Gain In Resolution Research Articles

Supercritical angle fluorescence for enhanced axial sectioning in STED microscopy.

We demonstrate subwavelength axial sectioning on biological samples with a stimulated emission depletion (STED) microscope combined with supercritical angle fluorescence (SAF) detection. SAF imaging is a powerful technique for imaging the membrane of the cell based on the direct exploitation of the fluorophore emission properties. Indeed, only when fluorophores are close to the interface can their evanescent near-field emission become propagative and be detected beyond the critical angle. Therefore, filtering out the SAF emission from the undercritical angle fluorescence (UAF) emission in the back focal plane of a high-NA objective lens permits nanometer axial sectioning of fluorescent emitters close to the coverslip. When combined with STED microscopy, a straightforward gain in axial resolution can be reached without any alteration of the STED beam path. Indeed, STED-SAF implementation only requires a modification in the detection path of the STED microscope and thus could be widely implemented.

Full-spectrum denoising of high-SNR hyperspectral images.

The high spectral redundancy of hyper/ultraspectral Earth-observation satellite imaging raises three challenges: (a)to design accurate noise estimation methods, (b)to denoise images with very high signal-to-noise ratio (SNR), and (c)to secure unbiased denoising. We solve (a)by a new noise estimation, (b)by a novel Bayesian algorithm exploiting spectral redundancy and spectral clustering, and (c)by accurate measurements of the interchannel correlation after denoising. We demonstrate the effectiveness of our method on two ultraspectral Earth imagers, IASI and IASI-NG, one flying and the other in project, and sketch the major resolution gain of future instruments entailed by such unbiased denoising.

Near-field Fourier ptychography: super-resolution phase retrieval via speckle illumination.

High spatial resolution is the goal of many imaging systems. While designing a high-resolution lens with diffraction-limited performance over a large field of view remains a difficult task, creating a complex speckle pattern with wavelength-limited spatial features is easily accomplished with a simple random diffuser. With this observation and the concept of near-field ptychography, we report a new imaging modality, termed near-field Fourier ptychography, to address high-resolution imaging challenges in both microscopic and macroscopic imaging settings. 'Near-field' refers to placing the object at a short defocus distance with a large Fresnel number. We project a speckle pattern with fine spatial features on the object instead of directly resolving the spatial features via a high-resolution lens. We then translate the object (or speckle) to different positions and acquire the corresponding images by using a low-resolution lens. A ptychographic phase retrieval process is used to recover the complex object, the unknown speckle pattern, and the coherent transfer function at the same time. In a microscopic imaging setup, we use a 0.12 numerical aperture (NA) lens to achieve an NA of 0.85 in the reconstruction process. In a macroscale photographic imaging setup, we achieve ~7-fold resolution gain by using a photographic lens. The collection optics do not determine the final achievable resolution; rather, the speckle pattern's feature size does. This is similar to our recent demonstration in fluorescence imaging settings (Guo et al., Biomed. Opt. Express, 9(1), 2018). The reported imaging modality can be employed in light, coherent X-ray, and transmission electron imaging systems to increase resolution and provide quantitative absorption and object phase contrast.

Capabilities of the Falcon III detector for single-particle structure determination

Direct electron detectors are an essential asset for the resolution revolution in electron cryo microscopy of biological objects. The direct detectors provide two modes of data acquisition; the counting mode in which single electrons are counted, and the integrating mode in which the signal that arises from the incident electrons is integrated. While counting mode leads to far higher detective quantum efficiency at all spatial frequencies, the integrating mode enables faster data acquisition at higher exposure rates. For optimal throughput at best possible resolution it is important to understand when the better performance in counting mode becomes essential for solving a structure and when the lower detective quantum efficiency in integrating mode can be compensated by increasing the number of particles in the data set. Here, we provide a case study of the Falcon III camera, which has counting mode capability at exposure rates of <0.9 e−/Px² and integrating mode capability at exposure rates above 10 e−/Px². We found that counting mode gives better resolution for medium sized complexes such as the β-galactosidase (465 kDa) (2.2 Å, 97% of Nyquist vs. 2.4 Å, 89% of Nyquist) with data sets of similar size. However, for larger particles such as Hepatitis B virus capsid like particles (4.8 MDa) we did not find any resolution gain in counting mode.

