Extended field-of-view ultrathin microendoscopes for high-resolution two-photon imaging with minimal invasiveness.

Imaging neuronal activity with high and homogeneous spatial resolution across the field-of-view (FOV) and limited invasiveness in deep brain regions is fundamental for the progress of neuroscience, yet is a major technical challenge. We achieved this goal by correcting optical aberrations in gradient index lens-based ultrathin (≤500 µm) microendoscopes using aspheric microlenses generated through 3D-microprinting. Corrected microendoscopes had extended FOV (eFOV) with homogeneous spatial resolution for two-photon fluorescence imaging and required no modification of the optical set-up. Synthetic calcium imaging data showed that, compared to uncorrected endoscopes, eFOV-microendoscopes led to improved signal-to-noise ratio and more precise evaluation of correlated neuronal activity. We experimentally validated these predictions in awake head-fixed mice. Moreover, using eFOV-microendoscopes we demonstrated cell-specific encoding of behavioral state-dependent information in distributed functional subnetworks in a primary somatosensory thalamic nucleus. eFOV-microendoscopes are, therefore, small-cross-section ready-to-use tools for deep two-photon functional imaging with unprecedentedly high and homogeneous spatial resolution.

Oblique scanning 2-photon light-sheet fluorescence microscopy for rapid volumetric imaging.

Light-sheet fluorescence microscopy (LSFM) is a powerful tool for biological studies because it allows for optical sectioning of dynamic samples with superior temporal resolution. However, LSFM using 2 orthogonally co-aligned objectives requires a special sample geometry, and volumetric imaging speed is limited due to physical sample translation. This paper describes an oblique scanning 2-photon LSFM (OS-2P-LSFM) that eliminates these limitations by using a single objective near the sample and a refractive scanning-descanning system. This system also provides improved light-sheet confinement against scattering by using a 2-photon Bessel beam. The OS-2P-LSFM hold promise for studying structural, functional and dynamic aspects of living tissues and organisms because it allows for high-speed, translation-free and scattering-robust 3D imaging of large biological specimens.

Ultrafast, large-field multiphoton microscopy based on an acousto-optic deflector and a spatial light modulator.

We present an ultrafast, large-field multiphoton excitation fluorescence microscope with high lateral and axial resolutions based on a two-dimensional (2-D) acousto-optical deflector (AOD) scanner and spatial light modulator (SLM). When a phase-only SLM is used to shape the near-infrared light from a mode-locked titanium:sapphire laser into a multifocus array including the 0-order beam, a 136 µm × 136 µm field of view is achieved with a 60× objective using a 2-D AOD scanner without any mechanical scan element. The two-photon fluorescence image of a neuronal network that was obtained using this system demonstrates that our microscopy permits observation of dynamic biological events in a large field with high-temporal and -spatial resolution.

Wide-band acousto-optic deflectors for large field of view two-photon microscope.

Acousto-optic deflector (AOD) is an attractive scanner for two-photon microscopy because it can provide fast and versatile laser scanning and does not involve any mechanical movements. However, due to the small scan range of available AOD, the field of view (FOV) of the AOD-based microscope is typically smaller than that of the conventional galvanometer-based microscope. Here, we developed a novel wide-band AOD to enlarge the scan angle. Considering the maximum acceptable acoustic attenuation in the acousto-optic crystal, relatively lower operating frequencies and moderate aperture were adopted. The custom AOD was able to provide 60 MHz 3-dB bandwidth and 80% peak diffraction efficiency at 840 nm wavelength. Based on a pair of such AOD, a large FOV two-photon microscope was built with a FOV up to 418.5 µm (40× objective). The spatiotemporal dispersion was compensated simultaneously with a single custom-made prism. By means of dynamic power modulation, the variation of laser intensity within the FOV was reduced below 5%. The lateral and axial resolution of the system were 0.58-2.12 µm and 2.17-3.07 µm, respectively. Pollen grain images acquired by this system were presented to demonstrate the imaging capability at different positions across the entire FOV.

Clinical study on the effects of a cosmetic product on dermal extracellular matrix components using a high-resolution multiphoton tomograph.

