Enhanced neuroimaging with a calcium sensor in ex-vivo Drosophila melanogaster brains using closed-loop adaptive optics light-sheet fluorescence microscopy.

Significance: Adaptive optics (AO) has been implemented on several microscopy setups and has proven its ability to increase both signal and resolution. However, reported configurations are not suited for fast imaging of live samples or are based on an invasive or complex implementation method. Aim: Provide a fast aberration correction method with an easy to implement AO module compatible with light-sheet fluorescence microscopy (LSFM) for enhanced imaging of live samples. Approach: Development of an AO add-on module for LSFM based on direct wavefront sensing without requiring a guide star using an extended-scene Shack-Hartmann wavefront sensor. The enhanced setup uses a two-color sample labeling strategy to optimize the photon budget. Results: Fast AO correction of in-depth aberrations in an ex-vivo adult Drosophila brain enables doubling the contrast when imaging with either cell reporters or calcium sensors for functional imaging. We quantify the gain in terms of image quality on different functional domains of sleep neurons in the Drosophila brain at various depths and discuss the optimization of key parameters driving AO. Conclusion: We developed a compact AO module that can be integrated into most of the reported light-sheet microscopy setups, provides significant improvement of image quality and is compatible with fast imaging requirements such as calcium imaging.

Single-shot quantitative aberration and scattering length measurements in mouse brain tissues using an extended-source Shack-Hartmann wavefront sensor.

Deep fluorescence imaging in mammalian brain tissues remains challenging due to scattering and optical aberration-induced loss in signal and resolution. Correction of aberrations using adaptive optics (AO) requires their reliable measurement in the tissues. Here, we show that an extended-source Shack-Hartmann wavefront sensor (ESSH) allows quantitative aberration measurements through fixed brain slices with a thickness up to four times their scattering length. We demonstrate in particular that this wavefront measurement method based on image correlation is more robust to scattering compared to the standard centroid-based approach. Finally, we obtain a measurement of the tissue scattering length taking advantage of the geometry of a Shack-Hartmann sensor.

Active image optimization for lattice light sheet microscopy in thick samples.

Lattice light-sheet microscopy (LLSM) is a very efficient technique for high resolution 3D imaging of dynamic phenomena in living biological samples. However, LLSM imaging remains limited in depth due to optical aberrations caused by sample-based refractive index mismatch. Here, we propose a simple and low-cost active image optimization (AIO) method to recover high resolution imaging inside thick biological samples. AIO is based on (1) a light-sheet autofocus step (AF) followed by (2) an adaptive optics image-based optimization. We determine the optimum AIO parameters to provide a fast, precise and robust aberration correction on biological samples. Finally, we demonstrate the performances of our approach on sub-micrometric structures in brain slices and plant roots.

EUV and Hard X-ray Hartmann Wavefront Sensing for Optical Metrology, Alignment and Phase Imaging.

For more than 15 years, Imagine Optic have developed Extreme Ultra Violet (EUV) and X-ray Hartmann wavefront sensors for metrology and imaging applications. These sensors are compatible with a wide range of X-ray sources: from synchrotrons, Free Electron Lasers, laser-driven betatron and plasma-based EUV lasers to High Harmonic Generation. In this paper, we first describe the principle of a Hartmann sensor and give some key parameters to design a high-performance sensor. We also present different applications from metrology (for manual or automatic alignment of optics), to soft X-ray source optimization and X-ray imaging.

Lensless microscopy platform for single cell and tissue visualization.

Today, 3D imaging techniques are emerging, not only as a new tool in early drug discovery but also for the development of potential therapeutics to treat disease. Particular efforts are directed towards in vivo physiology to avoid perturbing the system under study. Here, we assess non-invasive 3D lensless imaging and its impact on cell behavior and analysis. We test our concept on various bio-applications and present here the first results. The microscopy platform based on in-holography provides large fields of view images (several mm2 compared to several hundred µm2) with sub-micrometer spatial resolution. 3D image reconstructions are achieved using back propagation functions post-processing.

Study of contrast variations with depth in focused plenoptic cameras.