Development of a crystal collimation system for high-resolution ultra-small-angle X-ray scattering applications.

Crystal collimation offers a viable alternative to the commonly used pinhole collimation in small-angle X-ray scattering (SAXS) for specific applications requiring highest angular resolution. This scheme is not affected by the parasitic scattering and diffraction-limited beam broadening. The Darwin width of the rocking curve of the crystals mainly defines the ultimate beam divergence. For this purpose, a dispersive Si-111 crystal collimation set-up based on two well conditioned pseudo channel-cut crystals (pairs of well polished, independent parallel crystals) using a higher-order reflection (Si-333) has been developed. The gain in resolution is obtained at the expense of flux. The system has been installed at the TRUSAXS beamline ID02 (ESRF) for reducing the horizontal beam divergence in high-resolution mesurements. The precise mechanics of the system allows reproducible alignment of the Bragg condition. The high resolution achieved at a sample-detector distance of 31 m is demonstrated by ultra-small-angle X-ray scattering measurements on a model system consisting of micrometre-sized polystyrene latex particles with low polydispersity.

Computational Light Field Generation Using Deep Nonparametric Bayesian Learning

In this paper, we present a deep nonparametric Bayesian method to synthesize a light field from a single image. Conventionally, light-field capture requires special optical architecture, and the gain in angular resolution often comes at the expense of a reduction in spatial resolution. Techniques for computationally generating the light field from a single image can be expanded further to a variety of applications, ranging from microscopy and materials analysis to vision-based robotic control and autonomous vehicles. We treat the light field as multiple sub-aperture views, and to compute the novel viewpoints, our model contains three major components. First, a convolutional neural network is used for predicting the depth probability map from the image. Second, a multi-scale feature dictionary is constructed within a multi-layer dictionary learning network. Third, the novel views are synthesized taking into account both the probabilistic depth map and the multi-scale feature dictionary. The experiments show that our method outperforms several state-of-the-art novel view synthesis methods in delivering good image resolution.

High-Resolution Imaging of Multi-Channel Forward-Looking Synthetic Aperture Radar Under Curve Trajectory

Forward-looking synthetic aperture radar is an essential tool in modern remote sensing applications, but its imaging is a challenging task. Few existing methods consider the curve trajectory. Aiming at the difficulty of resolution and the Doppler ambiguity in forward-looking SAR under the curve trajectory, this paper establishes a forward-looking multi-channel SAR system model and proposes a novel approach for the forward-looking SAR imaging under the curve trajectory. The approach is implemented by a two-step process: 1) The Doppler ambiguity under forward-looking mode is solved by adaptive beam-forming processing; and 2) A sparsity-driven optimization imaging algorithm is developed to enhance the azimuth angle resolution in the forward-looking direction, where the resolution gain aroused by the curve trajectory is exploited. The extensive simulation confirms the validity of the proposed approach.

High-resolution polymer high performance liquid chromatography: Application of a saw tooth gradient for the separation of various polymers

Currently, a lot of research effort in polymer analysis by liquid chromatographic techniques, including size exclusion chromatography (SEC), polymer HPLC or liquid chromatography at critical conditions, is done aiming to improve separation performance. In this study, novel gradient protocols were investigated primarily based on gradient polymer elution chromatography (GPEC). Starting with linear gradients and stepwise gradients a new periodic saw tooth gradient profile was developed and optimized. Optimum settings for the saw tooth gradient design were evaluated by design of experiments (DoE) based on Taguchi’s methodology for various types of stationary phases. The gain of peak resolution was dependent on the effective gradient step height. The optimized protocol enabled high-resolution polymer HPLC (HRP-HPLC) separations with common HPLC instruments. The quality of separation was evaluated by heart-cut fraction collection of HRP-HPLC and subsequent determination of the individual fractions by SEC or MALDI-ToF mass spectrometry. Finally, different types of polymers, such as PVC, PDMS, PMMA, or PPG, were studied with the new method and a universal applicability was shown.

