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Updated: Jan 10, 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, Bart Krist2,3, Alok Dhawan4
1Sana HANSE-Klinikum Wismar, Clinic for Psychiatry, Wismar, Germany.
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 in somatic cells for its 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 signaling 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 have evolved DNA-damage response (DDR) mechanisms to repair genetic damage. This study presents a high-resolution microscopy method to quantitatively assess DDR events in preserved nuclei, aiding health and disease risk assessment.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- DNA damage from internal and external sources threatens genetic integrity in somatic and germ cells.
- The DNA-damage response (DDR) is a crucial cellular mechanism for sensing, signaling, and repairing DNA lesions.
- Dysfunctional DDR signaling is linked to various human diseases, including cancer, making DDR assessment a vital biomarker.
Purpose of the Study:
- To develop and describe experimental protocols for quantifying key DNA-damage response (DDR) events.
- To maintain spatial information within nuclei during quantitative assessment of DDR.
- To demonstrate the application of the method in both somatic and germ cells.
Main Methods:
- Utilizing laser scanning confocal immunofluorescence microscopy (LSCIM) on three-dimensionally preserved nuclei.
- Simultaneously detecting key DDR proteins: Rad50, phosphorylated H2AX, and ATM.
- Applying quantitative LSCIM for high-resolution analysis of DDR in intact nuclear architecture.
Main Results:
- Established protocols for high-resolution, quantitative assessment of individual DDR events.
- Demonstrated the ability of LSCIM to preserve spatial information within nuclei.
- Successfully applied the method to analyze DDR in both somatic and germ cells.
Conclusions:
- LSCIM provides a quantitative method to assess DDR events while preserving nuclear architecture and spatial information.
- This technique serves as a valuable tool for biomonitoring and assessing human health and disease risk.
- The described protocols enable detailed analysis of DDR in various cell types.
