Frequency domain photoacoustic and fluorescence microscopy.

Thomas Berer,Thomas A. Klar,Bianca Buchegger,Jaroslaw Jacak,Gregor Langer

doi:10.1364/boe.7.002692

Abstract

We report on simultaneous frequency domain optical-resolution photoacoustic and fluorescence microscopy with sub-µm lateral resolution. With the help of a blood smear, we show that photoacoustic and fluorescence images provide complementary information. Furthermore, we compare theoretically predicted signal-to-noise ratios of sinusoidal modulation in frequency domain with pulsed excitation in time domain.

Highlights

Fluorescence microscopy became a valuable tool for life sciences within the last century
We report on simultaneous frequency domain optical-resolution photoacoustic and fluorescence microscopy with sub-μm lateral resolution
We introduced simultaneous frequency domain optical-resolution photoacoustic and fluorescence microscopy using a continuous wave diode laser, which output power was sinusoidally modulated in the low MHz region

Summary

Introduction

Fluorescence microscopy became a valuable tool for life sciences within the last century. When the optically-excited molecules return into their ground state, the absorbed energy is either emitted radiatively via luminescence and/or is transferred into heat via non-radiative processes The latter process leads to thermal expansion and to the generation of ultrasonic waves, commonly known as the photoacoustic effect [3]. The axial resolution, which depends on a time-of-flight measurement, is determined by the bandwidth of the ultrasonic transducer and is, typically larger than 10 μm [4,8,15]. We excite photoacoustic and fluorescence signals using a cw diode laser with sinusoidal amplitude modulation. This is not necessarily a disadvantage as, e.g., microtome cuts and eukaryotic cells typically have thicknesses up to 10 μm For these kinds of specimens, time domain investigation does not provide sufficient axial resolution to gain additional information anyhow. A webcam (Logitech, C300) is used to get bright-field microscopic images of the sample

Determination of the excitation spot diameter

Multimodal imaging of a blood smear

Conclusion and outlook