Towards many colors in FISH on 3D-preserved interphase nuclei

Area of Science:

• Molecular Biology
• Cytogenetics
• Microscopy

Background:

• Multicolor Fluorescence In Situ Hybridization (FISH) is crucial for analyzing nuclear targets, particularly interphase chromosomes.
• Effective FISH relies on integrating probe labeling strategies with advanced image acquisition techniques.

Purpose of the Study:

• To review and compare existing methods for multicolor FISH on nuclear targets.
• To provide practical guidance for selecting optimal FISH and microscopy combinations for experimental design.

Main Methods:

• Discussion of M-FISH (combinatorial labeling, deconvolution, wide-field microscopy).
• Comparison of multicolor labeling with Structured Illumination Microscopy (SIM).
• Analysis of standard multicolor FISH with Confocal Laser Scanning Microscopy (CLSM), including variations with combinatorial labeling and linear unmixing.
• Consideration of image deconvolution and chromatic Z-shift correction for CLSM data.

Main Results:

• Detailed overview of five distinct multicolor FISH and microscopy approaches.
• Practical examples illustrating the application of each method.
• Identification of key factors influencing the choice of FISH and microscopy techniques.

Conclusions:

• The choice of multicolor FISH and microscopy method depends on specific experimental needs and target characteristics.
• Understanding the interplay between labeling strategies and imaging technologies is essential for successful nuclear target analysis.
• This review provides a framework for optimizing FISH experiments through informed selection of techniques.