Two‐Photon Singlet Oxygen Microscopy: The Challenges of Working with Single Cells

Abstract

A microscope is described in which singlet molecular oxygen, O2(a1deltag), is produced in a femtoliter focal volume via a nonlinear two-photon photosensitized process, and the 1270 nm phosphorescence from this population of O2(a1deltag) is detected in a photon counting experiment. Although two-photon excitation of a sensitizer is less efficient than excitation by a one-photon process, nonlinear excitation has several distinct advantages with respect to the spatial resolution accessible. Pertinent aspects of this two-photon O2(a1deltag) microscope were characterized using bulk solutions of photosensitizers. These data were compared to those obtained from a single biological cell upon linear one-photon excitation of a sensitizer incorporated in the cell. On the basis of the results obtained, we outline the challenges of using nonlinear optical techniques to create O2(aldeltag) at the single cell level and to then optically detect the O2(aldeltag) thus produced in a time-resolved experiment.

Solvent dependent photosensitized singlet oxygen production from an Ir(iii) complex: pointing to problems in studies of singlet-oxygen-mediated cell death

“…29,30 For the two-photon initiated cell kill experiments, the 800 nm (spectral fwhm ~ 12 nm) output of a fs laser operating at 80 MHz (Mai Tai from Spectra Physics) was coupled onto a microscope objective (60× Olympus LUMPlanFI/IR, water immersion, NA 0.9) in an inverted microscope (Olympus IX71). 11,12,25,31 The optics used result in a beam diameter at the focused waist of ~ 1 µm. 31 However, given that the probability of the two-photon transition depends on the square of the incident light intensity, only the light in a smaller spatial domain at the center of the focus results in the creation of excited states.…”

“…31 However, given that the probability of the two-photon transition depends on the square of the incident light intensity, only the light in a smaller spatial domain at the center of the focus results in the creation of excited states. 31 For all experiments, the average laser power Ltd. and used as received. De-oxygenated solutions were obtained by gentle bubbling with argon for 20 min prior to a given measurement.…”

Singlet Oxygen in a Cell: Spatially Dependent Lifetimes and Quenching Rate Constants

“…[12][13][14][15][16] A key aspect of our work is that, using a focused laser beam, sensitizer excitation can be confined to small sub-cellular spatial domains. 15,17,18 Using this approach, it is now possible to provide unique insight into singlet-oxygen-mediated processes that occur in a cell.…”

“…Although we can impart spatial resolution to our experiments by irradiating the sensitizer in the cell with a focused laser, our present beam waist (diameter ~ 1 µm) 15,18 is still large relative to the structures that define intracellular heterogeneity. As such, we chose to work 6 with both hydrophobic (chlorin, Chl) and hydrophilic (TMPyP) sensitizers (Figure 1), expecting that these molecules would localize in different domains of the cell.…”

Distribution and binding of novel photosensitizer 2-devinyl-2-(1-methoxyl-ethyl) chlorin f in human breast cancer cells MCF-7

“…Therefore, the three factors above has themselves incoordinate impact on the curative effect of PDT. The developments of modern optical technique establishes not only rock-firm technical elements for tumor PDT and non-invasive tumor diagnosis [15], but also provides potent research approaches for exploring their microcosmic mechanism and the key influencing the curative effect of PDT [11]. In the recent decade, a number of second generation photosensitizers especially related to chlorin derivatives have been reported in the literature [14,16].…”