A focused plenoptic camera has the ability to record and separate spatial and directional information of the incoming light. Combined with the appropriate algorithm, a 3D scene could be reconstructed from a single acquisition, over a depth range called plenoptic depth-of-field. In this Letter, we study the contrast variations with depth as a way to assess plenoptic depth-of-field. We take into account the impact of diffraction, defocus, and magnification on the resulting contrast. We measure the contrast directly on both simulated and acquired images. We demonstrate the importance of diffraction and magnification in the final contrast. Contrary to classical optics, the maximum of contrast is not centered around the main object plane, but around a shifted position, with a fast and nonsymmetric decrease of contrast.

Adaptive optics light-sheet microscopy based on direct wavefront sensing without any guide star.

We propose an adaptive optics light-sheet fluorescence microscope (AO-LSFM) for closed-loop aberrations' correction at the emission path, providing intrinsic instrumental simplicity and high accuracy when compared to previously reported schemes. The approach is based on direct wavefront sensing, i.e., not on time-consuming iterative algorithms, and does not require the use of any guide star, thus reducing instrumental complexity and/or sample preparation constraints. The design is based on a modified Shack-Hartmann wavefront sensor providing compatibility with extended sources such as images from optical sectioning microscopes. We report an AO-LSFM setup based on such sensors, including characterization of the sensor performance, and demonstrate for the first time to the best of our knowledge a significant contrast improvement on neuronal structures of the ex vivo adult drosophila brain in depth.

Dynamic full field optical coherence tomography: subcellular metabolic contrast revealed in tissues by interferometric signals temporal analysis.

We developed a new endogenous approach to reveal subcellular metabolic contrast in fresh ex vivo tissues taking advantage of the time dependence of the full field optical coherence tomography interferometric signals. This method reveals signals linked with local activity of the endogenous scattering elements which can reveal cells where other OCT-based techniques fail or need exogenous contrast agents. We benefit from the micrometric transverse resolution of full field OCT to image intracellular features. We used this time dependence to identify different dynamics at the millisecond scale on a wide range of organs in normal or pathological conditions.

High-resolution handheld rigid endomicroscope based on full-field optical coherence tomography.

Full-field optical coherence tomography (FF-OCT) is a powerful tool for nondestructive assessment of biological tissue, i.e., for the structural examination of tissue in depth at a cellular resolution. Mostly known as a microscopy device for ex vivo analysis, FF-OCT has also been adapted to endoscopy setups since it shows good potential for in situ cancer diagnosis and biopsy guidance. Nevertheless, all the attempts to perform endoscopic FF-OCT imaging did not go beyond lab setups. We describe here, to the best of our knowledge, the first handheld FF-OCT endoscope based on a tandem interferometry assembly using incoherent illumination. A common-path passive imaging interferometer at the tip of an optical probe makes it robust and insensitive to environmental perturbations, and a low finesse Fabry-Perot processing interferometer guarantees a compact system. A good resolution (2.7 µm transverse and 6 µm axial) is maintained through the long distance, small diameter relay optics of the probe, and a good signal-to-noise ratio is achieved in a limited 100 ms acquisition time. High-resolution images and a movie of a rat brain slice have been recorded by moving the contact endoscope over the surface of the sample, allowing for tissue microscopic exploration at 20 m under the surface. These promising ex vivo results open new perspectives for in vivo imaging of biological tissue, in particular, in the field of cancer and surgical margin assessment.

Assessment of Sentinel Node Biopsies With Full-Field Optical Coherence Tomography.

Current techniques for the intraoperative analysis of sentinel lymph nodes during breast cancer surgery present drawbacks such as time and tissue consumption. Full-field optical coherence tomography is a novel noninvasive, high-resolution, fast imaging technique. This study investigated the use of full-field optical coherence tomography as an alternative technique for the intraoperative analysis of sentinel lymph nodes. Seventy-one axillary lymph nodes from 38 patients at Tenon Hospital were imaged minutes after excision with full-field optical coherence tomography in the pathology laboratory, before being handled for histological analysis. A pathologist performed a blind diagnosis (benign/malignant), based on the full-field optical coherence tomography images alone, which resulted in a sensitivity of 92% and a specificity of 83% (n = 65 samples). Regular feedback was given during the blind diagnosis, with thorough analysis of the images, such that features of normal and suspect nodes were identified in the images and compared with histology. A nonmedically trained imaging expert also performed a blind diagnosis aided by the reading criteria defined by the pathologist, which resulted in 85% sensitivity and 90% specificity (n = 71 samples). The number of false positives of the pathologist was reduced by 3 in a second blind reading a few months later. These results indicate that following adequate training, full-field optical coherence tomography can be an effective noninvasive diagnostic tool for extemporaneous sentinel node biopsy qualification.

