Focal Plane Of Objective Research Articles

Tailoring bistability in optical tweezers with vortex beams and spherical aberration

We demonstrate a bistable optical trap by tightly focusing a vortex laser beam. The optical potential has the form of a Mexican hat with an additional minimum at the center. The bistable trapping corresponds to a nonequilibrium steady state, where the microsphere continually hops, due to thermal activation, between an axial equilibrium state and an orbital state driven by the optical torque. We develop a theoretical model for the optical force field, based entirely on experimentally accessible parameters, combining a Debye-type nonparaxial description of the focused vortex beam with Mie scattering by the microsphere. The theoretical prediction that the microsphere and the annular laser focal spot should have comparable sizes is confirmed experimentally by taking different values for the topological charge of the vortex beam. Spherical aberration introduced by refraction at the interface between the glass slide and the sample is considered and allows us to finetune between axial, bistable, and orbital states as the sample is shifted with respect to the objective focal plane. We find overall agreement between theory and experiment for a rather broad range of topological charges. Our results open the way for applications in stochastic thermodynamics, as they establish a control parameter—the height of the objective focal plane with respect to the glass slide—that allows us to shape the optical force field in real time and in a controllable way. Published by the American Physical Society 2024

Investigation of Galectins in Frozen Tissue and Mammalian Cell Culture Using Confocal Miccroscopy.

Galectins are multifunctional glycan-binding proteins present in various tissues that participate in multiple physiological and pathological processes and are considered as not only biomarkers of human diseases but also molecular targets for treating cancer and inflammatory illnesses in many organs. In the glycobiology field, it is crucial to determine the pattern of galectin expression and location in cells and tissues. Confocal microscopy is a powerful imaging technology that represents a unique approach to investigate the expression and location of biomolecules in various tissues and cells. The confocal microscope acquires images of the specimen through the reflected or fluorescent light from the objective's focal plane, using laser light focused on a small spot inside the tissue or cell. This technique provides high-resolution and high-contrast images without artifacts generated by conventional microscopy and enables reconstruction of virtual tridimensional images by acquiring multiple sections from several focal planes, which makes it possible to obtain the precise spatial location of any cellular structure or molecule. Furthermore, confocal microscopy is a non-invasive tissue imaging strategy used in clinical practices. We describe herein the immunofluorescence confocal method for examining galectins in frozen tissue sections and mammalian cell culture.

In vivo volumetric imaging of calcium and glutamate activity at synapses with high spatiotemporal resolution

Studying neuronal activity at synapses requires high spatiotemporal resolution. For high spatial resolution in vivo imaging at depth, adaptive optics (AO) is required to correct sample-induced aberrations. To improve temporal resolution, Bessel focus has been combined with two-photon fluorescence microscopy (2PFM) for fast volumetric imaging at subcellular lateral resolution. To achieve both high-spatial and high-temporal resolution at depth, we develop an efficient AO method that corrects the distorted wavefront of Bessel focus at the objective focal plane and recovers diffraction-limited imaging performance. Applying AO Bessel focus scanning 2PFM to volumetric imaging of zebrafish larval and mouse brains down to 500 µm depth, we demonstrate substantial improvements in the sensitivity and resolution of structural and functional measurements of synapses in vivo. This enables volumetric measurements of synaptic calcium and glutamate activity at high accuracy, including the simultaneous recording of glutamate activity of apical and basal dendritic spines in the mouse cortex.

Nonparaxial Mie Theory of Image Formation in Optical Microscopes and Characterization of Colloidal Particles

We derive an explicit partial-wave (Mie) series for the image of a dielectric microsphere collected by a typical infinity-corrected microscope. We model the propagation of the illumination and scattered vector fields through the optical components of the microscope by using the angular-spectrum theorem with the help of Wigner rotation matrix elements, allowing us to identify the contribution from spin-orbit helicity reversal. We consider a high numerical aperture objective well beyond the validity range of the paraxial approximation. The spherical aberration introduced by refraction at the planar interface between the sample and the glass slide is fully taken into account. By comparing our theoretical model with images of colloidal particles placed at different positions with respect to the objective focal plane, we characterize their radii and refractive index. We employ polystyrene microspheres with a known refractive index in order to fit the transverse attenuation length describing the transmission loss of the scattered field. As an application, we measure the radius and refractive index of individual silica beads. We compare the result for the radius with an independent measurement using high-resolution scanning electron microscopy. To validate the result for the refractive index, we develop a second method, independent of the theoretical model, based on the image contrast in glycerin-water solutions. In all cases we find very good agreement between our method and the validation procedures. In addition, the nonparaxial theory provides a reliable description of the images found for all focal-plane positions and for both polystyrene and silica microspheres. Our approach allows a common optical microscope to be used to measure the refractive index and radius of spherical particles covering the entire size range from the Rayleigh regime to the ray optics one.

