Fast repetition rate fs pulsed lasers for advanced PLIM microscopy

Sviatlana Kalinina,Alexander Jelzow,Angelika Rück,Tobias Plötzing

doi:10.1142/s1793545819400042

Abstract

Simultaneous metabolic and oxygen imaging is promising to follow up therapy response, disease development and to determine prognostic factors. FLIM of metabolic coenzymes is now widely accepted to be the most reliable method to determine cellular bioenergetics. Also, oxygen consumption has to be taken into account to understand treatment responses. The phosphorescence lifetime of oxygen sensors is able to indicate local oxygen changes. For phosphorescence lifetime imaging (PLIM) dyes based on ruthenium (II) coordination complexes are useful, in detail TLD1433 which possesses a variety of different triplet states, enables complex photochemistry and redox reactions. PLIM is usually reached by two photon excitation of the drug with a femtosecond (fs) pulsed Ti:Sapphire laser working at 80[Formula: see text]MHz repetition rate and (time-correlated single photon counting) (TCSPC) detection electronics. The interesting question was whether it is possible to follow up PLIM using faster repetition rates. Faster repetition rates could be advantageous for the induction of specific photochemical reactions because of similar light doses used normally in standard CW light treatments. For this, a default 2[Formula: see text]-FLIM–PLIM system was expanded by adding a second fs pulsed laser (“helixx”) which provides 50[Formula: see text]fs pulses at a repetition rate of 250[Formula: see text]MHz, more than 2.3[Formula: see text]W average power and tunable from 720[Formula: see text]nm to 920[Formula: see text]nm. The laser beam was coupled into the AOM instead of the default 80[Formula: see text]MHz laser. We demonstrated successful applications of the 250[Formula: see text]MHz laser for PLIM which correlates well with measurements done by excitation with the conventional 80[Formula: see text]MHz laser source.

Highlights

Luminescence lifetime imaging visualizes the decay of a molecule from therst excited singlet state or triplet state
FLIM of metabolic coenzymes is the basis for optical metabolic imaging (OMI)
Intersystem crossing (ISC) from therst excited singlet state to the triplet state is the prerequisite for a spin forbidden process called phosphorescence

Summary

Introduction

Luminescence lifetime imaging visualizes the decay of a molecule from therst excited singlet state or triplet state. Brie°y, in PDT, a photosensitizer (PS), used at nontoxic concentrations, undergoes ISC to the triplet excited state after excitation by light and subsequently transfers energy to ground state molecular oxygen This leadsnally to the production of reactive oxygen species (ROS), either singlet oxygen (1O2Þ by the so-called Type-II reaction or oxygen radicals (Type-I reaction).[1] direct oxygen-independent electron transfer reactions to biomolecules are feasible (Type-III reaction).[2] As oxygen is critical in PDT, tumor hypoxia is a major problem and could lead to treatment failure. The potential impact of fast repetition rate laser excitation for PDT treatment protocols and the development of novel diagnostic approaches towards personalized theranostic procedures will be discussed

PLIM measurements

Cell culture studies

PLIM data and statistical analysis

Results and Discussion

Conclusion