Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena

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Abstract

Quantitative colocalization analysis is an advanced digital imaging tool to examine antigens of interest in immunofluorescence images obtained using confocal microscopes. It employs specialized algorithms to estimate the degree of overlap of fluorescence signals and thus enables acquiring important new information not otherwise obtainable using qualitative approaches alone. As raw confocal images have high levels of background, they should be prepared to become suitable for reliable calculation of colocalization coefficients by correcting it. We provide concise theoretical basis of quantitative colocalization analysis, discuss its limitations, and describe proper use of the technique. The use of quantitative colocalization analysis is demonstrated by studying bile salt export pump and multidrug resistance associated protein 2 in the liver and major basic protein and platelet activating factor receptor antigens in conjunctiva. The review is focused on the applicability and correct interpretation of the results of colocalization coefficients calculations.

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Phospholipids undergo hop diffusion in compartmentalized cell membrane

Takahiro Fujiwara, Ken Ritchie, Hideji Murakoshi … (2002)

The diffusion rate of lipids in the cell membrane is reduced by a factor of 5–100 from that in artificial bilayers. This slowing mechanism has puzzled cell biologists for the last 25 yr. Here we address this issue by studying the movement of unsaturated phospholipids in rat kidney fibroblasts at the single molecule level at the temporal resolution of 25 μs. The cell membrane was found to be compartmentalized: phospholipids are confined within 230-nm-diameter (φ) compartments for 11 ms on average before hopping to adjacent compartments. These 230-nm compartments exist within greater 750-nm-φ compartments where these phospholipids are confined for 0.33 s on average. The diffusion rate within 230-nm compartments is 5.4 μm2/s, which is nearly as fast as that in large unilamellar vesicles, indicating that the diffusion in the cell membrane is reduced not because diffusion per se is slow, but because the cell membrane is compartmentalized with regard to lateral diffusion of phospholipids. Such compartmentalization depends on the actin-based membrane skeleton, but not on the extracellular matrix, extracellular domains of membrane proteins, or cholesterol-enriched rafts. We propose that various transmembrane proteins anchored to the actin-based membrane skeleton meshwork act as rows of pickets that temporarily confine phospholipids.

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Imaging biochemistry inside cells.

Fred Wouters, Peter Verveer, Philippe I.H. Bastiaens (2001)

Proteins provide the building blocks for multicomponent molecular units, or pathways, from which higher cellular functions emerge. These units consist of either assemblies of physically interacting proteins or dispersed biochemical activities connected by rapidly diffusing second messengers, metabolic intermediates, ions or other proteins. It will probably remain within the realm of genetics to identify the ensemble of proteins that constitute these functional units and to establish the first-order connectivity. The dynamics of interactions within these protein machines can be assessed in living cells by the application of fluorescence spectroscopy on a microscopic level, using fluorescent proteins that are introduced within these functional units. Fluorescence is sensitive, specific and non-invasive, and the spectroscopic properties of a fluorescent probe can be analysed to obtain information on its molecular environment. The development and use of sensors based on the genetically encoded variants of green-fluorescent proteins has facilitated the observation of 'live' biochemistry on a microscopic level, with the advantage of preserving the cellular context of biochemical connectivity, compartmentalization and spatial organization. Protein activities and interactions can be imaged and localized within a single cell, allowing correlation with phenomena such as the cell cycle, migration and morphogenesis.

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Seeing is believing? A beginners' guide to practical pitfalls in image acquisition

Alison North (2006)

Imaging can be thought of as the most direct of experiments. You see something; you report what you see. If only things were truly this simple. Modern imaging technology has brought about a revolution in the kinds of questions we can approach, but this comes at the price of increasingly complex equipment. Moreover, in an attempt to market competing systems, the microscopes have often been inappropriately described as easy to use and suitable for near-beginners. Insufficient understanding of the experimental manipulations and equipment set-up leads to the introduction of errors during image acquisition. In this feature, I review some of the most common practical pitfalls faced by researchers during image acquisition, and how they can affect the interpretation of the experimental data. This article is targeted neither to the microscopy gurus who push forward the frontiers of imaging technology nor to my imaging specialist colleagues who may wince at the overly simplistic comments and lack of detail. Instead, this is for beginners who gulp with alarm when they hear the word “confocal pinhole” or sigh as they watch their cells fade and die in front of their very eyes time and time again at the microscope. Take heart, beginners, if microscopes were actually so simple then many people (including myself) would suddenly be out of a job!

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Author and article information

Journal

Journal ID (nlm-ta): Acta Histochem Cytochem

Journal ID (publisher-id): AHC

Title: Acta Histochemica et Cytochemica

Publisher: Japan Society of Histochemistry and Cytochemistry (Tokyo, Japan )

ISSN (Print): 0044-5991

ISSN (Electronic): 1347-5800

Publication date (Print): 30 August 2007

Publication date (Electronic): 2 August 2007

Volume: 40

Issue: 4

Pages: 101-111

Affiliations

[1 ]Department of Anatomy and Cell Biology, Kochi University Faculty of Medicine

[2 ]Department of Ophthalmology, Kochi University Faculty of Medicine, Okoh-cho, Nankoku, Kochi 783–8505, Japan

Author notes

Correspondence to: Vadim Zinchuk, M.D., Ph.D., Department of Anatomy and Cell Biology, Kochi University Faculty of Medicine, Kohasu, Okoh-cho, Nankoku, Kochi 783–8505, Japan. E-mail: zinchuk@ 123456s.kochi-u.ac.jp

Article

Publisher ID: AHC07002

DOI: 10.1267/ahc.07002

PMC ID: 1993886

PubMed ID: 17898874

SO-VID: c78426a9-df16-4bed-afbe-36f5fd52bedf

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena

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Journal of Circulating Biomarkers

Most cited references 37

Phospholipids undergo hop diffusion in compartmentalized cell membrane

Imaging biochemistry inside cells.

Seeing is believing? A beginners' guide to practical pitfalls in image acquisition

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