Abstract
Fluorescence microscopy has become an essential tool in cell biology. This technique allows researchers to visualize the dynamics of tissue, cells, individual organelles, and macromolecular assemblies inside the cell. Unfortunately, fluorescence microscopy is not completely "noninvasive" as the high-intensity excitation light required for excitation of fluorophores is inherently toxic for live cells. Physiological changes induced by excessive illumination can lead to artifacts and abnormal responses. In this chapter, we review major factors that contribute to phototoxicity and discuss practical solutions for circumventing photodamage. These solutions include the proper choice of image acquisition parameters, optimization of filter sets, hardware synchronization, and the use of intelligent illumination to avoid unnecessary light exposure.
🔬 Techniques
🧬 Organisms
✨ Fluorophores
🧪 Sample Preparation
🔬 Cell Lines
🏭 Microscope Brands
📷 Detectors
🏛️ Research Organizations (ROR)
Affiliated research institutions:
📊 Figures
Figure 1
Photobleaching and photodamage effects in GFP-microscopy
(A) 3-D fluorescence time-lapse (30-s intervals) recorded on a standard research-grade imaging workstation. Fluorescence intensity gradually decreases (photobleaching) under these conditions. Concurre...
Figure 2
Dependence of photodamage on the spectrum of excitation
(A) Various states of fluorophore excitation. (1) ground-state fluorophore; (2) singlet-state excited molecule; (3) non-fluorescent triplet state; (4) higher-energy chemically reactive states. (B) Spe...
Figure 3
Disproportionate increase in photobleaching observed in multi-color recordings
(A) Absorption spectra of GFP (dashed line) and tdTomato (solid line). Arrows and arrowhead mark the efficiency of absorption for the wavelengths commonly used to excite these fluorophores. Notice tha...
Figure 4
Actual exposure times for different types of synchronization and different light sources
In all cases, nominal exposure time is set to 100-ms in the commercial software package that controls the imaging workstation. Actual exposure time is measured by a photo-detector placed near the foca...
Figure 5
Example of an adaptive illumination technique
(A) Selected frames from a 3-D time-lapse of centrosome movements during cell division in human (HeLa) cells. These cells become rounded during mitosis and the centrosomes move extensively in all thre...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
0 commentsNo comments yet. Be the first to start a discussion!
Leave a Comment