Enhanced optical resolution with photo-induced diffraction: Non-linear effects

Sequencing-based spatial transcriptomics (sST) enables transcriptome-wide gene expression mapping but falls short of reaching the optical resolution (200-300 nm) of imaging-based methods. Here, we present Seq-Scope-X (Seq-Scope-eXpanded), which empowers submicrometer-resolution Seq-Scope with tissue expansion to surpass this limitation. By physically enlarging tissues, Seq-Scope-X minimizes transcript diffusion effects and increases spatial feature density by an additional order of magnitude. In liver tissue, this approach resolves nuclear and cytoplasmic compartments in nearly every single cell, uncovering widespread differences between nuclear and cytoplasmic transcriptome patterns. Independently confirmed by imaging-based methods, these results suggest that individual hepatocytes can dynamically switch their metabolic roles. Seq-Scope-X also works in brain and colon, and can be adapted for spatial proteomics, profiling hundreds of barcode-tagged antibody stains at microscopic resolutions in mouse spleens and human tonsils. Together, these findings establish Seq-Scope-X as a powerful platform for ultra-high-resolution whole-transcriptome and proteome profiling, expanding the spatial precision achievable for studying cellular architecture, function, and disease mechanisms.

We discuss two methods for achieving optical resolution beyond the diffraction limit in air. The first method, the solid immersion lens (SIL), improves optical resolution by increasing the numerical aperture (NA) beyond 1.0, the usual limit in air, to a maximum of n, the refractive index of the SIL. We present experimental results with a scanning optical microscope (/spl lambda/=400 nm) based on a micromachined silicon nitride SIL that demonstrates optical resolution /spl sim/1.9 times better with the SIL than without. Specifically, the microfabricated silicon nitride SIL improves the optical edge response from /spl sim/190 nm to /spl sim/100 nm. The second method for improving resolution is based on the strongly enhanced electric field at the surface of a nanoparticle illuminated with light whose wavelength corresponds to the particle's plasmon resonance. We present finite difference time domain (FDTD) calculations showing that the electric field intensity at the sharp apex of a triangular nanoparticle is enhanced by more than two orders of magnitude over the incident intensity in a very small region (< 10 nm).

Introduction: Low-cost optical microscopes are under development by several groups to bring point-of-care malaria diagnosis services to under-served communities in low-resource settings. We describe a Fourier ptychography microscope (FPM) system based on an open-source design and a method of measuring spatial resolution in terms of the optical modulation transfer function (MTF) using images of the 1951 USAF resolution test device. <br/> <br clear="all"/> Methods: The FPM system uses a Raspberry Pi computer and 196-LED matrix light source. FPM images were reconstructed using in-house Python code. The MTF was determined from bar-patterns using a least-squares analysis to fit a square wave, including odd-harmonic terms, to image profiles through the bars. These were normalized to larger uniform regions of the pattern and combined to generate the MTF. <br/> <br clear="all"/> Results: Total component cost of the FPM was less than $200. The theoretical diffraction limit imposed by the pupil function of this system was 280 cycles/mm, slightly less than the measured MTF 10% frequency of 300 cycles/mm. The 10% frequency in FPM images was 550 cycles/mm. <br/> <br clear="all"/> Conclusions: The USAF test pattern provides a practical method for assessing FPM performance in terms of the achieved MTF of the $200 Raspberry Pi based Fourier ptychography microscope. The limiting frequency in FPM images, 550 cycles/mm, was slightly less than the Nyquist sampling cut-off frequency of 670 cycles/mm imposed by pixel spacing.

