ABSTRACT
Cell segmentation is the fundamental task. Only by segmenting, can we define the quantitative spatial unit for collecting measurements to draw biological conclusions. Deep learning has revolutionized 2D cell segmentation, enabling generalized solutions across cell types and imaging modalities. This has been driven by the ease of scaling up image acquisition, annotation and computation. However 3D cell segmentation, which requires dense annotation of 2D slices still poses significant challenges. Labelling every cell in every 2D slice is prohibitive. Moreover it is ambiguous, necessitating cross-referencing with other orthoviews. Lastly, there is limited ability to unambiguously record and visualize 1000's of annotated cells. Here we develop a theory and toolbox, u-Segment3D for 2D-to-3D segmentation, compatible with any 2D segmentation method. Given optimal 2D segmentations, u-Segment3D generates the optimal 3D segmentation without data training, as demonstrated on 11 real life datasets, >70,000 cells, spanning single cells, cell aggregates and tissue.
ABSTRACT
Adaptive optics (AO) can restore diffraction limited performance when imaging beyond superficial cell layers in vivo and in vitro, and as such is of interest for advanced 3D microscopy methods such as light-sheet fluorescence microscopy (LSFM). In a typical LSFM system, the illumination and detection paths are separate and subject to different optical aberrations. To achieve optimal microscope performance, it is necessary to sense and correct these aberrations in both light paths, resulting in a complex microscope system. Here, we show that in an oblique plane microscope (OPM), a type of LSFM with a single primary objective lens, the same deformable mirror can correct both the illumination and fluorescence detection. Besides reducing the complexity, we show that AO in OPM also restores the relative alignment of the light-sheet and focal plane, and that a projection imaging mode can stabilize and improve the wavefront correction in a sensorless AO format. We demonstrate OPM with AO on fluorescent nanospheres and by imaging the vasculature and cancer cells in zebrafish embryos embedded in a glass capillary, restoring diffraction limited resolution and improving the signal strength twofold.
ABSTRACT
We present a mechanically sheared image acquisition format for upright and open-top light-sheet microscopes that automatically places data in its proper spatial context. This approach, which reduces computational post-processing and eliminates unnecessary interpolation or duplication of the data, is demonstrated on an upright variant of Axially Swept Light-Sheet Microscopy (ASLM) that achieves a field of view, measuring 774 x 435 microns, that is 3.2-fold larger than previous models and a raw and isotropic resolution of â¼420 nm. Combined, we demonstrate the power of this approach by imaging sub-diffraction beads, cleared biological tissues, and expanded specimens.
ABSTRACT
Mapping the intricate spatial relationships between the many different molecules inside a cell is essential to understanding cellular functions in all their complexity. Super-resolution fluorescence microscopy offers the required spatial resolution but struggles to reveal more than four different targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging extends this number but is limited by its low throughput. Here, we present a method for rapid multiplexed super-resolution microscopy that can, in principle, be applied to a nearly unlimited number of molecular targets by leveraging fluorogenic labeling in conjunction with transient adapter-mediated switching for high-throughput DNA-PAINT (FLASH-PAINT). We demonstrate the versatility of FLASH-PAINT with four applications: mapping nine proteins in a single mammalian cell, elucidating the functional organization of primary cilia by nine-target imaging, revealing the changes in proximity of thirteen different targets in unperturbed and dissociated Golgi stacks, and investigating and quantifying inter-organelle contacts at 3D super-resolution.
Subject(s)
Microscopy, Fluorescence , Animals , DNA , Golgi Apparatus , Mammals , Microscopy, Fluorescence/methods , Oligonucleotides , ProteinsABSTRACT
navigate is a turnkey, open-source software solution designed to enhance light-sheet fluorescence microscopy (LSFM) by integrating smart microscopy techniques into a user-friendly framework. It enables automated, intelligent imaging with a Python-based control system that supports GUI-reconfigurable acquisition routines and the integration of diverse hardware sets. As a comprehensive package, navigate democratizes access to advanced LSFM capabilities, facilitating the development and implementation of smart microscopy workflows without requiring deep programming knowledge or specialized expertise in light-sheet microscopy.
ABSTRACT
The endoplasmic reticulum's (ER's) structure is directly linked to the many functions of the ER, but its formation is not fully understood. We investigate how the ER-membrane curving protein reticulon 4 (Rtn4) localizes to and organizes in the membrane and how that affects the local ER structure. We show a strong correlation between the local Rtn4 density and the local ER membrane curvature. Our data further reveal that the typical ER tubule possesses an elliptical cross-section with Rtn4 enriched at either end of the major axis. Rtn4 oligomers are linear shaped, contain about five copies of the protein, and preferentially orient parallel to the tubule axis. Our observations support a mechanism in which oligomerization leads to an increase of the local Rtn4 concentration with each molecule, increasing membrane curvature through a hairpin wedging mechanism. This quantitative analysis of Rtn4 and its effects on the ER membrane result in a new model of tubule shape as it relates to Rtn4.