Parallel optically detected magnetic resonance spectrometer for dozens of single nitrogen-vacancy centers using laser-spot lattice.

We develop a parallel optically detected magnetic resonance (PODMR) spectrometer to address, manipulate, and read out an array of single nitrogen-vacancy (NV) centers in diamond in parallel. In this spectrometer, we use an array of micro-lenses to generate a 20 × 20 laser-spot lattice (LSL) on the objective focal plane and then align the LSL with an array of single NV centers. The quantum states of NV centers are manipulated by a uniform microwave field from a Ω-shape coplanar coil. As an experimental demonstration, we observe 80 NV centers in the field of view. Among them, magnetic resonance (MR) spectra and Rabi oscillations of 18 NV centers along the external magnetic field are measured in parallel. These results can be directly used to realize parallel quantum sensing and multiple times speedup compared with the confocal technique. Regarding the nanoscale MR technique, PODMR will be crucial for a high throughput single molecular MR spectrum and imaging.

REFLECTION OF LIGHT FROM THE DIFFRACTION GRATING WITH TRIANGULAR PROFILE

we study the light diffraction at a reflective grating with triangular profile in scalar wave optics approximation. The case of 1D diffusively reflective rough surface can be described in the first approximation as a random set of reflective gratings with triangular profiles, which are characterised by normal (Gaussian) distribution of amplitude and spatial period. We have obtained the condition for amplitude and period of a diffraction grating in the case of absence of secondary reflections, and the dependence of optical path difference on amplitude and spatial period of the triangular profile. We have investigated the influence of triangular profile parameters on the intensity of reflected wave. We have measured the relative intensity (reflection coefficients) of rough surface reference samples in the case of normal incidence. The relative intensity of reflected wave in the case of normal incidence is used in the design of optical contactless methods for the characterisation of rough surfaces in range of 0.05…0.25 mm. We suggested an optical scheme for the surface roughness measurements, and tested it on the reference samples of rough surfaces. We investigated the dependence of reflected wave intensity on the diffraction angle, and simulated it using numerical solutions of Maxwell equation (FDTD methods). Therefore, we demonstrate that measurements of relative intensity of the reflected wave in the normal to sample plane direction in an objective focal plane allow for determination of the roughness of diffusely reflective surfaces

Scanless two-photon excitation with temporal focusing.

Temporal focusing, with its ability to focus light in time, enables scanless illumination of large surface areas at the sample with micrometer axial confinement and robust propagation through scattering tissue. In conventional two-photon microscopy, widely used for the investigation of intact tissue in live animals, images are formed by point scanning of a spatially focused pulsed laser beam, resulting in limited temporal resolution of the excitation. Replacing point scanning with temporally focused widefield illumination removes this limitation and represents an important milestone in two-photon microscopy. Temporal focusing uses a diffusive or dispersive optical element placed in a plane conjugate to the objective focal plane to generate position-dependent temporal pulse broadening that enables axially confined multiphoton absorption, without the need for tight spatial focusing. Many techniques have benefitted from temporal focusing, including scanless imaging, super-resolution imaging, photolithography, uncaging of caged neurotransmitters and control of neuronal activity via optogenetics.

Fluorescent Imaging and Microscopy for Dynamic Processes in Rats.

The rat is a favored model organism to study physiological function in vivo. This is largely due to the fact that it has been used for decades and is often more comparable to corresponding human conditions (both normal and pathologic) than mice. Although the development of genetic manipulations in rats has been slower than in mice, recent advances of new genomic editing tools allow for the generation of targeted global and specific cell type mutations in different rat strains. The rat is an ideal model for advancing imaging techniques like intravital multi-photon microscopy or IVMPM. Multi-photon excitation microscopy can be applied to visualize real-time physiologic events in multiple organs including the kidney. This imaging modality can generate four-dimensional high resolution images that are inherently confocal due to the fact that the photon density needed to excite fluorescence only occurs at the objective focal plane, not above or below. Additionally, longer excitation wavelengths allow for deeper penetration into tissue, improved excitation, and are inherently less phototoxic than shorter excitation wavelengths. Applying imaging tools to study physiology in rats has become a valuable scientific technique due to the relatively simple surgical procedures, improved quality of reagents, and reproducibility of established assays. In this chapter, the authors provide an example of the application of fluorescent techniques to study cardio-renal functions in rat models. Use of experimental procedures described here, together with multiple available genetically modified animal models, provide new prospective for the further application of multi-photon microscopy in basic and translational research.

