Multichannel Fluorescence Imaging Research Articles

Event Ordering in Live-Cell Imaging Determined from Temporal Cross-Correlation Asymmetry

We use the temporal asymmetry of the cross-correlation function to determine the temporal ordering of spatially localized cellular events in live-cell multichannel fluorescence imaging. The analysis is well suited to noisy, stochastic systems where the temporal order may not be apparent in the raw data. The approach is applicable to any biochemical reaction not in chemical equilibrium, including protein complex assembly, sequential enzymatic processes, gene regulation, and other cellular signaling events. As an automated quantitative measure, this approach allows the data to be readily interpreted statistically with minimal subjective biases. We first test the technique using simulations of simple biophysical models with a definite temporal ordering. We then demonstrate the approach by extracting the temporal ordering of three proteins—actin, sorting nexin 9, and clathrin—in the endocytic pathway.

Color Compensation of Multicolor FISH Images

Multicolor fluorescence in situ hybridization (M-FISH) techniques provide color karyotyping that allows simultaneous analysis of numerical and structural abnormalities of whole human chromosomes. Chromosomes are stained combinatorially in M-FISH. By analyzing the intensity combinations of each pixel, all chromosome pixels in an image are classified. Due to the overlap of excitation and emission spectra and the broad sensitivity of image sensors, the obtained images contain crosstalk between the color channels. The crosstalk complicates both visual and automatic image analysis and may eventually affect the classification accuracy in M-FISH. The removal of crosstalk is possible by finding the color compensation matrix, which quantifies the color spillover between channels. However, there exists no simple method of finding the color compensation matrix from multichannel fluorescence images whose specimens are combinatorially hybridized. In this paper, we present a method of calculating the color compensation matrix for multichannel fluorescence images whose specimens are combinatorially stained.

Activation of caspase-3 noninvolved in the bystander effect of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system

Use of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system is one of the promising approaches in the rapidly growing area of gene therapy. The "bystander effect," a phenomenon in which HSV-tk+ cells exposed to GCV are toxic to adjacent HSV-tk- cells, was reported to play an important role in suicide gene therapy. However, the mechanism by which HSV-tk/GCV induces the bystander effect is poorly understood. We monitored the activation of caspase-3 in living cells induced by the HSV-tk/GCV system using a genetically encoded fluorescence resonance energy transfer (FRET) probe CD3, , a caspase-3 recognition site fused with a cyan fluorescent protien (CFP) and a red fluorescent protein (DsRed) which we reported and named in a previous paper. Fluorescence protein (FP)-based multicolor cellular labeling, combined with the multichannel fluorescence imaging and FRET imaging techniques, provides a novel and improved approach to directly determine whether the activation of caspase-3 involved in the HSV-tk/GCV system induces cell apoptosis in tk gene-expressing cells and their neighboring cells. FRET ratio images of CD3, and fluorescence images of the fusion protein of thymidine kinase linked with green fluorescent protein (TK-GFP), indicated that HSV-tk/GCV system-induced apoptosis in human adenoid cystic carcinoma (ACC-M) cells was via a caspase-3 pathway, and the activation of caspase-3 was not involved in the bystander effect of HSV-tk/GCV system.

Automatic channel unmixing for high-throughput quantitative analysis of fluorescence images

Laser-scanning microscopy allows rapid acquisition of multi-channel data, paving the way for high-throughput, high-content analysis of large numbers of images. An inherent problem of using multiple fluorescent dyes is overlapping emission spectra, which results in channel cross-talk and reduces the ability to extract quantitative measurements. Traditional unmixing methods rely on measuring channel cross-talk and using fixed acquisition parameters, but these requirements are not suited to high-throughput processing. Here we present a simple automatic method to correct for channel cross-talk in multi-channel images using image data only. The method is independent of the acquisition parameters but requires some spatial separation between different dyes in the image. We evaluate the method by comparing the cross-talk levels it estimates to those measured directly from a standard fluorescent slide. The method is then applied to a high-throughput analysis pipeline that measures nuclear volumes and relative expression of gene products from three-dimensional, multi-channel fluorescence images of whole Drosophila embryos. Analysis of images before unmixing revealed an aberrant spatial correlation between measured nuclear volumes and the gene expression pattern in the shorter wavelength channel. Applying the unmixing algorithm before performing these analyses removed this correlation.

Immunocytochemistry and quantification of protein colocalization in cultured neurons

This protocol details a method to quantify the distribution of protein and colocalization in neuronal cultures. To that end, this protocol includes itemized steps and considerations for performing immunocytochemistry, acquiring fluorescence images and quantifying multichannel fluorescence images. Success in quantifying immunostained neurons relies on the accessibility of the proteins of interest, the sensitivity and specificity of antibodies, the signal-to-noise ratio of the collected image and the sensitivity of the quantification method. In contrast to other commonly employed methods for quantification, the protocol detailed here requires manual selection of punctae and subtraction of background selected for each neurite. This approach reliably and uniquely allows for detection of proteins in low signal-to-noise ratio images, which are characteristic of developing neurons. Thus, this method serves an important niche in image analysis poorly addressed by alternative published methods. In general, immunocytochemistry requires 3.5-7 h, and one triple-immunostained neuron can be quantified in 1.5 h.

Cellular screening assays using fluorescence microscopy

The recent development of automated fluorescence imaging systems has enabled fluorescence microscopy to be used for the purposes of compound screening. This information-rich technique has found various applications, including screening for the effect of kinase inhibitors on the cytoskeleton, agonist-stimulated receptor internalization, and protein phosphorylation and acetylation. The discovery of novel fluorescent proteins and new fluorescent dyes will find many applications in multichannel fluorescence imaging assays.

Use of confocal microscopy in examining fungi and bacteria in wood

When used in conjunction with digital image processing techniques, confocal laser scanning microscopy (CLSM) enables non‐invasive optical sectioning, allowing micromorphologies of wood decay to be examined at any depth within a relatively thick (0.05–0.1 mm) wood specimen without incision. In this study, the use of specially tailored multi‐fluorescent staining techniques with CLSM produced new information concerning spatial relationships between fungi and bacteria and the wood substrate, particularly in regard to their 3D characteristics. Glutaraldehyde fixation and a chitin fluorescent probe were used to locate fungal hyphae in wood. Bacteria colonising wood were examined using a fluorescent phospholipid probe. By counterstaining wood with this probe and a fluorescent dye specific for Gram‐positive bacteria, it was possible to clearly distinguish Gram types through simultaneous, multichannel fluorescent CLSM imaging. The combination of glutaraldehyde fixation and phospholipid probing proved to be reliable for detecting wood‐degrading bacteria in wood cell walls.