Chromophore-assisted laser inactivation in cell biology

Abstract

Chromophore-assisted laser inactivation (CALI) is a technique whereby engineered proteins and dye molecules that produce substantial amounts of reactive oxygen species upon absorption of light are used to perturb biological systems in a spatially and temporally defined manner. CALI is an important complement to conventional genetic and pharmacological manipulations. In this review, we examine the applications of CALI to cell biology and discuss the underlying photochemical mechanisms that mediate this powerful technique.

Figure 1

CALI of EGFP-CP induces the formation of dorsal ruffles and filopodia. (a) Fluorescence (left) and differential interference contrast (DIC) (right) images of Rat2 fibroblasts expressing EGFP-CP before CALI; the irradiated area is indicated with a black circle (diameter: 24 μm). (b) Fluorescence immediately after CALI (left) and DIC (middle) and dorsal surface F-actin (right) (stained by Alexa Fluor 568-labeled phalloidin) 3 min after CALI. Numerous actin-rich dorsal ruffles and filopodia are locally formed after irradiation.

Figure 2

Potential photodamage pathways in CALI.

Figure 3

ROS production in terms of simplified energy-level diagram. Excitation (denoted by absorption of a photon, hυ_a) occurs to one of several higher vibronic states followed by rapid internal conversion (compact squiggly arrow) resulting in the molecule dropping to the lowest excited singlet state (S₁) in which it exists for several nanoseconds. From this state, several fates are possible: the excited chromophore can emit (denoted by emission of a photon, hυ_f), transfer its energy to the solvent without emission (extended squiggly arrow), stimulate electron transfer from ³O₂ (depicted as the broken line from S₁ to ³O₂) to produce superoxide anion (O₂^•−) or undergo intersystem crossing (isc) to the triplet manifold. (Note, to simplify the diagram, generation of ¹O₂ from the chromophore single state is not depicted.) The triplet state is long lived and, for this reason, much photochemistry proceeds from it, including ROS production; emission from the triplet state is called phosphorescence (denoted by emission of a photon, hυ_p).