Dataset Information

Automating measurements of fluorescent signals in freely moving plant leaf specimens.

ABSTRACT: Existing methods to quantify fluorescent signals are primarily limited to non-moving objects or tracking a limited number of cells. These techniques, however, are unsuitable for measuring fluorescent signals in time-lapse experiments using plant specimens that move naturally during a course of imaging. We developed an automated method to measure fluorescent signal intensities in transgenic Arabidopsis plants using a stereomicroscope with standard microscopy software. The features of our technique include: 1) recognizing the shape of plant specimens using autofluorescent signals; 2) merging targeted fluorescent signals to specimen outlines; 3) extracting signals within the shape of specimens from their background signals. Our method facilitates the measurement of fluorescent signals on freely moving plant leaves that are physically unrestrained. The method we developed addresses the challenge of recognizing plant shapes without relying on: a) manual definition which is prone to subjectivity and human error; b) introducing stable fluorescent markers to define plant shapes; c) recognizing plant shapes from bright field images which include a wide range of colors and background noise; d) unnecessarily stressing plants by immobilizing them; e) the use of multiple software packages or software development expertise.

SUBMITTER: Kinoshita N

PROVIDER: S-EPMC6566008 | biostudies-other | 2019

REPOSITORIES: biostudies-other

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Automating measurements of fluorescent signals in freely moving plant leaf specimens.

Kinoshita Natsuko N Sugita Aki A Lustig Barry B Betsuyaku Shigeyuki S Fujikawa Tetsu T Morishita Tatsuji T

Plant biotechnology (Tokyo, Japan) 20190101 1

Existing methods to quantify fluorescent signals are primarily limited to non-moving objects or tracking a limited number of cells. These techniques, however, are unsuitable for measuring fluorescent signals in time-lapse experiments using plant specimens that move naturally during a course of imaging. We developed an automated method to measure fluorescent signal intensities in transgenic Arabidopsis plants using a stereomicroscope with standard microscopy software. The features of our techniqu ...[more]

PMID: 31275043

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Project description:Linking neural circuitry to behavior by mapping active neurons in vivo is a challenge. Both genetically encoded calcium indicators (GECIs) and intermediate early genes (IEGs) have been used to pinpoint active neurons during a stimulus or behavior but have drawbacks such as limiting the movement of the organism, requiring a priori knowledge of the active region or having poor temporal resolution. Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) was engineered to overcome these spatial-temporal challenges. CaMPARI is a photoconvertible protein that only converts from green to red fluorescence in the presence of high calcium concentration and 405 nm light. This allows the experimenter to precisely mark active neurons within defined temporal windows. The photoconversion can then be quantified by taking the ratio of the red fluorescence to the green. CaMPARI promises the ability to trace active neurons during a specific stimulus; however, CaMPARI's uses in adult Drosophila have been limited to photoconversion during fly immobilization. Here, we demonstrate a method that allows photoconversion of multiple freely-moving intact adult flies during a stimulus. Flies were placed in a dish with filter paper wet with acetic acid (pH = 2) or neutralized acetic acid (pH = 7) and exposed to photoconvertible light (60 mW) for 30 min (500 ms on, 200 ms off). Immediately following photoconversion, whole flies were fixed and imaged by confocal microscopy. The red:green ratio was quantified for the DC4 glomerulus, a bundle of neurons expressing Ir64a, an ionotropic receptor that senses acids in the Drosophila antennal lobe. Flies exposed to acetic acid showed 1.3-fold greater photoconversion than flies exposed to neutralized acetic acid. This finding was recapitulated using a more physiological stimulus of apple cider vinegar. These results indicate that CaMPARI can be used to label neurons in intact, freely-moving adult flies and will be useful for identifying the circuitry underlying complex behaviors.

Dataset Information

Automating measurements of fluorescent signals in freely moving plant leaf specimens.

Publications

Automating measurements of fluorescent signals in freely moving plant leaf specimens.

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