A brief historical outline of fluorescence fluctuation correlation techniques is presented, followed by an in-depth review of the theory and development of image correlation techniques, including: image correlation spectroscopy (ICS), temporal ICS (TICS), image cross-correlation spectroscopy (ICCS), spatiotemporal ICS (STICS), k-space ICS (kICS), raster ICS (RICS), and particle ICS (PICS). These techniques can be applied to analyze image series acquired on commercially available laser scanning or total internal reflection fluorescence microscopes, and are used to determine the number density, aggregation state, diffusion coefficient, velocity, and interaction fraction of fluorescently labeled molecules or particles. A comprehensive review of the application of ICS techniques to a number of systems, including cell adhesion, membrane receptor aggregation and dynamics, virus particle fusion, and fluorophore photophysics, is presented.
Mesonephric adenocarcinoma (MA) and mesonephric-like adenocarcinoma (MLA) are uncommon neoplasms of the gynecologic tract that have until recently been poorly understood. Although their morphologic, immunohistochemical, and molecular profiles have been recently defined, little is known about their clinical behavior. Small studies have demonstrated inconsistent findings and no large studies have examined the clinical behavior of these adenocarcinomas. In this multi-institutional study, representing the largest and most stringently defined cohort of cases to date, we examined the clinicopathologic features of 99 MAs and MLAs (30 MAs of the uterine cervix, 44 MLAs of the endometrium, and 25 MLAs of the ovary). Only tumors with characteristic mesonephric morphology and either immunohistochemical or molecular support were included. Our results demonstrate that the majority of mesonephric neoplasms presented at an advanced stage (II to IV) (15/25 [60%] MA of the cervix, 25/43 [58%] MLA of the endometrium, and 7/18 [39%] MLA of the ovary). The majority (46/89 [52%] overall, 12/24 [50%] MA of the cervix, 24/41 [59%] MLA of the endometrium, and 10/24 [42%] MLA of the ovary) developed recurrences, most commonly distant (9/12 [75%] MA of the cervix, 22/24 [92%] MLA of the endometrium, and 5/9 [56%] MLA of the ovary). The 5-year disease-specific survival was 74% (n=26) for MA of cervix, 72% (n=43) for MLA of endometrium, and 71% (n=23) for MLA of ovary. Our results confirm that mesonephric neoplasms are a clinically aggressive group of gynecologic carcinomas that typically present at an advanced stage, with a predilection for pulmonary recurrence.
Cell migration is regulated in part by the connection between the substratum and the actin cytoskeleton. However, the very large number of proteins involved in this linkage and their complex network of interactions make it difficult to assess their role in cell migration. We apply a novel image analysis tool, spatio-temporal image correlation spectroscopy (STICS), to quantify the directed movements of adhesion-related proteins and actin in protrusions of migrating cells. The STICS technique reveals protein dynamics even when protein densities are very low or very high, and works in the presence of large, static molecular complexes. Detailed protein velocity maps for actin and the adhesion-related proteins α-actinin, α5-integrin, talin, paxillin, vinculin and focal adhesion kinase are presented. The data show that there are differences in the efficiency of the linkage between integrin and actin among different cell types and on the same cell type grown on different substrate densities. We identify potential mechanisms that regulate efficiency of the linkage, or clutch, and identify two likely points of disconnect, one at the integrin and the other at α-actinin or actin. The data suggests that the efficiency of the linkage increases as actin and adhesions become more organized showing the importance of factors that regulate the efficiency in adhesion signaling and dynamics.
An unusual subset of endometrial carcinoma, the mesonephric-like adenocarcinomas, are morphologically and molecularly similar to mesonephric carcinoma, characterized by KRAS mutation and lack of microsatellite instability. They also have a unique immunohistochemical profile and are usually positive for GATA-3, CD10, TTF-1, and negative for ER and PR. This study implemented a combined morphologic and molecular approach to retrospectively identify mesonephric-like carcinomas of the endometrium. KRAS-mutated microsatellite stable (MSS) endometrial carcinomas were identified from a database of 570 endometrial carcinomas that had undergone massively parallel sequencing. MSS tumors with canonical KRAS mutations that lacked features diagnostic of endometrioid carcinoma (including squamous or mucinous differentiation), were re-reviewed for morphologic features of mesonephric-like adenocarcinomas. Ninty-eight of 570 endometrial carcinomas (17%) harbored canonical KRAS mutations. Of the KRAS-mutated cases, 80 (82%) were MSS and 18 (18%) had microsatellite instability. Of the KRAS-mutated MSS cases with morphology review, 39/61 (64%) had squamous and/or mucinous differentiation while 22 (36%) lacked these histotype-defining features. Eight of these 22 had PTEN mutations and lacked morphologic features of mesonephric-like adenocarcinoma, leaving 14 cases with a possible mesonephric-like adenocarcinoma-like molecular profile that underwent detailed morphologic re-review. Ten of 14 had morphology typical of serous (3), carcinosarcoma (4), or endometrioid (3) carcinoma. In 4 cases, there was striking morphologic, immunophenotypic, and molecular resemblance to mesonephric carcinoma, leading to re-classification as mesonephric-like adenocarcinoma. Two of the 4 cases presented at an advanced stage, and a third case later developed distant metastases. On the basis of this retrospective study, KRAS-mutated mesonephric-like adenocarcinoma represents ∼1% of all endometrial carcinomas. Future prospective recognition of this unusual variant of endometrial carcinoma may be important given its possible aggressive nature.
We present the theory and application of reciprocal space image correlation spectroscopy (kICS). This technique measures the number density, diffusion coefficient, and velocity of fluorescently labeled macromolecules in a cell membrane imaged on a confocal, two-photon, or total internal reflection fluorescence microscope. In contrast to r-space correlation techniques, we show kICS can recover accurate dynamics even in the presence of complex fluorophore photobleaching and/or "blinking". Furthermore, these quantities can be calculated without nonlinear curve fitting, or any knowledge of the beam radius of the exciting laser. The number densities calculated by kICS are less sensitive to spatial inhomogeneity of the fluorophore distribution than densities measured using image correlation spectroscopy. We use simulations as a proof-of-principle to show that number densities and transport coefficients can be extracted using this technique. We present calibration measurements with fluorescent microspheres imaged on a confocal microscope, which recover Stokes-Einstein diffusion coefficients, and flow velocities that agree with single particle tracking measurements. We also show the application of kICS to measurements of the transport dynamics of alpha5-integrin/enhanced green fluorescent protein constructs in a transfected CHO cell imaged on a total internal reflection fluorescence microscope using charge-coupled device area detection.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.