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Optical whispering-gallery-mode (WGM) microresonator-based sensors with high sensitivity and low detection limit down to single unlabeled biomolecules show high potential for disease diagnosis and clinical application. However, most WGM microresonator-based sensors, which are packed in a microfluidic cell, are a &ldquo, closed&rdquo, sensing configuration that prevents changing and sensing the surrounding liquid refractive index (RI) of the microresonator immediately. Here, we present an &ldquo, open&rdquo, sensing configuration in which the WGM microdisk laser is directly covered by a water droplet and pumped by a water-immersion-objective (WIO). This allows monitoring the chemical reaction progress in the water droplet by tracking the laser wavelength. A proof-of-concept demonstration of chemical sensor is performed by observing the process of salt dissolution in water and diffusion of two droplets with different RI. This WIO pumped sensing configuration provides a path towards an on-chip chemical sensor for studying chemical reaction kinetics in real time.

Significance Cryofluorescence imaging is of great interest in biological microscopy because vitrified samples (i.e., frozen without ice crystallization) are free from fixation artifacts, are highly photostable, and allow direct correlation with electron cryomicroscopy. Here, we show a concept for conducting cryo-light microscopy in immersion that provides a twofold to fivefold increase in image brightness and higher resolution than are attainable with air objectives. Spherical aberration at an imaging temperature below the glass transition of water (−135 °C) is corrected by our approach., Cryogenic fluorescent light microscopy of flash-frozen cells stands out by artifact-free fixation and very little photobleaching of the fluorophores used. To attain the highest level of resolution, aberration-free immersion objectives with accurately matched immersion media are required, but both do not exist for imaging below the glass-transition temperature of water. Here, we resolve this challenge by combining a cryoimmersion medium, HFE-7200, which matches the refractive index of room-temperature water, with a technological concept in which the body of the objective and the front lens are not in thermal equilibrium. We implemented this concept by replacing the metallic front-lens mount of a standard bioimaging water immersion objective with an insulating ceramic mount heated around its perimeter. In this way, the objective metal housing can be maintained at room temperature, while creating a thermally shielded cold microenvironment around the sample and front lens. To demonstrate the range of potential applications, we show that our method can provide superior contrast in Escherichia coli and yeast cells expressing fluorescent proteins and resolve submicrometer structures in multicolor immunolabeled human bone osteosarcoma epithelial (U2OS) cells at −140°C.

Most flying-spot optical coherence tomography and optical coherence microscopy (OCM) systems use a symmetric confocal geometry, where the detection path retraces the illumination path starting from and ending with the spatial mode of a single-mode optical fiber. Here we describe a visible light OCM instrument that breaks this symmetry to improve transverse resolution without sacrificing collection efficiency in scattering tissue. This was achieved by overfilling a water immersion objective on the illumination path while maintaining a conventional Gaussian mode detection path (1/e2 intensity diameter ∼0.82 Airy disks), enabling ∼1.1 μm full width at half-maximum (FWHM) transverse resolution. At the same time, a ∼0.9 μm FWHM axial resolution in tissue, achieved by a broadband visible light source, enabled femtoliter volume resolution. We characterized this instrument according to paraxial coherent microscopy theory and, finally, used it to image the meningeal layers, intravascular red blood cell-free layer, and myelinated axons in the mouse neocortex in vivo through the thinned skull.

The present protocol to visualize living bacteria at the pore level of cauliflower hydathodes is simple and trained users in confocal microscopy can execute it successfully. It can be easily adapted to capture images with other plant-microorganism interactions at the leaf surface and should be useful to obtain important information on pore and stomatal biology. A critical limitation to methods used to observe plant-microorganism interactions in the pore is the application of too much pressure to the sample during observations and z-stack acquisitions. To solve this issue, we recommend the use of a long working-distance water immersion objective lens that allows observations even with thick samples.

