microscopes to image the structure and function of not just single cells but of entire networks of cells. Along with novel strategies to image over exceptionally long durations and with increased depth penetration in living brains, these advances are opening new opportunities in neurobiology that were previously unavailable.

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Autoconfocal microscopy with nonlinear transmitted light detection

T. Pons and J. Mertz,

Journal of the Optical Society of America B - Optical Physics

We describe a simple and robust technique for transmission confocal laser scanning microscopy wherein the detection pinhole is replaced by a thin second-harmonic generation crystal. The advantage of this technique is that self-aligned confocality is achieved without the need for signal descanning. We derive the point-spread function of our instrument and quantify both signal degradation and background rejection when imaging deep within a turbid slab. As an example, we consider a slab whose index of refraction fluctuations exhibit Gaussian statistics. Our model is corroborated by experiment.

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Enhanced two-photon absorption with novel octupolar propeller-shaped fluorophores derived from triphenylamine

L. Porrès, O. Mongin, C. Katan, M. Charlot, T. Pons, J. Mertz and M. Blanchard-Desce,

Organic Letters

Novel octupolar fluorophores derived from the symmetrical functionalization of a triphenylamine core with strong acceptor peripheral groups via phenylene-ethynylene linkers have been synthesized and shown to exhibit high fluorescence quantum yields, very large TPA cross-sections in the red−NIR region, and suitable photostability.

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