scholarly journals Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy.

1992 ◽  
Vol 119 (5) ◽  
pp. 1245-1260 ◽  
Author(s):  
K L Taneja ◽  
L M Lifshitz ◽  
F S Fay ◽  
R H Singer
Keyword(s):  
In Situ Hybridization ◽  
Digital Imaging ◽  
Actin Filaments ◽  
Fluorescent In Situ Hybridization ◽  
Single Cells ◽  
Ribonuclease A ◽  
Structural Elements ◽  
Digital Imaging Microscopy ◽  
Cellular Compartments

The distribution of poly(A) RNA has been visualized in single cells using high-resolution fluorescent in situ hybridization. Digital imaging microscopy was used to quantitate the signal in various cellular compartments. Most of the poly(A) signal remained associated with the cellular filament systems after solubilization of membranes with Triton, dissociation of ribosomes with puromycin, and digestion of non-poly(A) RNA with ribonuclease A and T1. The actin filaments were shown to be the predominant cellular structural elements associating with the poly(A) because low doses of cytochalasin released about two-thirds of the poly(A). An approach to assess the extent of colocalization of two images was devised using in situ hybridization to poly(A) in combination with probes for ribosomes, membranes, or F-actin. Digital imaging microscopy showed that most poly(A) spatially distributes most significantly with ribosomes, slightly less with F-actin, and least of all with membranes. The results suggest a mechanism for anchoring (and perhaps moving) much of the cellular mRNA utilizing the interaction between actin filaments and poly(A).

scholarly journals Fluorescence ratio measurements of double-labeled probes for multiple in situ hybridization by digital imaging microscopy

Cytometry ◽  
1992 ◽  
Vol 13 (8) ◽  
pp. 839-845 ◽  
Author(s):  
P. M. Nederlof ◽  
S. van der Flier ◽  
J. Vrolijk ◽  
H. J. Tanke ◽  
A. K. Raap
Keyword(s):  
In Situ Hybridization ◽  
Digital Imaging ◽  
Fluorescence Ratio ◽  
Digital Imaging Microscopy

scholarly journals smiFISH and embryo segmentation for single-cell multi-gene RNA quantification in arthropods

2020 ◽  
Author(s):  
Llilians Calvo ◽  
Matthew Ronshaugen ◽  
Tom Pettini
Keyword(s):  
In Situ Hybridization ◽  
Single Cell ◽  
Single Molecule ◽  
Fluorescent In Situ Hybridization ◽  
Single Cells ◽  
Fano Factor ◽  
Confocal Imaging ◽  
Cell Segmentation ◽  
Expression Variability

ABSTRACTRecently, advances in fluorescent in-situ hybridization techniques and in imaging technology have enabled visualisation and counting of individual RNA molecules in single cells. This has greatly enhanced the resolution in our understanding of transcriptional processes. Here, we adapt a recently published smiFISH protocol (single-molecule inexpensive fluorescent in-situ hybridization) to whole embryos across a range of arthropod model species, and also to non-embryonic tissues. Using multiple fluorophores with distinct spectra and white light laser confocal imaging, we simultaneously detect and separate single RNAs from up to eight different genes in a whole embryo. We also combine smiFISH with cell membrane immunofluorescence, and present an imaging and analysis pipeline for 3D cell segmentation and single-cell RNA counting in whole blastoderm embryos. Finally, using whole embryo single-cell RNA count data, we propose two alternative single-cell variability measures to the commonly used Fano factor, and compare the capacity of these three measures to address different aspects of single-cell expression variability.

scholarly journals smiFISH and embryo segmentation for single-cell multi-gene RNA quantification in arthropods

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Llilians Calvo ◽  
Matthew Ronshaugen ◽  
Tom Pettini
Keyword(s):  
In Situ Hybridization ◽  
Single Cell ◽  
Single Molecule ◽  
Fluorescent In Situ Hybridization ◽  
Single Cells ◽  
Fano Factor ◽  
Confocal Imaging ◽  
Cell Segmentation ◽  
Expression Variability

AbstractRecently, advances in fluorescent in-situ hybridization techniques and in imaging technology have enabled visualization and counting of individual RNA molecules in single cells. This has greatly enhanced the resolution in our understanding of transcriptional processes. Here, we adapt a recently published smiFISH protocol (single-molecule inexpensive fluorescent in-situ hybridization) to whole embryos across a range of arthropod model species, and also to non-embryonic tissues. Using multiple fluorophores with distinct spectra and white light laser confocal imaging, we simultaneously detect and separate single RNAs from up to eight different genes in a whole embryo. We also combine smiFISH with cell membrane immunofluorescence, and present an imaging and analysis pipeline for 3D cell segmentation and single-cell RNA counting in whole blastoderm embryos. Finally, using whole embryo single-cell RNA count data, we propose two alternative single-cell variability measures to the commonly used Fano factor, and compare the capacity of these three measures to address different aspects of single-cell expression variability.

1796: Analysis of Chromosomal Aneuploidy in Patients with Oligoasthenoteratozoospermia(OAT) Using Fluorescent in-situ Hybridization

2007 ◽  
Vol 177 (4S) ◽  
pp. 596-597
Author(s):  
Joseph P. Alukal ◽  
Bobby B. Najari ◽  
Wilson Chuang ◽  
Lata Murthy ◽  
Monica Lopez-Perdomo ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Chromosomal Aneuploidy

Fluorescent in situ hybridization (FISH): A useful diagnostic tool for childhood conjunctival melanoma

2021 ◽  
pp. 112067212110307
Author(s):  
Raquel María Moral ◽  
Carlos Monteagudo ◽  
Javier Muriel ◽  
Lucía Moreno ◽  
Ana María Peiró
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Pediatric Population ◽  
Diagnostic Technique ◽  
Histopathological Diagnosis ◽  
Fish Analysis ◽  
Conjunctival Melanoma ◽  
Adequate Treatment ◽  
First Case

Introduction: Conjunctival melanoma is extremely rare in children and has low rates of resolution. Definitive histopathological diagnosis based exclusively on microscopic findings is sometimes difficult. Thus, early diagnosis and adequate treatment are essential to improve clinical outcomes. Clinical case: We present the first case in which the fluorescent in situ hybridization (FISH) diagnostic technique was applied to a 10-year-old boy initially suspected of having amelanotic nevi in his right eye. Based on the 65% of tumor cells with 11q13 (CCND1) copy number gain and 33% with 6p25 (RREB1) gain as measured by the FISH analysis, and on supporting histopathological findings, the diagnosis of conjunctival melanoma could be made. Following a larger re-excision, adjuvant therapy with Mitomycin C (MMC), cryotherapy and an amniotic membrane graft, the patient has remained disease-free during 9 years of long-term follow-up. Case discussion: Every ophthalmologist should remember to consider and not forget the possibility of using FISH analyses during the differential diagnosis of any suspicious conjunctival lesions. Genetic techniques, such as FISH, have led to great advances in the classification of ambiguous lesions. Evidence-based guidelines for diagnosing conjunctival melanoma in the pediatric population are needed to determine the most appropriate strategy for this age group.

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