A compact, high-speed variable-focus liquid lens using acoustic radiation force is proposed. The lens consists of an annular piezoelectric ultrasound transducer and an aluminum cell (height: 3 mm; diameter: 6 mm) filled with degassed water and silicone oil. The profile of the oil-water interface can be rapidly varied by applying acoustic radiation force from the transducer, allowing the liquid lens to be operated as a variable-focus lens. A theoretical model based on a spring-mass-dashpot model is proposed for the vibration of the lens. The sound pressure distribution in the lens was calculated by finite element analysis and it suggests that an acoustic standing wave is generated in the lens. The fastest response time of 6.7 ms was obtained with silicone oil with a kinematic viscosity of 100 cSt.

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (>200 µm), localization precision of 2-3 nm can be achieved.

Using the immersion lensing technique, the resolution of a conventional spherical lens can be improved by a factor of 1/n over its value in air (n, the refractive index of the immersion medium). Depending on the relative position between an object and a lens, either a real or a virtual image is formed. Here we report a new physical phenomenon experimentally observed in the microscale lens imaging. We find that when a microscale spherical lens is semi-immersed in a medium, the resolution of the lens is improved as it can intercept more fine details of the object. However, the microscale lens has two image channels for the fine and coarse details and two images corresponding to the two components can be formed simultaneously. Our findings will advance the understanding of the super-resolution imaging mechanisms in microscale lenses.

Optical Coherence Microscopy (OCM) combines coherence gating, high numerical aperture optics, and a fiber core pinhole to provide high axial and lateral resolution with relatively large depth of imaging. We present a handheld rigid OCM endoscope with a 6 mm diameter tip, 1 mm scan width, and 1 mm imaging depth. This probe will allow noninvasive imaging of fine structural detail in vivo . X-Y scanning is performed distally with mirrors mounted to micro galvonometer scanners incorporated into the endoscope handle. Two scanning doublet lenses relay the stop from the galvonometers to the afocal relay stop. The endoscope optical design consists of an afocal Hopkins relay lens system and a 0.4 NA objective. To allow focusing at various depths in the tissue, the endoscope housing is designed in two pieces screwed together with a fine pitch threads. A small rotation of the outer housing moves the lenses proximal and distal relative to the window, causing the focal location in the tissue to change. The space between the final objective lens and the window is filled with distilled water to avoid misalignment of the focus and coherence gate. A knife edge test was performed and the line spread function FWHM was measured to be 2.25 μm. The MTF has at least 0.3 contrast at a 5 μm line pair. This rigid handheld OCM endoscope will be useful for application ranging from minimally invasive surgical imaging to assessing dysplasia and sun damage in skin.