Subject(s)
Endoplasmic Reticulum , Nogo Proteins , Endoplasmic Reticulum/ultrastructure , Nogo Proteins/chemistryABSTRACT
Membrane surface reconstruction at the nanometer scale is required for understanding mechanisms of subcellular shape change. This historically has been the domain of electron microscopy, but extraction of surfaces from specific labels is a difficult task in this imaging modality. Existing methods for extracting surfaces from fluorescence microscopy have poor resolution or require high-quality super-resolution data that are manually cleaned and curated. Here, we present NanoWrap, a new method for extracting surfaces from generalized single-molecule localization microscopy data. This makes it possible to study the shape of specifically labeled membranous structures inside cells. We validate NanoWrap using simulations and demonstrate its reconstruction capabilities on single-molecule localization microscopy data of the endoplasmic reticulum and mitochondria. NanoWrap is implemented in the open-source Python Microscopy Environment.
Subject(s)
Mitochondria , Nanotechnology , Membranes , Endoplasmic Reticulum , Microscopy, Fluorescence/methodsABSTRACT
Single-molecule localization microscopy enables three-dimensional fluorescence imaging at tens-of-nanometer resolution, but requires many camera frames to reconstruct a super-resolved image. This limits the typical throughput to tens of cells per day. While frame rates can now be increased by over an order of magnitude, the large data volumes become limiting in existing workflows. Here we present an integrated acquisition and analysis platform leveraging microscopy-specific data compression, distributed storage and distributed analysis to enable an acquisition and analysis throughput of 10,000 cells per day. The platform facilitates graphically reconfigurable analyses to be automatically initiated from the microscope during acquisition and remotely executed, and can even feed back and queue new acquisition tasks on the microscope. We demonstrate the utility of this framework by imaging hundreds of cells per well in multi-well sample formats. Our platform, implemented within the PYthon-Microscopy Environment (PYME), is easily configurable to control custom microscopes, and includes a plugin framework for user-defined extensions.
Subject(s)
Imaging, Three-Dimensional , Software , Microscopy, Fluorescence/methods , Single Molecule Imaging/methodsABSTRACT
Membrane surface reconstruction at the nanometer scale is required for understanding mechanisms of subcellular shape change. This historically has been the domain of electron microscopy, but extraction of surfaces from specific labels is a difficult task in this imaging modality. Existing methods for extracting surfaces from fluorescence microscopy have poor resolution or require high-quality super-resolution data that is manually cleaned and curated. Here we present NanoWrap, a new method for extracting surfaces from generalized single-molecule localization microscopy (SMLM) data. This makes it possible to study the shape of specifically-labelled membraneous structures inside of cells. We validate NanoWrap using simulations and demonstrate its reconstruction capabilities on SMLM data of the endoplasmic reticulum and mitochondria. NanoWrap is implemented in the open-source Python Microscopy Environment.
ABSTRACT
The endoplasmic reticulumâ™s (ER) structure is directly linked to the many functions of the ER but its formation is not fully understood. We investigate how the ER-membrane curving protein reticulon 4 (Rtn4) localizes to and organizes in the membrane and how that affects local ER structure. We show a strong correlation between the local Rtn4 density and the local ER membrane curvature. Our data further reveal that the typical ER tubule possesses an elliptical cross-section with Rtn4 enriched at either end of the major axis. Rtn4 oligomers are linear-shaped, contain about five copies of the protein, and preferentially orient parallel to the tubule axis. Our observations support a mechanism in which oligomerization leads to an increase of the local Rtn4 concentration with each molecule increasing membrane curvature through a hairpin wedging mechanism. This quantitative analysis of Rtn4 and its effects on the ER membrane result in a new model of tubule shape as it relates to Rtn4. Summary: Rtn4 forms linear-shaped oligomers that contain an average of five Rtn4 proteins, localize to the sides of elliptical tubules, prefer orientations near parallel to the tubule axis, and increase local curvature of the ER membrane by increasing local Rtn4 density.
ABSTRACT
We propose an automated wavelet-based method of tracking particles in unreconstructed off-axis holograms to provide rough estimates of the presence of motion and particle trajectories in digital holographic microscopy (DHM) time series. The wavelet transform modulus maxima segmentation method is adapted and tailored to extract Airy-like diffraction disks, which represent bacteria, from DHM time series. In this exploratory analysis, the method shows potential for estimating bacterial tracks in low-particle-density time series, based on a preliminary analysis of both living and dead Serratia marcescens, and for rapidly providing a single-bit answer to whether a sample chamber contains living or dead microbes or is empty.
