Combined scanning optical coherence and two-photon-excited fluorescence microscopy

We demonstrate simultaneous imaging by optical coherence microscopy (OCM) and two-photon-excited (TPE) fluorescence microscopy. A mode-locked Ti:sapphire laser is focused and scanned in three dimensions through a fixed sample, generating both backscattered light and fluorescence light, which are independently detected. Both imaging modes provide rapid en-face imaging with submicrometer resolution. High-power delivery into the sample yields an OCM sensitivity in excess of 130 dB at 100-kHz pixel rates. Simultaneous imaging of cell nuclei with OCM and TPE is demonstrated in live drosophila embryos.     

En face imaging of single cell layers by differential phase-contrast optical coherence microscopy

Optical coherence microscopy (OCM) is capable of imaging the backscattering potential of a sample with high transversal and axial resolution. We report on a combination of OCM with a differential phase-contrast technique that permits imaging of the subwavelength optical path differences that occur between a narrow beam probing a sample and its surrounding. This technique allows small transversal refractive-index variations close to a selected interface to be seen. We report on the method and present first images of a test sample and a single cell layer. The cells act as phase objects; imaging the phase properties improves the contrast compared with that of intensity images.     

Quantitative morphological evaluation of laser ablation on calculus using full-field optical coherence microscopy

The quantitative morphological evaluation at high resolution is of significance for the study of laser-tissue interaction. In this paper, a full-field optical coherence microscopy (OCM) system with high resolution of ∼2 μm was developed to investigate the ablation on urinary calculus by a free-running Er:YAG laser. We studied the morphological variation quantitatively corresponding to change of energy setting of the Er:YAG laser. The experimental results show that the full-field OCM enables quantitative evaluation of the morphological shape of craters and material removal, and particularly the fine structure. We also built a heat conduction model to simulate the process of laser-calculus interaction by using finite element method. Through the simulation, the removal region of the calculus was calculated according to the temperature distribution. As a result, the depth, width, volume, and the cross-sectional profile of the crater in calculus measured by full-field OCM matched well with the theoretical results based on the heat conduction model. Both experimental and theoretical results confirm that the thermal interaction is the dominant effect in the ablation of calculus by Er:YAG laser, demonstrating the effectiveness of full-field OCM in studying laser-tissue interactions.     

Imaging interferometric microscopy

We introduce and demonstrate a new microscopy concept: imaging interferometric microscopy (IIM), which is related to holography, synthetic-aperture imaging, and off-axis-dark-field illumination techniques. IIM is a wavelength-division multiplex approach to image formation that combines multiple images covering different spatial-frequency regions to form a composite image with a resolution much greater than that permitted by the same optical system using conventional techniques. This new type of microscopy involves both off-axis coherent illumination and reinjection of appropriate zero-order reference beams. Images demonstrate high resolution, comparable with that of a high-numerical-aperture (NA) objective, while they retain the long working distance, the large depth of field, and the large field of view of a low-NA objective. A Fourier-optics model of IIM is in good agreement with the experiment.     

Wide-field high-resolution structured illumination solid immersion fluorescence microscopy

The use of aplanatic solid immersion lenses (ASILs) made of high-refractive-index optical materials provides a route to wide-field high-resolution optical microscopy. Structured illumination microscopy (SIM) can double the spatial bandwidth of a microscope to also achieve high-resolution imaging. We investigate the combination of ASILs and SIM in fluorescence microscopy, which we call structured illumination solid immersion fluorescence microscopy (SISIM), to pursue a microscopic system with very large NA and high lateral resolution. We demonstrate that the combination can produce a wide-field high-resolution microscopic system with bandwidth corresponding to an NA of 3. Future developments of the SISIM system to make it achieve even higher resolution are proposed.     

Comparison of the Optical Connectivity Method to X-Ray spray measurements in the near field of a diesel injector

For diesel sprays, the primary breakup processes are only rarely understood due to the high optical density and the resulting difficulties to measure them with extremely high spatial and sufficient temporal resolution. The Optical Connectivity Method (OCM) has been proposed in the last years to allow the determination of the breakup length of the connected liquid core, thus giving a measurement quantity of the primary breakup. In this work, an improved optical setup of the OCM is applied to a three-hole test injector nozzle where several measurement techniques are compared currently under well-defined conditions up to 100?MPa injection pressure. In this work, the direct comparison with X-Ray measurements done at the Advanced Photon Source of the Argonne National Laboratory will be described. This allows an evaluation of the OCM technique and a comparison of the different measurement quantities in the first 500?µm range of the spray. The structure of the spray is measured by X-Ray phase contrast imaging and the fuel mass distribution is measured by X-Ray absorption imaging. A detailed comparison of the two X-Ray techniques and the OCM technique has been possible for the first time. It is found that the measurement data of the spray near field are very congruent with all three methods. Due to this comparison, the measurement of the non-perturbed length, which describes the distance from the nozzle orifice up to the point where the formation of surface disturbances is starting, by the OCM is validated for the first time. Within this non-perturbed length of the spray, the OCM signal is weak before it starts to illuminate from the scattering of the perturbed surface. Thus, the OCM technique can deliver two characteristic length scales, the non-perturbed length and the breakup length, characterizing the primary spray breakup.     

