Scattering of exciting light by live cells in fluorescence confocal imaging: phototoxic effects and relevance for FRAP studies

Abstract

As exciting light in a scanning confocal microscope encounters a cell and its subcellular components, it is refracted and scattered. A question arises as to what proportion of the exciting light is scattered by subcellular structures and whether cells in the vicinity of the imaged area, i.e., cells that are not directly illuminated by the laser beam, can be affected by either an exposure to scattered light and ensuing phototoxic reactions, or by the products of photoactivated reactions diffusing out of the directly illuminated area. We have designed a technique, which allows us to detect subtle cell photodamage and estimate the extent and range of phototoxic effects inflicted by interaction between scattered exciting light and fluorescent probes in the vicinity of the illuminated area. The technique is based on detecting an increased influx of acridine orange into photodamaged cells, which is manifested by a change of color. We demonstrate that phototoxic effects can be exerted not only on the illuminated cell, but also on fluorescently labeled neighboring cells. The damage inflicted on neighbors is due to exposure to light scattered by the imaged (i.e., directly illuminated) cell, but not phototoxic products diffusing out of the directly illuminated area. When light encounters a cell nucleus, scattering is so intense that photodamage can be inflicted even on fluorescently labeled cells located within a radius of approximately 90 microm, i.e., several cell diameters away. This range of scattering is comparable with that caused by the glass bead resting on a coverslip (up to 120 microm). The intense scattering of exciting light imposes limits on FRAP, FLIP, and other techniques employing high intensity laser beams.