Large Depth-of-Field Integral Microscopy by Use of a Liquid Lens.

Integral microscopy is a 3D imaging technique that permits the recording of spatial and angular information of microscopic samples. From this information it is possible to calculate a collection of orthographic views with full parallax and to refocus computationally, at will, through the 3D specimen. An important drawback of integral microscopy, especially when dealing with thick samples, is the limited depth of field (DOF) of the perspective views. This imposes a significant limitation on the depth range of computationally refocused images. To overcome this problem, we propose here a new method that is based on the insertion, at the pupil plane of the microscope objective, of an electrically controlled liquid lens (LL) whose optical power can be changed by simply tuning the voltage. This new apparatus has the advantage of controlling the axial position of the objective focal plane while keeping constant the essential parameters of the integral microscope, that is, the magnification, the numerical aperture and the amount of parallax. Thus, given a 3D sample, the new microscope can provide a stack of integral images with complementary depth ranges. The fusion of the set of refocused images permits to enlarge the reconstruction range, obtaining images in focus over the whole region.

Compact self-illuminated image upconversion system based on intracavity second-harmonic generation.

We present an image upconversion system based on intracavity Type II second-harmonic generation to create an image in the visible spectrum of a target illuminated by an infrared laser. The system has the novelty of being self-illuminated. It uses some fractional leaking power of the infrared laser to illuminate a target located in the object focal plane of the system, and to couple back a created infrared target image to an intracavity nonlinear crystal, where it mixes with the cavity laser beam to obtain a second-harmonic image, visible with a silicon CCD camera. For a proof of concept, we have built a system based on a diode-pumped Nd3+:YVO4 continuous-wave (cw) laser and an intracavity KTP crystal to upconvert 1342nm target images to 671nm. The upconverted cw power allowed us to capture real-time video in a standard nonintensified CCD camera, with 2.5W of a diode pump.

LITE microscopy: Tilted light-sheet excitation of model organisms offers high resolution and low photobleaching.

Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.

Automatic Five-Dimensional Single Particle Tracking in Live Cells

A novel single particle tracking (SPT) technique has been developed to visualize the intracellular transport of cargos in five dimensions (5D): the three spatial coordinates (x, y, z) and two orientation angles (azimuthal angle and polar angle) of a probe's transition dipole over a series of time steps. Tracking rotational probes, such as gold nanorods, in the z-axis is challenging because translational motions in the z-axis can be easily confused with rotational motions as both types of motion result in similar changes in signal intensity. The 5D-SPT method overcomes this long-standing challenge by utilizing the concept of parallax to sense the axial movement of the target object and employs an automatic feedback loop algorithm to control the objective's focal plane and bring the target object back to focus repetitively. In such an implementation, the target object is kept in focus to provide the highest possible S/N for 2D localization and orientation determination, while the z positions are recorded from the vertical movement of a high-precision objective scanner. Through the live-cell SPT experiments, we demonstrate that the 5D-SPT technique has potential to significantly improve the capacity to study the intracellular transport of cargos in a cellular environment.

High-Throughput Contact Flow Lithography.

High-throughput fabrication of graphically encoded hydrogel microparticles is achieved by combining flow contact lithography in a multichannel microfluidic device and a high capacity 25 mm LED UV source. Production rates of chemically homogeneous particles are improved by two orders of magnitude. Additionally, the custom-built contact lithography instrument provides an affordable solution for patterning complex microstructures on surfaces.

Three-dimensional particle tracking in microfluidic channel flow using in and out of focus diffraction

Three-dimensional particle tracking is important to accurately understand the motion of particles within complex flow fields. We show that three-dimensional trajectories of particles within microfluidic flow can be extracted from two-dimensional bright field video microscopy. The method utilizes the defocusing that occurs as particles move out of the objective focal plane when viewed through a high numerical aperture objective lens. A fast and simple algorithm based on cross correlation to a set of reference images taken at prescribed amounts of defocus is used to extract out-of-plane particle position. In-plane particle position is determined through center point detection and therefore the particle position in all three dimensions can be constructed at each time point. Particle trajectories at high flow velocity of greater than 2mm/s can be tracked by utilizing a high speed camera to obtain unblurred images. Three dimensional computational fluid simulations are used to validate the particle tracking methods.