Nanohybrids investigation by waveguide SNOM illumination mode

We demonstrate how optical waveguides can serve as a high resolution illumination source to image hybrid nano-objects in near field optical microscopy. Nanohybrid particles deposited on the surface of glass waveguides are imaged in terms of topography, scattering and fluorescence.     

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

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.     

Theoretical assessment of optical resolution enhancement and background fluorescence reduction by three-dimensional nonlinear structured illumination microscopy using stimulated emission depletion

Three-dimensional structured illumination microscopy (SIM) enlarges frequency cutoff laterally and axially by a factor of two, compared with conventional microscopy. However, its optical resolution is still fundamentally limited. It is necessary to introduce nonlinearity to enlarge frequency cutoff further. We propose three-dimensional nonlinear structured illumination microscopy based on stimulated emission depletion (STED) effect, which has a structured excitation pattern and a structured STED pattern, and both three-dimensional illumination patterns have the same lateral pitch and orientation. Theoretical analysis showed that nonlinearity induced by STED effect, which causes harmonics and contributes to enlarging frequency cutoff, depends on the phase difference between two structured illuminations and that the phase difference of π is the most efficient to increase nonlinearity. We also found that undesirable background fluorescence, which degenerates the contrast of structured pattern and limits the ability of SIM, can be reduced by our method. These results revealed that optical resolution improvement and background fluorescence reduction would be compatible. The feasibility study showed that our method will be realized with commercially available laser, having 3.5 times larger frequency cutoff compared with conventional microscopy.     

Image formation in structured illumination wide-field fluorescence microscopy

We present a theoretical analysis of the image formation in structured illumination wide-field fluorescence microscopy (SIWFFM). We show that the optically sectioned images obtained with this approach possess the optical sectioning strengths comparable to those obtained with the confocal microscope. We further show that the transfer function behaviour is directly comparable to that of the true confocal instrument. The theoretical considerations are compared with and confirmed by experimental results.     

In vivo dark-field reflection-mode photoacoustic microscopy

Reflection-mode photoacoustic microscopy with dark-field laser pulse illumination and high-numerical-aperture ultrasonic detection is designed and implemented in noninvasively imaged blood vessels in the skin in vivo. Dark-field optical illumination minimizes the interference caused by strong photoacoustic signals from superficial structures. A high-numerical-aperture acoustic lens provides high lateral resolution, 45-120 microm in this system. A broadband ultrasonic detection system provides high axial resolution, estimated to be approximately 15 microm. The optical illumination and ultrasonic detection are in a coaxial confocal configuration for optimal image quality. The system is capable of imaging optical-absorption contrast as deep as 3 mm in biological tissue.     

Phase contrast enhancement in microscopy using spiral phase filtering

We demonstrate two methods based on Fourier plane filtering using (a) a fractional spiral phase plate (SPP) and (b) an off-axial SPP for phase contrast enhancement in optical microscopy. In comparison to previous works, a spatially incoherent LED is used in the Köhler illumination as the light source to illuminate the biological specimen. We demonstrate that both these methods can transform the phase specimen into a relief-like view even under such illumination. The degree and orientation of enhancement can be controlled by changing the phase structure of the filter. The SPP is displayed on a phase-only spatial light modulator, and can be integrated into the optical path of standard microscopes.     

Quantum spatial superresolution by optical centroid measurements

Quantum lithography (QL) has been suggested as a means of achieving enhanced spatial resolution for optical imaging, but its realization has been held back by the low multiphoton detection rates of recording materials. Recently, an optical centroid measurement (OCM) procedure was proposed as a way to obtain spatial resolution enhancement identical to that of QL but with higher detection efficiency (M. Tsang, Phys. Rev. Lett. 102, 253601 (2009)). Here we describe a variation of the OCM method with still higher detection efficiency based on the use of photon-number-resolving detection. We also report laboratory results for two-photon interference. We compare these results with those of the standard QL method based on multiphoton detection and show that the new method leads to superresolution but with higher detection efficiency.     