BACKGROUND/PURPOSE: The aim of this study was to demonstrate the effects of selected plant extracts in a cosmetic cream on the dermal network components after a 3-month treatment using an in vivo multiphoton tomographic device. METHODS: Twenty-four Caucasian women aged between 45 and 65 applied randomly a cosmetic emulsion B containing active ingredients (soy and jasmine) twice a day on one arm and its vehicle A (without active ingredients) on the other arm during 3 months. Measurements were performed on the internal side of the forearm before starting the treatment (T0), after 4 week (T4) and 12 weeks (T12) treatment. Measurements consisted of a multi-layers acquisitions using a multiphoton tomograph with subcellular resolution. Optical sections (about 6 microm thick) were recorded from 0 to about 200 microm using two different wavelengths: 760 and 820 nm. To compare the series of images and obtain an objective quantification of the signal of second harmonic generation (SHG) and autofluorescence, the method used consisted of taking the integrated brightness of an image (same rectangular area for all images) as a measure of the signal. Following this step, a ratio between brightness of images from the area treated with cream A or B and brightness of untreated area was calculated and used as an assessment of treatment efficacy. The parameter used for statistical analysis (variance analysis) is the difference before and after 12 weeks of treatment by either cream A or B of the signal ratios calculated in the upper dermis (118-130 microm) and those from a deeper region of the upper dermis (165-178 microm). RESULTS: Signals (autofluorescence+SHG) of extracellular matrix do not change significantly with time (weeks 0, 4 and 12) when cream A (vehicle with no active ingredient) is applied. Treatment with cream B results in an enhancement in the signal level of extracellular matrix at week 12. The comparison of signals, in both areas (118-130 microm and 160-178 microm), show an higher increase in the deeper region than in the more superficial one for product B while we do not notice any change with product A. CONCLUSION: The multiphoton tomograph provided excellent high-resolution images, which describe clearly the different skin layers, single cells and extracellular matrix components in all the 24 volunteers. Statistic analyses reveal a real effect for product B with selected plant extracts, known to increase collagen synthesis. Changes observed are characteristics of modifications in dermal collagen and elastin content. To our knowledge, it is the first time that it was possible to demonstrate in vivo the effect of a cosmetic product on the superficial dermal layer, in a non-invasive and non-destructive process, i.e. without cutting the skin.

Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging.

The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near-infrared laser into the scan head and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon EZ-C1 software without modification.

Multi-photon excitation of intrinsic protein fluorescence and its application to pharmaceutical drug screening.

The majority of proteins contain intrinsic fluorophores as natural sensors of molecular structures, dynamics, and interactions. The intrinsic protein fluorescence signal allows for the label-free and, hence, undisturbed and rapid study of protein-ligand interactions. Ultraviolet-based drug screening is hampered by the background, photobleaching, light scattering, inner filter effects, and interfering assay compounds. Such problems can be overcome by means of molecular three-photon excitation (3PE) with infrared femtosecond light pulses since longer excitation wavelengths result in less Raleigh scattering, and the subfemtoliter (confocal-like) 3PE volume minimizes out-of-focus photobleaching, background generation, and inner filter effects. We demonstrate the general feasibility of 3PE for protein spectroscopy and illustrate the technique's excellent potential for high-throughput screening. By using the intrinsic fluorescence intensity of a protein-substrate, we were able to discriminate between ligands of different affinities in binding assays.

Miniaturized two-photon microscope based on a flexible coherent fiber bundle and a gradient-index lens objective.

We present a miniature, flexible two-photon microscope consisting of a fused coherent optical fiber bundle with 30,000 cores and a gradient-index lens objective. The laser focus of a standard two-photon laser-scanning microscope was scanned over the entrance surface of the fiber bundle, resulting in sequential coupling into individual cores. Fluorescent light was detected through the fiber bundle. Micrometer-sized fluorescent beads and pollen grains were readily resolved. In addition, fluorescently labeled blood vessels were imaged through the fiber bundle in rat brain in vivo.

Imaging plant cells by two-photon excitation.

Along the past recent years, two-photon excitation (TPE) microscopy has moved from the realms of technical curiosity to be a standard application in many advanced cell biology laboratories. The growing body of literature covered in this review points out the obvious advantages of TPE over any other imaging method based on fluorescence, clearly improving signal-to-noise ratio and thick-tissue penetration and showing added potential for vital imaging. Like any new technology that has to gain its own space, TPE microscopy is still going through the growing pains in which reproducible protocols, probes, and applications are scarce. Yet, the published reports and unpublished results covered in this review point out that TPE can eventually accommodate most available protocols and probes, most of the times with evident advantages. Further, the potential for plant sciences is obvious, as plant cells possess many absorbing molecules and structures and are routinely more opaque than tissues of other organisms. Since prices make it one of the most expensive microscopies, TPE is coming slow to be a generalised technology, but enough data are emerging to establish it as a method with no alternative for some objectives.