Microsphere-mediated optical contrast tuning for designing imaging systems with adjustable resolution gain

Upon illumination, a dielectric microsphere (μS) can generate a photonic nanojet (PNJ), which plays a role in the super-resolution imaging of a sample placed in the μS’s immediate proximity. Recent microscopy implementations pioneered this concept but, despite the experimental characterization and theoretical modeling of the PNJ, the key physical factors that enable optimization of such imaging systems are still debated. Here, we systematically analyzed the parameters that govern the resolution increase in the case of large-diameter (>20 µm) μS-assisted incoherent microscopy by studying both the illumination and the detection light paths. We determined the enhanced-resolution zone created by the μS, in which the detection system has a net resolution gain that we calculated theoretically and subsequently confirmed experimentally. Our results quantitatively describe the resolution enhancement mediated by the optical contrast between the μS and its surrounding medium, and provide concrete means for designing μS-enhanced imaging systems for several application requirements.

Resolution gain in space-time digital holography by self-assembling of the object frequencies.

Space-time digital holography (STDH) exploits the object motion to record the hologram in a hybrid ST domain. This representation adds new capabilities to conventional DH, e.g.,unlimited field of view and variable phase shifting. This is the best candidate for imaging biological samples flowing in microfluidic channels. Here, we show that STDH is able to improve the spatial resolution as well. Different from other super-resolution approaches, stitching between holograms or their spectra is no longer required. Moreover, we introduce a new oblique STDH modality to record and process hybrid ST representations. This allows improving resolution in 2D with one single object scan, paving the way to the use of STDH for super-resolution imaging onboard microfluidic devices.

3D functional ultrasound imaging of pigeons

Recent advances in ultrasound Doppler imaging have facilitated the technique of functional ultrasound (fUS) which enables visualization of brain-activity due to neurovascular coupling. As of yet, this technique has been applied to rodents as well as to human subjects during awake craniotomy surgery and human newborns. Here we demonstrate the first successful fUS studies on awake pigeons subjected to auditory and visual stimulation. To allow successful fUS on pigeons we improved the temporal resolution of fUS up to 20,000 frames per second with real-time visualization and continuous recording. We show that this gain in temporal resolution significantly increases the sensitivity for detecting small fluctuations in cerebral blood flow and volume which may reflect increased local neural activity. Through this increased sensitivity we were able to capture the elaborate 3D neural activity pattern evoked by a complex stimulation pattern, such as a moving light source. By pushing the limits of fUS further, we have reaffirmed the enormous potential of this technique as a new standard in functional brain imaging with the capacity to unravel unknown, stimulus related hemodynamics with excellent spatiotemporal resolution with a wide field of view.

Bubble-echo based deconvolution of contrast-enhanced ultrasound imaging: Simulation and experimental validations.

Improvement of both the imaging resolution and the contrast-to-tissue ratio (CTR) is a current emphasis of contrast-enhanced ultrasound (CEUS) for microvascular perfusion imaging. Based on the strong nonlinear characteristics of contrast agents, the CTRs have been significantly enhanced using various advanced CEUS methods. However, the imaging resolution of these methods is limited by the finite bandwidth of the imaging process. This study aimed to propose a bubble-echo based deconvolution (BED) method for CEUS with both improved resolution and CTR. The method is built on a modified convolution model and uses novel bubble-echo based point-spread-functions to reconstruct the images by regularized inverse Wiener filtering. Performances of the proposed BED for three CEUS modes are investigated through simulations and in vivo perfusion experiments. BED of fundamental imaging was found to have the highest improvement in imaging resolution with the resolution gain up to 2.0 ± 0.2 times, which was comparable to the approved cepstrum-based deconvolution (CED). BED of second-harmonic imaging had the best performance in CTR with an enhancement of 9.8 ± 2.3 dB, which was much higher than CED. Pulse inversion BED had both a better resolution and a higher CTR. All results indicate that BED could obtain CEUS image with both an improved resolution and a high CTR, which has important significance to microvascular perfusion evaluation in deep tissue.