Map-free line-scanning tomographic optical microscope

Line-scanning tomographic optical microscopy (LSTOM) requires precise rotation of the scanning line. We demonstrate a method that applies translation-invariant optical elements (polarizer and birefringent plate) to minimize the rotation error. An astigmatic line produced by means of a focused beam through a birefringent plate is used as line illumination. A comparative theoretical and experimental study is presented using an LSTOM system.     

Application of a femtosecond self-sustaining mode-locked Ti:sapphire laser to the field of laser scanning confocal microscopy

Developments in ultrafast Ti:sapphire laser technology can be applied in the investigation of nonlinear optical processes. We describe the application of a self-sustaining femtosecond Ti:sapphire laser as an illumination source in the field of confocal laser scanning fluorescence microscopy (LSM). We present spectra for various fluorescent stains under two-photon excitation and present LSM images of stained samples under mode-locked illumination. The potential for such a system as a non-destructive technique for studying live cells in biomedical research is discussed.     

Reciprocity in scanning near-field optical microscopy: illumination and collection modes of transmission measurements

We experimentally investigated the reciprocity of scanning near-field optical microscopy between illumination and collection modes. Near-field transmission images of single gold spheres and nanorods observed by the two modes are found to be equivalent to each other in the region from visible to near infrared. This result shows that reciprocity holds for the near-field scattering problems. We found that the conventional optical selection rule for far-field excitations does not apply not only under illumination mode but also with collection-mode arrangements. The possible origin of this observation might be the near-field probe.     

Optically tunable surfaces with trapped particles in microcavities

We introduce optically tunable surfaces based upon metallic gold nanoparticles trapped in open, water-filled gold cavities. The optical properties of the surfaces change dramatically with the presence and location of the particles inside the cavities. The precise position of the particles is shown to be controllable through optical forces exerted by external illumination, thus leading to all-optical tunability, whereby the optical response of the surfaces is tuned through externally applied light. We discuss the performance of the cavity-particle complex in detail and provide theoretical support for its application as a novel concept of a large-scale optically tunable system.     

Near-Field Optical Microscope with Tapping Illumination and Synchronous Detection

We use the tapping illumination and synchronous detection in a scanning near-field optical microscopy to obtain a near-field optical signal that is separated from the far-field signal. The illumination light was irradiated from the bent fiber tip vibrating normal to the sample surface. The transmitted light synchronized with the tapping vibration was observed. The obtained image of an organic film shows that this technique is effective for the weak contrast samples.This paper was originally presented at the 5th International Conference on NEAR FIELD OPTICS and RELATED TECHNOLOGIES (NFO-5), which was held on December 6–10, 1998 at Coganoi Bay Hotel, Shirahama, Japan, in cooperation with the Japan Society of Applied Physics and Mombusho Grant-in Aid for Scientific Research on Priority Areas “Near-field Nano-optics” Project, sponsored by Japan Society for the Promotion of Science.     

Synthetic aperture fourier holographic optical microscopy

We report a new synthetic aperture optical microscopy in which high-resolution, wide-field amplitude and phase images are synthesized from a set of Fourier holograms. Each hologram records a region of the complex two-dimensional spatial frequency spectrum of an object, determined by the illumination field's spatial and spectral properties and the collection angle and solid angle. We demonstrate synthetic microscopic imaging in which spatial frequencies that are well outside the modulation transfer function of the collection optical system are recorded while maintaining the long working distance and wide field of view.     

Spatial and temporal coherence effects in interference microscopy and full‐field optical coherence tomography

Low‐coherence optical microscopy or optical coherence microscopy uses light with short coherence length. The well‐known case is: “white‐light interferometry”, which became recently more known as: “optical coherence tomography”. However, when lenses and microscope objectives are used to create interferometric images, in what is known classically as “interference microscopy” or today as “full‐field optical coherence tomography” the spatial coherence starts to play a critical role. In this article the coherence effects in low‐coherence optical microscopy are reviewed. As this technology is becoming increasingly publicized due to its importance in three‐dimensional imaging, particularly of scattering biological media and optical metrology, the understanding of the fundamental physics behind it is essential. The interplay between longitudinal spatial coherence and temporal coherence and the effects associated with them are discussed in detail particularly when high numerical apertures are used. An important conclusion of this study is that a high‐contrast, high‐resolution system for imaging of multilayered samples is the one that uses narrowband illumination and high‐NA objectives with an index‐matching fluid. Such a system, when combined with frequency‐domain operation, can reveal nearly real‐time three‐dimensional images, and is thus competitive with confocal microscopy.