Full-field optical coherence tomography of human donor and pathological corneas.

PURPOSE: To evaluate the performance of a full-field optical coherence tomography (FF-OCT) system in the study of human donor and pathological corneas and assess its suitability for use in eye banks. METHODS: Our study was carried out using an FF-OCT system developed for non-invasive imaging of tissue structures in depth with ultrahigh resolution (1 µm in all directions). Images were acquired from eight stored human donor corneas (either edematous or after deswelling) and five surgical specimens of corneas with various diseases (bullous keratopathy, lattice corneal dystrophy, stromal scar after keratitis, keratoconus and Fuchs dystrophy). They were compared with standard histology and pre-operative spectral domain OCT. RESULTS: The FF-OCT device enabled a precise visualization of the cells and the different structures (epithelium, basement membrane, Bowman's layer, stroma, Descemet's membrane and endothelium) in normal corneas. Specific lesions in various corneal diseases could also be easily identified, such as corneal edema, epithelium and Bowman's layer irregularities, breaks, or scars (keratoconus), stromal opacities, deposits, fibrosis (stromal corneal scar, bullous keratopathy, lattice corneal dystrophy) and Descemet's membrane thickening and guttae (Fuchs dystrophy). FF-OCT image features were comparable to the details provided by conventional histology. Higher resolution could be demonstrated with FF-OCT when compared with spectral domain OCT. CONCLUSION: FF-OCT is a powerful non-invasive imaging tool that allows detailed study of corneal structures. Images correlate well with conventional histology. Further studies should evaluate the benefit of this technique as a complement to current assessment methods of human donor corneas.

Large field, high resolution full-field optical coherence tomography: a pre-clinical study of human breast tissue and cancer assessment.

We present a benchmark pilot study in which high-resolution Full-Field Optical Coherence Tomography (FF-OCT) was used to image human breast tissue and is evaluated to assess its ability to aid the pathologist's management of intra-operative diagnoses. FF-OCT imaging safety was investigated and agreement between FF-OCT and routinely prepared histopathological images was evaluated. The compact setup used for this study provides 1 mm3 resolution and 200 mm imaging depth, and a 2.25 cm2 specimen is scanned in about 7 minutes. 75 breast specimens were imaged from 22 patients (21 women, 1 man) with a mean age of 58 (range: 25-83). Pathologists blind diagnosed normal/benign or malignant tissue based on FF-OCT images alone, diagnosis from histopathology followed for comparison. The contrast in the FF-OCT images is generated by intrinsic tissue scattering properties, meaning that no tissue staining or preparation is required. Major architectural features and tissue structures of benign breast tissue, including adipocytes, fibrous stroma, lobules and ducts were characterized. Subsequently, features resulting from pathological modification were characterized and a diagnosis decision tree was developed. Using FF-OCT images, two breast pathologists were able to distinguish normal/benign tissue from lesional with a sensitivity of 94% and 90%, and specificity of 75% and 79% respectively.

Imaging of non-tumorous and tumorous human brain tissues with full-field optical coherence tomography.

A prospective study was performed on neurosurgical samples from 18 patients to evaluate the use of full-field optical coherence tomography (FF-OCT) in brain tumor diagnosis. FF-OCT captures en face slices of tissue samples at 1 µm resolution in 3D to a penetration depth of around 200 µm. A 1 cm(2) specimen is scanned at a single depth and processed in about 5 min. This rapid imaging process is non-invasive and requires neither contrast agent injection nor tissue preparation, which makes it particularly well suited to medical imaging applications. Temporal chronic epileptic parenchyma and brain tumors such as meningiomas, low-grade and high-grade gliomas, and choroid plexus papilloma were imaged. A subpopulation of neurons, myelin fibers and CNS vasculature were clearly identified. Cortex could be discriminated from white matter, but individual glial cells such as astrocytes (normal or reactive) or oligodendrocytes were not observable. This study reports for the first time on the feasibility of using FF-OCT in a real-time manner as a label-free non-invasive imaging technique in an intraoperative neurosurgical clinical setting to assess tumorous glial and epileptic margins.

