Journal Article

Multiplexed Spectral Imaging of 120 Different Fluorescent Labels

Valm, Alex M., Oldenbourg, Rudolf, Borisy, Gary G.

2016

Overview

The number of fluorescent labels that can unambiguously be distinguished in a single image when acquired through band pass filters is severely limited by the spectral overlap of available fluorophores. The recent development of spectral microscopy and the application of linear unmixing algorithms to spectrally recorded image data have allowed simultaneous imaging of fluorophores with highly overlapping spectra. However, the number of distinguishable fluorophores is still limited by the unavoidable decrease in signal to noise ratio when fluorescence signals are fractionated over multiple wavelength bins. Here we present a spectral image analysis algorithm to greatly expand the number of distinguishable objects labeled with binary combinations of fluorophores. Our algorithm utilizes a priori knowledge about labeled specimens and imposes a binary label constraint on the unmixing solution. We have applied our labeling and analysis strategy to identify microbes labeled by fluorescence in situ hybridization and here demonstrate the ability to distinguish 120 differently labeled microbes in a single image.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Algorithms / Analysis / Bandpass filters / Biofilms / Chemical compounds / Data processing / Engineering and Technology / Fluorescence / Fluorescence in situ hybridization / Fluorescent Dyes - chemistry / Fluorescent proteins / Fluorophores / Identification and classification / Image acquisition / Image analysis / Image processing / In Situ Hybridization, Fluorescence / Labeling / Labels / Laboratories / Medical imaging / Methods / Microorganisms / Microscopy / Microscopy, Fluorescence - methods / Optical Imaging / Physical Sciences / Physiological aspects / Proteins / Research and Analysis Methods / Signal to noise ratio / Spectra / Spectral analysis (Signal analysis)