Femtosecond Optical Trap-Assisted Nanopatterning through Microspheres by a Single Ti:Sapphire Oscillator

A new approach for fabricating a range of patterns using femtosecond optical trap-assisted nanopatterning is presented. We report how a single Gaussian laser beam from a 55 fs, 80 MHz, 780 nm Ti:sapphire oscillator trapping dielectric microspheres near surfaces can be used to enable near-field, direct-write, subwavelength ∼λ/6 (∼130 nm), two-dimensional nanopatterning of a polymer surface. We discuss the stability conditions for effective manipulation of the particle by the pulsed beam. Klein–Kramers and Brownian motion models were used to analyze the positional accuracy of femtosecond tweezers. We studied effects of the microsphere size, pulsed laser energy and light polarization, and spacing between objective focal plane and polymer surface on the pattern size experimentally and theoretically. Microspheres with a diameter of about 1 μm provide the smallest patterns. The experimental results are reasonably matched by generalized Lorentz–Mie theory.

Three-dimensional DNA image cytometry by optical projection tomographic microscopy for early cancer diagnosis.

Aneuploidy is typically assessed by flow cytometry (FCM) and image cytometry (ICM). We used optical projection tomographic microscopy (OPTM) for assessing cellular DNA content using absorption and fluorescence stains. OPTM combines some of the attributes of both FCM and ICM and generates isometric high-resolution three-dimensional (3-D) images of single cells. Although the depth of field of the microscope objective was in the submicron range, it was extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. These projections were later reconstructed using computed tomography methods to form a 3-D image. We also present an automated method for 3-D nuclear segmentation. Nuclei of chicken, trout, and triploid trout erythrocyte were used to calibrate OPTM. Ratios of integrated optical densities extracted from 50 images of each standard were compared to ratios of DNA indices from FCM. A comparison of mean square errors with thionin, hematoxylin, Feulgen, and SYTOX green was done. Feulgen technique was preferred as it showed highest stoichiometry, least variance, and preserved nuclear morphology in 3-D. The addition of this quantitative biomarker could further strengthen existing classifiers and improve early diagnosis of cancer using 3-D microscopy.

Projection two-photon polymerization using a spatial light modulator

The development of a high-efficiency projection two-photon polymerization (P2PP) process by using a liquid crystal spatial light modulator (SLM) is presented. Rapid fabrication of 2D patterned microstructures with P2PP is demonstrated, and the effect of laser pattern and exposure dose on the surface roughness of the fabricated microstructures is investigated. It is found that the distribution of laser intensity at the focal plane of objective has a significant effect on the profiles of microstructures. This unique technology has a promising approach to increase the efficiency of two-photon polymerization (2PP) and a parallel fabrication of complex 2D and 3D microstructures.

Experimental and theoretical analysis for improved microscope design of optical projection tomographic microscopy

We present theoretical and experimental results of axial displacement of objects relative to a fixed condenser focal plane (FP) in optical projection tomographic microscopy (OPTM). OPTM produces three-dimensional, reconstructed images of single cells from two-dimensional projections. The cell rotates in a microcapillary to acquire projections from different perspectives where the objective FP is scanned through the cell while the condenser FP remains fixed at the center of the microcapillary. This work uses a combination of experimental and theoretical methods to improve the OPTM instrument design.

Reconstruction of objects above and below the objective focal plane with dimensional fidelity by FINCH fluorescence microscopy

Fresnel Incoherent Correlation Holography (FINCH) can faithfully reproduce objects above and below the optical plane of focus. However, as in optical imaging, the transverse magnification and optimal reconstruction depth changes based on the longitudinal distance of objects from the focal plane of the input lens with the exception that objects above and below the focal plane are in focus with FINCH and out of focus by standard optical imaging. We have analyzed these effects both theoretically and experimentally for two configurations of a FINCH fluorescence microscopy system. This information has been used to reconstruct a test planar object placed above or below the optical plane of focus with high dimensional and image fidelity. Because FINCH is inherently a super-resolving system, this advance makes it possible to create super-resolved 3D images from FINCH holograms.

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets, monitor dynamics in living cells, and visualize the three dimensional evolution of entire organisms. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo and are currently being applied to disease imaging and diagnosis in clinical settings. Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples. Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location. Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists. In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.