Real-Time Quantification of Cellular Photodamage via In Situ Raman Imaging by the Cytochrome c Marker Band.

In situ Raman imaging of living cells is a pivotal technique for probing cellular activities since it can faithfully demonstrate the bioevents without changing the intracellular microenvironment. However, the laser exposure may lead to irreversible damage to cells during long-term optical imaging. Here, we developed a sensitive method to quantify and limit the photodamage during long-term in situ live-cell imaging by using the Raman band of cytochrome c (cyt c). This approach enables the detection of subtle chemical composition changes in the early stages of photodamage with over 5 times higher sensitivity than that of conventional morphological evaluation and mitochondrial fluorescence staining. By calibrating the cyt c Raman response against delivered light dosage across cells, we derive a dose-damage relationship with a well-defined linear regime, enabling real-time quantification of photodamage during acquisition. Under the experimental conditions tested in this work, the model suggests that the cell can maintain good viability when cyt c fluctuations remain within a conservative tolerance (∼15-30%). Using this dosimetry as a decision rule, we implement practical mitigation strategies, i.e., interval time-lapse imaging to facilitate cellular self-recovery and prevent irreversible damage. With this method, we demonstrated that the lipid change during cell starvation can be faithfully recorded for up to 8 h. This work provides an efficient method to maintain cell activity during long-term in situ Raman imaging, which was difficult to achieve previously.