BackgroundTwo of the key parameters to be monitored during cellcultivation processes are cell concentration and viability.Until today, this is very often done off-line by sterilesampling and subsequent counting using a hemocyt-ometer or an electronic cell counter. Cell biology lacks ameasurable quantity by which single cells in suspensioncan be non-invasively diagnosed as dead or alive. How-ever, it would be of significance for process monitoringand in the light of initiatives like PAT if cell density aswell as viability could be determined directly and on-line.Optical measurement of cell density byin situ micro-scopy eliminates the need for sampling and allows forcontinuous monitoring of this key parameter; see e.g.[1,2]; Guez et al. [1] describe an in situ microscope(ISM) which does not use any moving mechanical partswithin or outside the fermentation vessel. It transmits inreal time images taken directly in the stirred suspensionwithin the bioreactor. Image data is processed and eval-uated to provide monitoring of cell-density and mor-phological parameters, e.g. cell size, by means ofassessing the obtained in situ cell-micrographs.Previously, we have extendedin situ microscopytowards viability assessment of suspended cells [3,5].Now, we present new findings on this topic and showthat in cultures of suspended cells, cell-death corre-sponds to measurable changes in morphometric para-meters as e.g. variance, contrast or entropy of thegreyvalues of in situ cell-micrographs. As an example,here we show viability determination via greyvaluedispersion.Material and methodsWe use a custom built high resolution ISM (HS Man-nheim) with water immersion objective, 40x magnifica-tion, numerical aperture 0.75 equipped with optical fiberillumination. Data acquisition is at 0.3– 15 frames persecond, frames have 1293x1040 pixels; primary dataanalysis results in cell micrographs (Figure 1a). We haveapplied the system to bench top and larger bioreactors(see e.g. [4]) and worked mainly with Jurkat, CHO andHybridoma cells.For the experiment presented in Figure 1a/b, cells arecultivated in a Biostat C30 (Sartorius BBI Systems) withthe ISM inserted in one of the existing probe ports. Pro-prietary hybridoma cells (InVivo BioTech Services) arecultured in serum free ISF-1 (InVivo BioTech Services;Biochrom AG) and monitored over the full length ofthe fermentation (not shown).For the experiment presented in Figure 1c, cells arecultivated in a custom built autoclavable steel bench topreactor(HSMannheim)with25mmporttoaccommo-date the ISM and a working volume of 0.7 L. To testreal time and viability determination capabilities of thesystem over a wide range of viabilities in a short time,cells were challenged with 3% Ethanol at 42 hours. Cellcounts (not shown) and viability were determined by insitu microscopy and, as reference, by means of a ViCellcell viability analyser (Beckman Coulter) and Flow Cyto-metry (Partec) using Annexin V / FITC and PI staining.Jurkat cells (DSMZ ACC 282) were cultured in 90%RPMI 1640 + 10% FBS.

New projection- type X-ray microscope with a compound refractive lens as the optical element is presented. The microscope consists of an X-ray source that is 1-2 mm in diameter, compound X-ray lens and X-ray camera that are placed in-line to satisfy the lens formula. The lens forms an image of the X-ray source at camera sensitive plate. An object is placed between the X-ray source and the lens as close as possible to the source, and the camera shows a shadow image of the object. Spatial resolution of the microscope depends on the lens focal length, lens aperture and the distance from the source to the object. One to two micron resolution may be achieved by placing the object at a distance of 1-5 mm from the source. The X-ray source may be designed with the target deposited on a 200-µm thick Be window, which permits the object to be placed very close to the emitting surface. The tube focal spot is equal to 1-2 mm. Results of imaging experiments with an ordinary copper anode X-ray tube and a 10-cm focal length spherical compound refractive X-ray lens are discussed.

International audience; The use of a confocal detection scheme in a dual-beam thermal-lens microscope is presented. It allows the measurement of absorption factors smaller than 10(-6) using a heating laser power of 100 mW incident on the sample. A simple mathematical model based on gaussian beam propagation is presented that takes into account the main features of the instrument: the confocal detection scheme and the presence of chromatic aberrations in the objective lenses, and that allows to estimate the sensitivity and resolution of the instrument. This model is validated by experimental measurements on different test samples. Results are presented that prove a 450 nm axial resolution when using a NA = 1.2 water immersion objective lens.

International audience; The use of a confocal detection scheme in a dual-beam thermal-lens microscope is presented. The scheme allows the measurement of absorption factors down to 1.2×10-7 in a 0.35-μm3 volume by use of a heating laser power of 100 mW incident upon the sample. Results are presented that prove that a 450-nm axial resolution is possible when a 1.2 water immersion objective lens with a N.A. of 1.2 is used

A high-speed variable-focus liquid optical lens using acoustic radiation force is proposed. The lens consists of a piezoelectric ring and a lens cell filled with degassed water and silicone oil. The profile of the oil-water interface can be rapidly varied by applying acoustic radiation force from the ultrasonic transducer, allowing the liquid lens to be operated as a variable-focus lens. The fastest response time of 6.7 ms was obtained with silicone oil with a kinematic viscosity of 100 cSt. The lens could scan at focal lengths of from 7.0 to 14.5 mm at 1 kHz in the axial direction.