Abstract LB-383: Towards automation of germline variant curation in cancer genetics

Abstract Cancer care professionals are now increasingly asked to interpret multiplexed gene sequencing of patients at hereditary risk for cancer. Assessments for variant classification now require orthogonal data searches, requiring aggregation of multiple lines of evidence from diverse resources. The burden of evidence required for each variant to meet thresholds for pathogenicity or actionability now poses a growing challenge for those seeking to counsel patients and families following germline genetic testing. A computational tool that automates, provides uniformity and significantly accelerates this interpretive process is required. The automated tool described here, Pathogenicity of Mutation Analyzer (PathoMAN) automates germline genomic variant curation from clinical sequencing based on ACMG guidelines. PathoMAN aggregates multiple tracks of genomic, protein and disease specific information from public sources. When compared against expert curation aggregated from three groups - (i) Prostate Cancer Study; (ii) Breast Cancer Study and (iii) ClinVar. PathoMAN achieves high concordance (83.1% pathogenic, 75.5% benign) and negligible discordance (0.04% pathogenic, 0.9% benign) when contrasted against expert curation. Loss of resolution (8.6% pathogenic, 23.64% benign) and gain of resolution (6.6% pathogenic, 1.6% benign) are observed. We highlight the advantages and weaknesses related to the use of programmable automation of variant classification. PathoMAN provides a substantial advance in rapid classification of genetic variants by generation of robust models using a knowledge-base of diverse genetic data. It is an easily accessible, web-based resource that allows rapid pathogenicity assessment of a large number of variants. We also propose a new nosology for the five ACMG classes to facilitate more accurate reporting to ClinVar. The proposed refinements will better structure ClinVar to allow automation in cancer susceptibility reporting. Such bioinformatics tools are an essential prerequisite to reduce the manual workload of domain level experts. Citation Format: Vignesh Ravichandran, Zarina Shameer, Kenneth Offit, Vijai Joseph. Towards automation of germline variant curation in cancer genetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-383.

Ultrasonic tissue reflectivity function estimation using correlation constrained multichannel flms algorithm with missing rf data

Poor resolution of ultrasound images due to convolution of the tissue reflectivity function (TRF) with the system point spread function (PSF) is a major issue in medical ultrasound imaging. In this paper, we propose a correlation constrained missing-data estimation based blind multichannel frequency-domain least-mean-squares (md-bMCFLMS) algorithm to undo the effect of PSF on the ultrasound radio-frequency (RF) data. In the first step, a block-based MCFLMS (bMCFLMS) algorithm is proposed to estimate the TRFs and the PSF, which are used in the second step to estimate the missing data. This missing data is used in the md-bMCFLMS algorithm to construct a modified cost function for further improvement of the image resolution. To account for the nonstationarity of the PSF, unlike the blocking approach described in the literature, we introduce a time-efficient blocking method in this paper. The blocking approach described here uses a block position independent fixed size matrix and can be implemented parallely. The bMCFLMS algorithm, however, shows misconvergence due to both channel noise and propagation of TRF estimation error from the previous blocks. This phenomenon is more intense in the case of md-bMCFLMS algorithm because of increased estimation error. To address this problem, a novel constraint based on the correlation between the measured RF data and estimated TRF is proposed in this paper. The efficacy of our proposed blind deconvolution algorithm is measured using simulation phantom and in-vivo data. The proposed md-bMCFLMS algorithm shows normalised projection misalignment (NPM) improvement of about dB and resolution gain (RG) improvement of 1.14 ∼ 6.4 dB compared to other techniques in the literature. Moreover, because of the frequency-domain implementation it is computationally more efficient, fast converging and robust than its time-domain counterpart l1-bMCLMS algorithm reported in the literature.

Preferred image quality metric for shifted superimposition-based resolution-enhanced images

Shifted superimposition is a resolution-enhancement method that has gained popularity in the projector industry the last couple of years. This method consists of shifting every other projected frame spatially with subpixel precision, and by doing so creating a new pixel grid on the projected surface with smaller effective pixel pitch. There is still an open question of how well this technique performs in comparison with the native resolution, and how high the effective resolution gain really is. To help investigate these questions, we have developed a framework for simulating different superimposition methods over different image contents, and evaluate the result using several image quality metrics (IQMs). We have also performed a subjective experiment with observers who rate the simulated image content, and calculated the correlation between the subjective results and the IQMs. We found that the visual information fidelity metric is the most suitable to evaluate natural superimposed images when subjective match is desired. However, this metric does not detect the distortion in synthetic images. The multiscale structural similarity metric which is based on the analysis of image structure is better at detecting this distortion.