FIGURE 1

(I) Efficiency of drug efflux can be estimated by the color of cytoplasmic AO. In all panels extracellular concentration of AO was 8 μg/ml, i.e., a concentration when ds and ss nucleic acids are stained differentially, but the access of AO to cell interior was different, as described: (A) image of AO luminescence in a cell fixed with formaldehyde. Because internal and plasma membranes are fully permeable, the intracellular and extracellular concentrations of AO are equal. Single stranded RNA in cytoplasm and nucleoli stain red, double stranded DNA in nucleus stains green. Bar, 20 μm. (B) Image of AO fluorescence in the cytoplasm of live, intact cells. Cells pump out AO, thus, intracellular concentration of AO is lower than extracellular and AO stacks are not formed. The cytoplasm as well as nucleus (N) and nucleoli (NC) emit green fluorescence. Acidic endosomes (ES) accumulate AO and the high intraendosomal concentration of AO promotes formation of stacks, resulting in red luminescence. (C) Image of AO luminescence following mechanical damage to plasma membranes. The cell monolayer was scratched with a needle resulting in mechanical damage to some cells (arrowheads) located at the edge of the wound (arrow). This damage caused a rapid entry of AO into cells and an increase of intracellular AO concentration to the level of the surrounding medium. As a result, the cytoplasm emits red luminescence in damaged cells (arrowheads). Intact cells (at a distance from the wound) maintain the AO gradient and still emit only green fluorescence. (D) Image of AO in cells treated with verapamil, a drug, which inhibits drug efflux. The intracellular concentration of AO increases following exposure to verapamil and cytoplasm assumes orange color, derived from the presence of both, AO stacks (red) and AO monomers (green). The presence of stacks indicates that the intracellular concentration of AO has increased above the level of ∼5 μg/ml. (E) Image of AO in cells deprived of energy by maintaining in culture without medium replenishment for 4 days. Drug efflux is less efficient and intracellular concentration of AO rises. The increasing intracellular concentration of AO leads to formation of stacks and a relative increase of red luminescence. (F) Image of AO in a cell (arrow) after exposure to a high intensity exciting light (458 nm, 50 μW beam, pixel 30 nm, 0.3 scan/s, 10 scans). Interaction of light with AO (and presumably subsequent reactions with oxygen) leads to various phototoxic effects. These effects are manifested by the inhibition of drug efflux and increased permeability of plasma membranes. As a consequence, the intracellular concentration of AO increases and stacks are formed on RNA. AO in cytoplasm emits not only green fluorescence, as in a control, but red luminescence as well. Endosomes are damaged and lose the accumulated AO. Neighboring cells also suffer subtle damage, as demonstrated by red emission of cytoplasm; this damage is less severe as shown by the presence of endosomes that are still capable of maintaining accumulated AO. (II) Subtle photodamage is manifested by less efficient drug efflux and can be assessed on the basis of the relative increase of the intracellular concentration of AO; PI exclusion can only detect heavy photodamage. The images describe photodamage in fluorescently labeled cells illuminated by exciting light of various intensities. Top rows, AO luminescence; bottom rows, fluorescence of propidium. (A) Control, unilluminated cells with efficient drug efflux. AO in cytoplasm and nucleus emits green fluorescence (except for acidic endosomes); red luminescence of AO bound to RNA is undetectable. Propidium is excluded from cells. A slight bleed-through of the intense red luminescence of AO from endosomes is detected in the red channel dedicated to propidium. Bar, 20 μm. (B and C) AO in cells exposed to excitation light of a low intensity (B) All cells in the field of view were exposed to low light levels and suffered subtle photodamage; (C) the damaged cell is marked with an arrow. Green AO luminescence and a detectable red component indicates that drug efflux is less efficient than in cells not subjected to illumination. The lack of intracellular fluorescence of PI indicates that the integrity of plasma membrane is not compromised. (D–F) AO and PI luminescence in cells exposed to high doses of exciting light (arrow). Cell damage is manifested by red luminescence. Integrity of plasma membrane is lost and the cells are unable to exclude propidium. (Bottom) A graph showing the parameter γ as a function of the doses of exciting light used in B–F above. Higher light doses cause more pronounced damage to drug efflux mechanisms (and presumably the structure of plasma membrane) and the damage is reflected in a higher concentration of cytoplasmic AO. In the most heavily damaged cells plasma membrane integrity is lost and cells no longer exclude propidium (the gray area in the graph). The test with AO can detect subtle damage, whereas PI exclusion detects only heavy, lethal damage manifested by a complete loss of plasma membrane integrity. (III) Sublethal damage can be inflicted on neighbors of the illuminated cell due to interaction between the scattered light and fluorescent labels residing in these cells. The damage is manifested by impairment of drug efflux. (Left column) Schematics of the illuminated sample, (center left) a graph showing values of the parameter γ as a function of the distance from the exciting beam (blue bars, control; pink bars, cells affected by scattered light); (center right and far right) images before and after exposure to exciting light (458 nm, 50 μW laser beam, 10 scans, 0.3 scan/s). The illuminated area (17 × 17 μm) is marked with an arrow. (A) Exciting light is incident on a glass outside of cells; neighboring cells are intact. Bar, 20 μm. (B) Exciting light is incident on cytoplasm of two adjacent cells in an area with few acidic endosomes. Only a small area of the cytoplasm shows signs of damage; neighbors remain intact. (C) Exciting light is incident on nucleus; neighbors within 70–90 μm are damaged. (D) Exciting light is incident on a glass bead (diameter 100 μm) resting on a coverslip. The point of contact of the bead with the coverslip is marked with an arrow. Neighbors within a radius of 120 μm (beyond the field of view shown) are damaged.

FIGURE 2

Photodamage does not spread throughout the AO stained cell (see also Supplementary Material, section 2). Low intensity exciting light was incident on a selected small area (5 × 5 μm) of cytoplasm (arrow points at the region that was illuminated and acquired red luminescence). After 2 min still only a small area of the cytoplasm shows signs of damage. Red staining expanded by no more than 2 μm beyond the originally illuminated region. This may be a result of light scatter and limited range of diffusion of toxic products of reactions activated by exciting light and AO. Bar, 10 μm.

FIGURE 3

Phototoxic effects of eGFP bound to histone H2B in cell nucleus are not detectable. Exciting blue light (488 nm, 40 μW laser beam reaching the specimen, 10 scans, area 10 × 10 μm, 512 × 512 pixels) was incident on a rectangular area within the cell nucleus (the bleached region). Following illumination, the cells were submerged in medium containing AO to detect cell damage. No increased drug influx has been detected, demonstrating that eGFP did not cause detectable impairment of drug efflux or breach of plasma membrane integrity. Bar, 20 μm.