A Simple Method for Creating a High‐Content Microscope for Imaging Multiplexed Tissue Microarrays

Abstract

High-throughput, high-content imaging technologies and multiplex slide scanning have become widely used. Advantages of these approaches include the ability to archive digital copies of slides, review slides as teams using virtual microscopy software, and standardize analytical approaches. The cost and hardware and software inflexibility of dedicated slide scanning devices can, however, complicate implementation. Here, we describe a simple method that allows any microscope to be used for slide scanning. The only requirements are that the microscope be equipped with a motorized filter turret or wheels (for multi-channel fluorescence) and a motorized xyz stage. This example uses MetaMorph software, but the same principles can be used with any microscope control software that includes a few standard functions and allows programming of simple command routines, or journals. The series of journals that implement the method perform key functions, including assistance in defining an unlimited number of regions of interest (ROI) and imaging parameters. Fully automated acquisition is rapid, taking less than 3 hr to image fifty 2.5-mm ROIs in four channels. Following acquisition, images can be easily stitched and displayed using open-source or commercial image-processing and virtual microscope applications.

Mechanisms underlying distinct subcellular localization and regulation of epithelial long myosin light-chain kinase splice variants

“…Sections were deparaffinized and rehydrated and underwent antigen retrieval using Trilogy (Sigma-Aldrich) solution and a pressure cooker. After cooling, sections were incubated in bleaching buffer under a bright, broad spectrum light for 1 h at room temperature ( 49 ). After replenishing with fresh bleaching buffer, incubation under light was continued for an additional 1 h. After incubation with blocking buffer (Agilent), antigen affinity-purified rabbit anti-long MLCK (AB_2921292, 10 μg/ml), antigen affinity-purified rat anti-MLCK1 (AB_2909525, 20 μg/ml), monoclonal rat anti-ZO-1 (AB_2783858, 1 μg/ml), or monoclonal mouse anti-γ-actin (Santa Cruz, AB_2890619, 0.4 μg/ml) prepared in antibody diluent (Agilent).…”

“…Tiled tissue microarray images (widefield) were collected using a DM4000 widefield microscope with 20X NA 0.7 HC PLAN APO objective (Leica), ORCA-Flash 4.0 LT+ CMOS camera (Hamamatsu), motorized emission filter wheel and xyz stage (Ludl), 5-channel Aura light engine (Lumencor), and multichannel dichroic matched to single band excitation filters (Semrock), as described ( 49 ).…”

Distinct steady-state properties and TNF responses of epithelial long myosin light chain kinase (MLCK) splice variants

“…Sections were deparaffinized and rehydrated and underwent antigen retrieval using Trilogy (Sigma-Aldrich) solution and a pressure cooker. After cooling, sections were incubated in bleaching buffer under a bright LED (broad spectrum) light for 1 hour at room temperature (44). After replenishing with fresh bleaching buffer, incubation under light was continued for an additional 1 hour.…”

“…Tiled tissue microarray images were collected using a DM4000 widefield microscope with 20X NA 0.7 HC PLAN APO objective (Leica), ORCA-Flash 4.0 LT+ CMOS camera (Hamamatsu), motorized emission filter wheel and xyz stage (Ludl), 5 channel Aura light engine (Lumencor), and multichannel dichroic matched to single band excitation filters (Semrock), controlled by MetaMorph 7, as described (44).…”