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy.

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

Parametric bandwidth synthesis for active sonar

This talk presents an unconventional range processing technique applicable to linear FM pulsed and continuous active sonar. Parametric bandwidth synthesis (BWS) involves performing linear-predictive extrapolation using a low-order parametric autoregressive (AR) model to extend the bandwidth of received sonar returns. Synthetically increasing the bandwidth of echoes results in improved range resolution and greater pulse compression gain compared to standard matched filtering. It should be noted that despite the resolution improvement, BWS does not improve ranging accuracy beyond that of the transmitted signal. Using a low-order AR model for BWS enhances specular target returns and discrete scatterers, while not enhancing diffuse multipath, reverberation, and noise returns. BWS performance was verified in simulation and with sea trial data from the Littoral Continuous Active Sonar 2015 (LCAS-15) experiment. Processed results confirm that BWS improves range resolution of the echo repeater target, while suppressing multipath, diffuse reverberation, and noise. Analysis of the practical limits of BWS are also included.This talk presents an unconventional range processing technique applicable to linear FM pulsed and continuous active sonar. Parametric bandwidth synthesis (BWS) involves performing linear-predictive extrapolation using a low-order parametric autoregressive (AR) model to extend the bandwidth of received sonar returns. Synthetically increasing the bandwidth of echoes results in improved range resolution and greater pulse compression gain compared to standard matched filtering. It should be noted that despite the resolution improvement, BWS does not improve ranging accuracy beyond that of the transmitted signal. Using a low-order AR model for BWS enhances specular target returns and discrete scatterers, while not enhancing diffuse multipath, reverberation, and noise returns. BWS performance was verified in simulation and with sea trial data from the Littoral Continuous Active Sonar 2015 (LCAS-15) experiment. Processed results confirm that BWS improves range resolution of the echo repeater target, while suppr...

Numerical calculation and experimental study on the small-size streak tube

The streak tube imaging lidar (STIL) community requires the streak tube with characteristics of small-size, high edge spatial resolution, high luminance gain, and large working area. In this work, with the aid of the computer simulation technology software, a streak camera with high edge spatial resolution and high luminance gain is designed, in which there are adopted 1) a spherical photocathode and screen to increase the edge spatial resolution and detection area, further enlarging the field of view for the STIL; 2) a slit accelerating electrode instead of the mesh one favorable for improving the electrical resistance and reliability for streak camera; 3) a streak tube with lower magnification combining with -15 kV working voltage to be able to achieve high luminance gain, thus further increasing the detection distance for STIL. Some static and dynamic properties of the tube are analyzed by observing different electron trajectories emitted from a number of different points on the photocathode. As for the spatial resolution, spatial modulation transfer function method is used to evaluate the spatial resolution characteristics of the streak tube. The 36.9 lp/mm at MTF=5% in static mode and 23 lp/mm at MTF=5% in dynamic mode of the high resolution across the 16 mm long slit on the photocathode can be obtained. As for the temporal resolution, three electron pulses at intervals of 54.6 ps can be well resolved by the streak tube in the dynamic mode. Thus, the temporal resolution of the streak tube is better than 54.6 ps. Furthermore, the influence of shape of the photocathode and screen on spatial resolution are analyzed. Compared with the P-streak tube (streak tube with plane photocathode and plane screen), S-streak tube (steak tube with spherical photocathode and spherical screen) can greatly improve the spatial resolution. The slit image of the spherical and plane photocathode are simulated. The spatial dispersion of the off-axis 8 mm slit image along the scanning direction is analyzed. The experimental results demonstrate that the spatial resolution of the small-size streak tube is 29.3 lp/mm at MTF=5% over the whole working area 16 mm2 mm, and the luminance gain is higher than 39.4. The static spatial resolution of the small-size streak tube is much higher than 15 lp/mm at CTF = 11.64%; the dynamic spatial resolution is higher than 9.8 lp/mm at CTF=5.51%; the temporal resolution is higher than 54.6 ps at Tscreen=4.3 ns and has good consistency on the whole photocathode, and the dynamic range is 345:1 at 54.6 ps. The streak camera contains 6 scanning levels for different depth of field and detection accuracy to achieve ultrafast signal diagnosis at different scanning speeds. The streak tube has a smaller dimension of 40 mm140 mm. It is of great significance in unmanned aerial and spaceborne laser imaging lidar detection.

