From LipidomicsWiki

Contents 1 Introduction 2 Platforms 2.1 SPP for the Evotec Opera automated microscope 2.2 SPP for the analysis of lipid droplets on the Discovery-1 automated microscope of Molecular Devices 2.3 SPP for the analysis of lipid droplet volume using the Volocity software from Improvision (Perkin Elmer) 3 Implementation 4 Processing Pipeline

Introduction

Much more so than other experimental platforms, high-content imaging comes with SPP's that are tightly linked to the acquisition platforms. For this reason, the SPP's for high-content imaging are given per platform used in the consortium.

Platforms

SPP for the Evotec Opera automated microscope

• SPP for the Evotec Opera automated microscope

SPP for the analysis of lipid droplets on the Discovery-1 automated microscope of Molecular Devices

• SPP for the analysis of lipid droplets for the Discovery-1 automated microscope of Molecular Devices

SPP for the analysis of lipid droplet volume using the Volocity software from Improvision (Perkin Elmer)

Because the lipid droplets were not evenly distributed in the same focal plane we acquired z-stacks in 30 sections of cells of each treatment using the Leica SP5 confocal microscope. To achieve the required spatial resolution, we used a 40x (NA 0.90) objective. Using the Volocity software, images were then automatically projected into a single focal plane with all objects appearing in focus. This allowed segmentation and identification of objects within the cells using a customized image analysis protocol. In this protocol objects were first identified by intensity and subsequently not touching objects were separated by a size of 0.08 µm3. In addition objects with a size smaller than 0.05 µm3 and greater than 0.8 µm3 were excluded. Using this protocol a good separation of solitary lipid droplets within the cells could be achieved.

Figure shows the creation of a measurement protocol in the Volocity software for 3D analysis of lipid droplets. Different colours of lipid droplets in the image represent different lipid droplet volumes.

First the required measurement protocol tasks are dragged off from the list into the measurement protocol and values are added. The image preview will show the feedbyck as measurements are made and the measurements are subsequently ahown as a table or histogram.

Implementation

Implementation of High-Content Imaging SPPs

Processing Pipeline

Processing Pipeline for High-Content Imaging SPPs

Back to Standard Processing Procedures