SUMMARYThe resolution of modern transmission electron microscopes reaches the physical limits imposed by lens aberrations and energy width. One of the many conditions to be fulfilled, the alignment of illuminating and imaging beam onto the coma‐free objective axis, is particularly discussed here since axial coma cannot be detected by the usual resolution‐checking methods. Space consumption of specimen stages prevents the full utilization of the magnetic saturation limit only in the 100 keV range. With higher energies, this handicap is obviated, and some additional advantages can be gained which promote material investigations at atomic resolution, and which are presently utilized in instrumental research projects. High resolution with biological specimens has up to now been unsuccessful because of radiation damage. Optimum utilization of all electrons scattered at the specimen must thus be given priority over optical resolution. Important instrumental requirements are minimum exposure beam control, imaging modes with high collection efficiency, and recording devices with high detection quantum efficiency connected on‐line to image processors. A remarkable decrease in beam sensitivity of organic crystals, by more than one order, has been found by cooling the specimen down to 4 K which, by the use of superconducting lenses, can be combined with both ultra high vacuum and the stability requirements for high resolution. Yet up to now, such protection has not been achieved with He cryostates in conventional lenses, perhaps because a temperature increase even of only a few degree K is harmful.Purely magnetic imaging energy filters are about to be developed to a high optical quality but have been employed so far in only a few high resolution instruments. Such filters allow removal of the inelastic background and thus improvement of contrast of images of low‐Z specimens, particularly in the dark field mode.Finally, some ‘non‐conventional’ projects have made progress. Correction of spherical and chromatic aberration by multipole lenses offers a chance to improve remarkably the resolution in the 100 keV range, to extend the bandwidth of phase contrast transfer and to obtain highly resolved information about inelastic images when an energy filter is also applied. Electron holography provides possibly useful large area phase contrast, particularly if the electron energy is decreased, which may be of great benefit in investigations of unstained specimens.

This research was to determine the effect of Cerium and cooling rate on the microstructure of eutectic Al-11Si-Cu-Mg cast alloy. The Cerium was added to produce Al-11Si-Cu-Mg-1.5Ce alloys. The microstructure was characterized by optical microscope. The Si structure was modified with the increase of the cooling rate, where smaller area was achieved. Ce formed rich intermetallic phase, such as Al-Si-Ce.

Superresolution microscopy of the volume phase transition of pNIPAM microgels

Microscopic optical image density and resolution performance are discussed for laser thermal transfer printing of the dye sublimation type. This printing method provides high resolution because of the high converging property of the laser beam. In this study, optical density profiles of line images printed by dye sublimation type laser thermal transfer printing were measured and compared with images of the wax type in order to clarify the spread of the printed dots. These profiles were explained numerically, using tone reproduction curves. Resolution performance was evaluated, quantitatively, and the maximum resolution was found to be 10,160 dpi. This value was confirmed by the printed microcharacters. Laser dye transfer printing is feasible for printing not only full-color images, but also fine patterns and microcharacters.

Real-time monitoring of lactate release from brain slices has been studied with an optical two-dimensional (2D) imaging biosensor. The 2D biosensor is prepared by direct immobilization of lactate dehydrogenase (LDH) molecules onto a flat silica glass surface through a covalent binding mechanism. The biosensor is able to spatially differentiate lactate concentration variations with conventional optical microscopic spatial resolution. This biosensor has the capability to effectively detect lactate down to a concentration of 100 nM. The 2D biosensor responds uniformly with 2.5% RSD from pixel to pixel. With a 100 ms response time, this 2D biosensor has the capability of monitoring simultaneously many cells in one image. We have studied the impact of KCI on lactate release from brain slices. Clear differences have been observed in lactate release for different regions of the tissue. The real-time determination of the newly released lactate from the mouse brain slices clearly demonstrates the feasibility of monitoring lactate release from living specimens. The 2D biosensor will enable us to study cellular communications and possibly other biological processes that require simultaneous temporal and spatial resolution.

Scanning near-field optical microscopy (SNOM) employs many different forms of optical probes to achieve sub-diffraction limited imaging. The first commonly used probes utilized optical fibers pulled or etched to a small end diameter. This technique has successfully demonstrated spatial optical resolution better than 100 nm. These original near-field probes utilized a coating of aluminum on the sidewalls to achieve field confinement. The fabrication process had problems with irreproducible coatings (leading to blockage or leakage of light), insensitive scanning surface interaction mechanisms, or taper issues leading to low throughput. To overcome these issues, a probe design which involved illumination of sharp metals with optimal polarization was developed to achieve higher topographic and optical spatial resolution. This technique has been termed tip enhanced near-field scanning optical microscopy (TENOM). Although this technique overcomes many of the issues with using fibers, it introduces other issues. This work will cover how one overcome some of the issues with metal probes as well as presenting show our latest results.

The synergy of scanning probe microscope and optical microscope realizes a promising sensing technique in surface characterization in the nanoscale with a high chemical sensitivity, which is not attainable by a simple topographic contrast. This paper introduces one of the synergy tools, that is, near-field scanning optical microscope particularly the use of a sharp metallic probe tip. While optical microscope exhibits a high chemical sensitivity, the spatial resolution of conventional optical microscopes using lens is diffraction limited due to wave nature of light. The key technique to break through this limitation is surface plasmon polariton excitation at the tip, which is promising for both high spatial resolution and high sensitivity. This technique has been recently recognized as tip-enhanced near-field spectroscopy.

Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetero)aryl- or (hetero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413-480 and 527-668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyridyl)ethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.

Overcoming the spatial resolution limitations in optical microscopy due to the diffraction of light has become a major challenge in modern microscopy. To overcome this limit, we demonstrate here the upconversion process in rare-earth-doped fluoride nanoparticles (RE:NPs) that may serve as novel sources for fluorescent microscopy. RE:NPs have been reported to serve as efficient biolabels for cellular and small animal imaging [1]. While almost all work devoted to RE doped nanocrystals in biological applications had investigated NaYF <inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</inf> , here we performed experiments with LaF <inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</inf> and KY <inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</inf> F <inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">10</inf> , other possible materials to host upconversion. Specifically, (2at.%) Er <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3+</sup> , (2at.%) Yb <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3+</sup> : KY <inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</inf> F <inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">10</inf> , (1at.%) Tm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3+</sup> , (6at.%) Yb <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3+</sup> : LaF <inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</inf> and (0.5at.%) Tm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3+</sup> , (12at.%) Yb <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3+</sup> : KY <inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</inf> F <inf xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">10</inf> 20–50 nm-sized crystals had been characterized using AFM, STEM, X-ray diffraction and dynamic light scattering techniques and investigated by luminescence spectroscopy and nonlinear optical microscopy [2]. The latter was done on NP's dispersed in PEG and spun-coated on a glass cover slip. One of the fluorescence images produced with upconversion process in these nanoparticles is presented in Fig. 1. The increase of the photon process order and the corresponding decrease in the full width of half-maximum (FWHM) of intensity point spread function (IPSF) value can be clearly observed. As the result of the photon process order, N, increase, the instrument's optical resolution becomes: Δx, ΔY ≅ 0.61λ/NA√N (1) where λ is the excitation wavelength and NA is the objective's numerical aperture.

Kidney glomerular diseases, such as the minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), and other nephrotic syndromes, are typically diagnosed or confirmed via electron microscopy. Although optical microscopy has been a vital tool to examine clinical specimens for diagnoses in pathology for decades, the optical resolution is constricted by the physical diffraction limit of the optical microscope, which prevents high-resolution investigation of subcellular anatomy, such as of the podocyte tertiary foot processes. Here, we describe a simple, fast, and inexpensive protocol for nanoscale optical imaging of kidney glomeruli. The protocol is based on Expansion Pathology (ExPath), a new principle of microscopy that overcomes optical diffraction limit by chemically embedding specimens into a swellable polymer and physically expanding it homogenously prior to imaging. Our method uses only commercially available reagents, a conventional fluorescence microscope and it can be applied to both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. It requires minimal operative experience in a wet lab, optical microscopy and imaging processing. Finally, we also discuss challenges, limitations and prospective applications for ExPath-based imaging of glomeruli.

A novel combined photoacoustic tomography (PAT), optical resolution photoacoustic microscopy (ORPAM) and optical coherence tomography (OCT) instrument has been developed for imaging biological tissues. The system is based on the use of a Fabry-Perot (FP) polymer film ultrasound sensor. This is designed to be transparent to wavelengths between 590nm and 1200nm so that photoacoustic excitation laser pulses in this spectral range can be transmitted through the sensor into the underlying tissue to allow backward mode operation. The dual PAT-ORPAM capability of the system was demonstrated by imaging a tissue phantom composed of 7&mu;m diameter carbon fibres immersed in an optically scattering liquid. The lateral and vertical spatial resolutions in ORPAM mode are approximately 7&mu;m and 10&mu;m respectively for sub-mm depths. In PAT mode, the lateral spatial resolution is less than 50&mu;m for depths up to 5mm and the vertical resolution is approximately 10&mu;m. The transparent nature of the FP polymer film ultrasound sensor offers a convenient platform for combining other optical imaging modalities with PAT and ORPAM. To illustrate this, a frequency-domain OCT system operating at 1060nm was integrated into the system and combined PAT/OCT images of the skin of a mouse were obtained in vivo.

Photoacoustic microscopy for cells in a 3D microenvironment: Systems and applications