SIAL: A simple image analysis library for wet-lab scientists

Tyrpak, David R.; Li, Yaocun; Lei, Siqi; MacKay, J. Andrew

doi:10.21105/joss.02689

Cited by 3 publications

(6 citation statements)

References 3 publications

(3 reference statements)

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“…In addition to providing a detailed protocol to estimate the intracellular T t of non-fluorescent ELP fusions, we also developed an easily installed FIJI plugin named SIAL . SIAL contains programs for image randomization and blinding, phenotype scoring, and ROI selection.…”

Section: Discussionmentioning

confidence: 99%

“…To blind and randomize our datasets, we developed a Java program that copies a specified directory of images, renames each image with a random three-digit number, and then places the renamed images in a new directory, along with a key which matches each original filename with its corresponding three digit number. This program is called File Randomizer and is part of a larger plugin named Simple Image Analysis Library (or SIAL) . SIAL also contains plugins to assist in phenotype scoring and ROI recording.…”

Section: Methodsmentioning

confidence: 99%

“…This program is called File Randomizer and is part of a larger plugin named Simple Image Analysis Library (or SIAL). 29 SIAL also contains plugins to assist in phenotype scoring and ROI recording. To download SIAL, researchers should open FIJI, go to "Help > Update..." and then update FIJI.…”

Section: ■ Introductionmentioning

confidence: 99%

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Single-Cell Quantification of the Transition Temperature of Intracellular Elastin-like Polypeptides

Tyrpak

Lei

et al. 2021

ACS Biomater. Sci. Eng.

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Elastin-like polypeptides (ELPs) are modular, stimuli-responsive materials that self-assemble into protein-rich microdomains in response to heating. By cloning ELPs to effector proteins, expressed intracellular fusions can even modulate cellular pathways. A critical step in engineering these fusions is to determine and control their intracellular phase transition temperature (T t ). To do so, this Method paper describes a simple live-cell imaging technique to estimate the T t of non-fluorescent ELP fusion proteins by co-transfection with a fluorescent ELP marker. Intracellular microdomain formation can then be visualized in live cells through the co-assembly of the non-fluorescent and fluorescent ELP fusion proteins. If the two ELP fusions have different T t , the intracellular ELP mixture phase separates at the temperature corresponding to the fusion with the lower T t . In addition, co-assembled ELP microdomains often exhibit pronounced differences in size or number, compared to single transfected treatments. These features enable live-cell imaging experiments and image analysis to determine the intracellular T t of a library of related ELP fusions. As a case study, we employ the recently reported Caveolin1-ELP library (CAV1-ELPs). In addition to providing a detailed protocol, we also report the development of a useful FIJI plugin named SIAL (Simple Image Analysis Library), which contains programs for image randomization and blinding, phenotype scoring, and ROI selection. These tasks are important parts of the protocol detailed here and are also commonly employed in other image analysis workflows.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Single-Cell Quantification of the Transition Temperature of Intracellular Elastin-like Polypeptides

Tyrpak

Lei

et al. 2021

ACS Biomater. Sci. Eng.

Self Cite

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“…Confocal microscopy imaging of indirect immunofluorescence demonstrated that Rab27a-enriched vesicles were present in both male BALB/c and NOD mouse LGAC near apical and basolateral membranes (Figure 2a). Quantification of Rab27a immunofluorescence per acinar cell using the corrected total cell fluorescence (CTCF) method [46,47] revealed that Rab27 accumulation was significantly elevated in NOD mouse LGAC, as shown in Figure 2b. Moreover, the increase was apparent, by visual inspection to be more prominent in the subapical area.…”

Section: In Male Nod Mouse Lg Rab27a-enriched Vesicles Are Subapically Increased and Their Size Decreased Relative To Rab27a-enriched Vesmentioning

confidence: 99%

“…Images were first assigned with random codes to achieve a blinded analysis. Corrected total cell fluorescence quantification was performed using a reported image processing pipeline using ImageJ version 2.1.0 (U. S. National Institutes of Health, Bethesda, MD, USA) [47]. 3D % volume colocalization within regions of interest was carried out using Bitplane Imaris software version 9.0.1 (Andor Technology Ltd., Belfast, UK) according to the manufacturer's instructions.…”

Section: Tissue Processing and Confocal Fluorescence Microscopymentioning

confidence: 99%

Rab27a Contributes to Cathepsin S Secretion in Lacrimal Gland Acinar Cells

Edman

2021

IJMS

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Altered lacrimal gland (LG) secretion is a feature of autoimmune dacryoadenitis in Sjögren’s syndrome (SS). Cathepsin S (CTSS) is increased in tears of SS patients, which may contribute to disease. Rab3D and Rab27a/b isoforms are effectors of exocytosis in LG, but Rab27a is poorly studied. To investigate whether Rab27a mediates CTSS secretion, we utilized quantitative confocal fluorescence microscopy of LG from SS-model male NOD and control male BALB/c mice, showing that Rab27a-enriched vesicles containing CTSS were increased in NOD mouse LG. Live-cell imaging of cultured lacrimal gland acinar cells (LGAC) transduced with adenovirus encoding wild-type (WT) mCFP-Rab27a revealed carbachol-stimulated fusion and depletion of mCFP-Rab27a-enriched vesicles. LGAC transduced with dominant-negative (DN) mCFP-Rab27a exhibited significantly reduced carbachol-stimulated CTSS secretion by 0.5-fold and β-hexosaminidase by 0.3-fold, relative to stimulated LGAC transduced with WT mCFP-Rab27a. Colocalization of Rab27a and endolysosomal markers (Rab7, Lamp2) with the apical membrane was increased in both stimulated BALB/c and NOD mouse LG, but the extent of colocalization was much greater in NOD mouse LG. Following stimulation, Rab27a colocalization with endolysosomal membranes was decreased. In conclusion, Rab27a participates in CTSS secretion in LGAC though the major regulated pathway, and through a novel endolysosomal pathway that is increased in SS.

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Phenylephrine increases tear cathepsin S secretion in healthy murine lacrimal gland acinar cells through an alternative secretory pathway

Janga

Edman

2021

Experimental Eye Research

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SIAL: A simple image analysis library for wet-lab scientists

Cited by 3 publications

References 3 publications

Single-Cell Quantification of the Transition Temperature of Intracellular Elastin-like Polypeptides

Single-Cell Quantification of the Transition Temperature of Intracellular Elastin-like Polypeptides

Rab27a Contributes to Cathepsin S Secretion in Lacrimal Gland Acinar Cells

Phenylephrine increases tear cathepsin S secretion in healthy murine lacrimal gland acinar cells through an alternative secretory pathway

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