Blinking fluorescent probes for tubulin nanoscopy in living and fixed cells

Here we report a small molecule tubulin probe for single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy and MINFLUX nanoscopy, which can be used in living and fixed cells. We explored a series of taxane derivatives containing spontaneously blinking far-red dye hydroxymethyl silicon–rhodamine (HMSiR) and found that the linker length profoundly affects the probe permeability and off-targeting in living cells. The best performing probe, HMSiR-tubulin, is composed of cabazitaxel and the 6′-regioisomer of HMSiR bridged by a C6 linker. Microtubule diameter of ≤50 nm was routinely measured in SMLM experiments on living and fixed cells. HMSiR-tubulin allows a complementary use of different nanoscopy techniques for investigating microtubule functions and developing imaging methods. For the first time, we resolved the inner microtubule diameter of 16 ± 5 nm by optical nanoscopy and thereby demonstrated the utility of a self-blinking dye for MINFLUX imaging

Carboxylated photoswitchable diarylethenes for biolabeling and super-resolution RESOLFT microscopy.

Reversibly photoswitchable 1,2-bis(2-ethyl-6-phenyl-1-benzothiophene-1,1-dioxide-3-yl)perfluorocyclopentenes (EBT) having fluorescent "closed" forms were decorated with four or eight carboxylic groups and attached to antibodies. Low aggregation, efficient photoswitching in aqueous buffers, specific staining of cellular structures, and good photophysical properties were demonstrated. Alternating light pulses of UV and blue light induce numerous reversible photochemical transformations between two stables states with distinct structures. Using relatively low light intensities, EBTs were applied in biology-related super-resolution microscopy based on the reversible saturable (switchable) optical linear fluorescence transitions (RESOLFT) and demonstrated optical resolution of 75 nm

Carboxylierte photoschaltbare Diarylethene als Biomarkierungen für hochauflösende RESOLFT-Mikroskopie.

Reversibel photoschaltbares 1,2-Bis(2-ethyl-6-phenyl-1-benzothiophen-1,1-dioxid-3-yl)perfluorcyclopenten (EBT) mit fluoreszierender “geschlossener” Form wurde mit vier oder acht Carboxygruppen versehen und an Antikörper gebunden. Die carboxylierten Derivate wiesen geringe Aggregation, effizientes Photoschalten in wässrigen Puffern, gezieltes Färben von zellulären Strukturen und gute photophysikalische Eigenschaften auf. Abwechselnde Bestrahlung mit UV und blauem Licht relativ geringer Intensität führte zu reversibler photochemischer Isomerisierung zwischen zwei stabilen Strukturen über mehrere dutzend Schaltzyklen. Dies ermöglichte die Verwendung der Farbstoffe für hochauflösende RESOLFT-Mikroskopie (“reversible switchable optical linear fluorescence transitions”). Hierbei konnte eine optische Auflösung von 75 nm an zellulären Tubulin-Filamenten erzielt werden

Large-scale computations on histology images reveal grade-differentiating parameters for breast cancer

BACKGROUND: Tumor classification is inexact and largely dependent on the qualitative pathological examination of the images of the tumor tissue slides. In this study, our aim was to develop an automated computational method to classify Hematoxylin and Eosin (H&E) stained tissue sections based on cancer tissue texture features. METHODS: Image processing of histology slide images was used to detect and identify adipose tissue, extracellular matrix, morphologically distinct cell nuclei types, and the tubular architecture. The texture parameters derived from image analysis were then applied to classify images in a supervised classification scheme using histologic grade of a testing set as guidance. RESULTS: The histologic grade assigned by pathologists to invasive breast carcinoma images strongly correlated with both the presence and extent of cell nuclei with dispersed chromatin and the architecture, specifically the extent of presence of tubular cross sections. The two parameters that differentiated tumor grade found in this study were (1) the number density of cell nuclei with dispersed chromatin and (2) the number density of tubular cross sections identified through image processing as white blobs that were surrounded by a continuous string of cell nuclei. Classification based on subdivisions of a whole slide image containing a high concentration of cancer cell nuclei consistently agreed with the grade classification of the entire slide. CONCLUSION: The automated image analysis and classification presented in this study demonstrate the feasibility of developing clinically relevant classification of histology images based on micro- texture. This method provides pathologists an invaluable quantitative tool for evaluation of the components of the Nottingham system for breast tumor grading and avoid intra-observer variability thus increasing the consistency of the decision-making process

Utility of multispectral imaging for nuclear classification of routine clinical histopathology imagery

<p>Abstract</p> <p>Background</p> <p>We present an analysis of the utility of multispectral versus standard RGB imagery for routine H&E stained histopathology images, in particular for pixel-level classification of nuclei. Our multispectral imagery has 29 spectral bands, spaced 10 nm within the visual range of 420–700 nm. It has been hypothesized that the additional spectral bands contain further information useful for classification as compared to the 3 standard bands of RGB imagery. We present analyses of our data designed to test this hypothesis.</p> <p>Results</p> <p>For classification using all available image bands, we find the best performance (equal tradeoff between detection rate and false alarm rate) is obtained from either the multispectral or our "ccd" RGB imagery, with an overall increase in performance of 0.79% compared to the next best performing image type. For classification using single image bands, the single best multispectral band (in the red portion of the spectrum) gave a performance increase of 0.57%, compared to performance of the single best RGB band (red). Additionally, red bands had the highest coefficients/preference in our classifiers. Principal components analysis of the multispectral imagery indicates only two significant image bands, which is not surprising given the presence of two stains.</p> <p>Conclusion</p> <p>Our results indicate that multispectral imagery for routine H&E stained histopathology provides minimal additional spectral information for a pixel-level nuclear classification task than would standard RGB imagery.</p