Subject(s)
Bacteria/isolation & purification , Holography/methods , Microscopy/methods , Bacteria/ultrastructure , Cell Tracking/methods , Particle Size , Serratia marcescens/isolation & purification , Serratia marcescens/ultrastructureABSTRACT
PURPOSE: The microenvironment of breast tumors plays a critical role in tumorigenesis. As long as the structural integrity of the microenvironment is upheld, the tumor is suppressed. If tissue structure is lost through disruptions in the normal cell cycle, the microenvironment may act as a tumor promoter. Therefore, the properties that distinguish between healthy and tumorous tissues may not be solely in the tumor characteristics but rather in surrounding non-tumor tissue. The goal of this paper was to show preliminary evidence that tissue disruption and loss of homeostasis in breast tissue microenvironment and breast bilateral asymmetry can be quantitatively and objectively assessed from mammography via a localized, wavelet-based analysis of the whole breast. METHODS: A wavelet-based multifractal formalism called the 2D Wavelet Transform Modulus Maxima (WTMM) method was used to quantitate density fluctuations from mammographic breast tissue via the Hurst exponent (H). Each entire mammogram was cut in hundreds of 360 × 360 pixel subregions in a gridding scheme of overlapping sliding windows, with each window boundary separated by 32 pixels. The 2D WTMM method was applied to each subregion individually. A data mining approach was set up to determine which metrics best discriminated between normal vs. cancer cases. These same metrics were then used, without modification, to discriminate between normal vs. benign and benign vs. cancer cases. RESULTS: The density fluctuations in healthy mammographic breast tissue are either monofractal anti-correlated (H < 1/2) for fatty tissue or monofractal long-range correlated (H>1/2) for dense tissue. However, tissue regions with H~1/2, as well as left vs. right breast asymetries, were found preferably in tumorous (benign or cancer) breasts vs. normal breasts, as quantified via a combination metric yielding a P-value ~ 0.0006. No metric considered showed significant differences between cancer vs. benign breasts. CONCLUSIONS: Since mammographic tissue regions associated with uncorrelated (H~1/2) density fluctuations were predominantly in tumorous breasts, and since the underlying physical processes associated with a H~1/2 signature are those of randomness, lack of spatial correlation, and free diffusion, it is hypothesized that this signature is also associated with tissue disruption and loss of tissue homeostasis.
Subject(s)
Breast Neoplasms/diagnostic imaging , Breast Neoplasms/pathology , Mammography , Tumor Microenvironment , Adult , Aged , Aged, 80 and over , Breast/diagnostic imaging , Breast/pathology , Homeostasis , Humans , Image Processing, Computer-Assisted , Middle Aged , Wavelet AnalysisABSTRACT
There is growing evidence that the microenvironment surrounding a tumor plays a special role in cancer development and cancer therapeutic resistance. Tumors arise from the dysregulation and alteration of both the malignant cells and their environment. By providing tumor-repressing signals, the microenvironment can impose and sustain normal tissue architecture. Once tissue homeostasis is lost, the altered microenvironment can create a niche favoring the tumorigenic transformation process. A major challenge in early breast cancer diagnosis is thus to show that these physiological and architectural alterations can be detected with currently used screening techniques. In a recent study, we used a 1D wavelet-based multi-scale method to analyze breast skin temperature temporal fluctuations collected with an IR thermography camera in patients with breast cancer. This study reveals that the multifractal complexity of temperature fluctuations superimposed on cardiogenic and vasomotor perfusion oscillations observed in healthy breasts is lost in malignant tumor foci in cancerous breasts. Here we use a 2D wavelet-based multifractal method to analyze the spatial fluctuations of breast density in the X-ray mammograms of the same panel of patients. As compared to the long-range correlations and anti-correlations in roughness fluctuations, respectively observed in dense and fatty breast areas, some significant change in the nature of breast density fluctuations with some clear loss of correlations is detected in the neighborhood of malignant tumors. This attests to some architectural disorganization that may deeply affect heat transfer and related thermomechanics in breast tissues, corroborating the change to homogeneous monofractal temperature fluctuations recorded in cancerous breasts with the IR camera. These results open new perspectives in computer-aided methods to assist in early breast cancer diagnosis.
ABSTRACT
BACKGROUND: When screening for breast cancer, the radiological interpretation of mammograms is a difficult task, particularly when classifying precancerous growth such as microcalcifications (MCs). Biophysical modeling of benign vs. malignant growth of MCs in simulated mammographic backgrounds may improve characterization of these structures METHODS: A mathematical model based on crystal growth rules for calcium oxide (benign) and hydroxyapatite (malignant) was used in conjunction with simulated mammographic backgrounds, which were generated by fractional Brownian motion of varying roughness and quantified by the Hurst exponent to mimic tissue of varying density. Simulated MC clusters were compared by fractal dimension, average circularity of individual MCs, average number of MCs per cluster, and average cluster area. RESULTS: Benign and malignant clusters were distinguishable by average circularity, average number of MCs per cluster, and average cluster area with p<0.01 across all Hurst exponent values considered. Clusters were distinguishable by fractal dimension with p<0.05 in low Hurst exponent environments. As the Hurst exponent increased (tissue density increased) benign and malignant MCs became indistinguishable by fractal dimension. CONCLUSIONS: The fractal dimension of MCs changes with breast tissue density, which suggests tissue environment plays a role in regulating MC growth. Benign and malignant MCs are distinguishable in all types of tissue by shape, size, and area, which is consistent with findings in the literature. These results may help to better understand the effects of the tissue environment on tumor progression, and improve classification of MCs in mammograms via computer-aided diagnosis.