Adaptive optics with pupil tracking for high resolution retinal imaging.

Adaptive optics, when integrated into retinal imaging systems, compensates for rapidly changing ocular aberrations in real time and results in improved high resolution images that reveal the photoreceptor mosaic. Imaging the retina at high resolution has numerous potential medical applications, and yet for the development of commercial products that can be used in the clinic, the complexity and high cost of the present research systems have to be addressed. We present a new method to control the deformable mirror in real time based on pupil tracking measurements which uses the default camera for the alignment of the eye in the retinal imaging system and requires no extra cost or hardware. We also present the first experiments done with a compact adaptive optics flood illumination fundus camera where it was possible to compensate for the higher order aberrations of a moving model eye and in vivo in real time based on pupil tracking measurements, without the real time contribution of a wavefront sensor. As an outcome of this research, we showed that pupil tracking can be effectively used as a low cost and practical adaptive optics tool for high resolution retinal imaging because eye movements constitute an important part of the ocular wavefront dynamics.

Cellular-resolution in vivo imaging of the feline retina using adaptive optics: preliminary results.

PURPOSE: To perform cellular-level in vivo imaging of the feline retina using an adaptive optics flood illumination fundus camera (AO FIFC) designed for the human eye. MATERIALS AND METHODS: Cellular-level images were obtained from three eyes of two normal sedated cats. Ocular aberrations were corrected using an AO system based on a 52-acuator electromagnetic deformable mirror and a 1024 lenslet Hartmann-Shack sensor (both Imagine Eyes, Orsay, France). A square 3°×3° area of the ocular fundus was flood-illuminated by a pulsed LED emitting at 850 nm and imaged onto a low-noise, high-resolution CCD camera. The animal's pupils were dilated and the effective pupil size was set to 7.5 mm. Conjunctival atraumatic clips were used to avoid eyeball movements and eyelid closure. The cornea was artificially hydrated throughout the experiments. Each acquisition consisted of 20 consecutive images, out of which 10 were numerically averaged to produce an enhanced final image. RESULTS: The total amount of ocular aberrations was greatly reduced by the AO correction, from 2.4 to 0.21 microns root mean square on average. The resulting images presented white dots distributed at a density similar to that of cone photoreceptors and they allowed us to visualize small blood vessels and nerve fiber bundles at a higher resolution than classically obtained with conventional fundus photography. CONCLUSION: Retinal imaging with cellular resolution was feasible in cats under sedation using an AO FIFC designed for human eyes without any optical modification. The AO FIFC technology could find new applications in clinical, pharmacological, and toxicological investigations.

Effects of Zernike wavefront aberrations on visual acuity measured using electromagnetic adaptive optics technology.

PURPOSE: This study measured the changes in visual acuity induced by individual Zernike ocular aberrations of various root-mean-square (RMS) magnitudes. METHODS: A crx1 Adaptive Optics Visual Simulator (Imagine Eyes) was used to modify the wavefront aberrations in nine eyes. After measuring ocular aberrations, the device was programmed to compensate for the eye's wavefront error up to the 4th order and successively apply different individual Zernike aberrations using a 5-mm pupil. The generated aberrations included defocus, astigmatism, coma, trefoil, and spherical aberration at a level of 0.1, 0.3, and 0.9 microm. Monocular visual acuity was assessed using computer-generated Landolt-C optotypes. RESULTS: Correction of the patients' aberrations improved visual acuity by a mean of 1 line (-0.1 logMAR) compared to best sphero-cylinder correction. Aberrations of 0.1 microm RMS resulted in a limited decrease in visual acuity (mean +0.05 logMAR), whereas aberrations of 0.3 microm RMS induced significant visual acuity losses with a mean reduction of 1.5 lines (+0.15 logMAR). Larger aberrations of 0.9 microm RMS resulted in greater visual acuity losses that were more pronounced with spherical aberration (+0.64 logMAR) and defocus (+0.62 logMAR), whereas trefoil (+0.22 logMAR) was found to be better tolerated. CONCLUSIONS: The electromagnetic adaptive optics visual simulator effectively corrected and generated wavefront aberrations up to the 4th order. Custom wavefront correction significantly improved visual acuity compared to best-spectacle correction. Symmetric aberrations (eg, defocus and spherical aberration) were more detrimental to visual performance.