13-fold resolution gain through turbid layer via translated unknown speckle illumination.

Fluorescence imaging through a turbid layer holds great promise for various biophotonics applications. Conventional wavefront shaping techniques aim to create and scan a focus spot through the turbid layer. Finding the correct input wavefront without direct access to the target plane remains a critical challenge. In this paper, we explore a new strategy for imaging through turbid layer with a large field of view. In our setup, a fluorescence sample is sandwiched between two turbid layers. Instead of generating one focus spot via wavefront shaping, we use an unshaped beam to illuminate the turbid layer and generate an unknown speckle pattern at the target plane over a wide field of view. By tilting the input wavefront, we raster scan the unknown speckle pattern via the memory effect and capture the corresponding low-resolution fluorescence images through the turbid layer. Different from the wavefront-shaping-based single-spot scanning, the proposed approach employs many spots (i.e., speckles) in parallel for extending the field of view. Based on all captured images, we jointly recover the fluorescence object, the unknown optical transfer function of the turbid layer, the translated step size, and the unknown speckle pattern. Without direct access to the object plane or knowledge of the turbid layer, we demonstrate a 13-fold resolution gain through the turbid layer using the reported strategy. We also demonstrate the use of this technique to improve the resolution of a low numerical aperture objective lens allowing to obtain both large field of view and high resolution at the same time. The reported method provides insight for developing new fluorescence imaging platforms and may find applications in deep-tissue imaging.

Performance optimization of ultra high-resolution recycling liquid chromatography

The optimization of a twin-column recycling separation process (TCRSP) for maximum resolution or maximum speed-resolution was investigated. The general optimization method was based on the construction of kinetic plots by assuming an ideal TCRSP (no efficiency loss upon recycling). For the optimization, we examined three chromatographic parameters: operation pressure (3000, 6000, 9000, and 12,000psi), column length (10, 15, and 25cm), and column inner diameter (i.d.) (2.1, 3.0, and 4.6mm). Accordingly, the highest TCRSP resolution level is expected for 25cm long columns packed with 2.5, 2.0, 1.7, and 1.6μm particles at pressures of 3000, 6000, 9000, and 12,000psi, respectively. The maximum speed-resolution performance is expected for 10cm columns packed with 3.7, 3.0, 2.6, and 2.4μm particles. 3.0mm i.d. columns are best to minimize the negative impacts of thermal and inter-column dispersion effects on the TCRSP performance. The method was illustrated for the challenging separation (selectivity factor α<1.02) of small molecules in RPLC at a maximum pressure of 6000psi using commercially available columns. Accordingly, 3.0×150mm columns packed with 2.5μm cellulose-1 Trefoil particles (chiral separation, γ-phenylbutyrolactone, α=1.01, efficiency N=4500) and 2.7μm Cortecs-C18 particles (isotope separation, α=1.02, N=14, 500) particles were found to be the most suitable columns to maximize speed-resolution performance. Further optimization of the TCRSP performance was required by reducing the inter-column sample dispersion that could cause undesirable peak tailing. A standard 2.4μL Rheodyne valve and 100μm i.d. tubes were replaced with a home-made 0.5μL low-dispersion prototype valve and 75μm i.d. perfect connection tubes. As a result, the experimental resolution factors were increased by +60% (γ-phenylbutyrolactone, 25 cycles, Rs=0.7→1.1) and +80% (deuterated benzenes, 22 cycles, Rs=1.1→2.0). Direct comparison between the experimental and the predicted TCRSP performance unambiguously demonstrated that the resolution gain was explained by the significant reduction of the peak tailing after a large number of cycles (n>20).