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Updated: May 9, 2026
Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage
Published on: January 31, 2018
Multicolor laser scanning confocal immunofluorescence microscopy of DNA damage response biomarkers
Julian Laubenthal1, Michal R Gdula, Alok Dhawan
1Medical Sciences Division, School of Life Sciences, University of Bradford, Bradford, UK.
Abstract:
DNA damage through endogenous and environmental toxicants is a constant threat to both a human's ability to pass on intact genetic information to its offspring as well as somatic cells for their own survival. To counter these threats posed by DNA damage, cells have evolved a series of highly choreographed mechanisms--collectively defined as the DNA damage response (DDR)--to sense DNA lesions, signal their presence, and mediate their repair. Thus, regular DDR signalling cascades are vital to prevent the initiation and progression of many human diseases including cancer. Consequently, quantitative assessment of DNA damage and response became an important biomarker for assessment of human health and disease risk in biomonitoring studies. However, most quantitative DNA damage biomarker techniques require dissolution of the nuclear architecture and hence loss of spatial information. Laser scanning confocal immunofluorescence microscopy (LSCIM) of three-dimensionally preserved nuclei can be quantitative and maintain the spatial information. Here we describe the experimental protocols to quantify individual key events of the DDR cascade in three-dimensionally preserved nuclei by LSCIM with high resolution, using the simultaneous detection of Rad50 as well as phosphorylated H2AX and ATM and in somatic and germ cells as an example.
Insights
Cells possess DNA damage response (DDR) mechanisms to repair genetic damage from toxins. This study presents a microscopy method to quantitatively assess DDR events in preserved cell nuclei, aiding biomonitoring.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- DNA damage from endogenous and environmental factors threatens genetic integrity and cell survival.
- The DNA damage response (DDR) is a crucial cellular mechanism for sensing, signaling, and repairing DNA lesions.
- Dysfunctional DDR signaling is implicated in human diseases, including cancer.
Purpose of the Study:
- To develop and describe protocols for the quantitative assessment of key DNA damage response (DDR) events.
- To maintain spatial information within nuclei during quantitative analysis of DDR.
- To utilize Laser Scanning Confocal Immunofluorescence Microscopy (LSCIM) for high-resolution DDR event quantification.
Main Methods:
- Utilized Laser Scanning Confocal Immunofluorescence Microscopy (LSCIM) on three-dimensionally preserved nuclei.
- Quantified individual DDR cascade events, including Rad50, phosphorylated H2AX, and ATM.
- Applied the method to both somatic and germ cells to assess DDR in different cell types.
Main Results:
- Demonstrated the ability of LSCIM to quantitatively assess DDR events in preserved nuclei.
- Successfully maintained spatial information, allowing for detailed analysis of DDR localization.
- Showcased the simultaneous detection of multiple DDR proteins (Rad50, p-H2AX, ATM) as a viable approach.
Conclusions:
- LSCIM provides a high-resolution, quantitative method for assessing DDR events while preserving nuclear architecture.
- This technique offers a valuable biomarker for human health and disease risk assessment in biomonitoring.
- The described protocols enable detailed spatial analysis of DDR in various cell types.
