In this work, we theoretically and experimentally deal with photoacoustic resolution enhancement by means of saturated modulation quenching. It is shown that experimental systems for resolution enhancement with saturated modulation quenching in fluorescence microscopy are not necessarily suited for photoacoustic modulation quenching. Here, we show that modulation quenching is not limited to fluorescent dyes but can be also applied to metallic nanoparticles. For modulation quenching in photoacoustic microscopy it is sufficient that the signal saturates with increasing excitation intensity.
In this work, we demonstrate a new technique which has the potential for super-resolution fluorescence imaging. In this technique, similar to STED microscopy, a tightly focused intensity-modulated excitation beam and a donut shaped cw beam are confocally raster-scanned over the sample. In contrast to STED microscopy, both beams need to be absorbed by the fluorophore. A lock-in amplifier is used to measure only the modulated fluorescence. Sufficiently high cw donut beam intensities lead to saturation of the fluorophores in the outer rim of the modulated point spread function which enables resolution enhancement. Theoretically, sub-diffraction resolution can be achieved.
In this work, we employ frequency-domain photoacoustic microscopy to obtain photoacoustic images of labeled and unlabeled cells. The photoacoustic microscope is based on an intensity-modulated diode laser in combination with a focused piezo-composite transducer and allows imaging of labeled cells without severe photo-bleaching. We demonstrate that frequency-domain photoacoustic microscopy realized with a diode laser is capable of recording photoacoustic images of single cells with sub-µm resolution. As examples, we present images of undyed human red blood cells, stained human epithelial cells, and stained yeast cells.
In this paper, multimodal optical-resolution frequency-domain photoacoustic and fluorescence scanning microscopy is presented on labeled and unlabeled cells. In many molecules, excited electrons relax radiatively and non-radiatively, leading to fluorescence and photoacoustic signals, respectively. Both signals can then be detected simultaneously. There also exist molecules, e.g. hemoglobin, which do not exhibit fluorescence, but provide photoacoustic signals solely. Other molecules, especially fluorescent dyes, preferentially exhibit fluorescence. The fluorescence quantum yield of a molecule and with it the strength of photoacoustic and fluorescence signals depends on the local environment, e.g. on the pH. Therefore, the local distribution of the simultaneously recorded photoacoustic and fluorescence signals may be used in order to obtain information about the local chemistry.
Proc. SPIE. 10064, Photons Plus Ultrasound: Imaging and Sensing 2017
KEYWORDS: Signal to noise ratio, Modulation, Blood, Glasses, Microscopy, Luminescence, Laser induced fluorescence, Monte Carlo methods, Transducers, Optical resolution, Photoacoustic microscopy, Signal detection
In this paper a multimodal optical-resolution photoacoustic and fluorescence microscope in frequency domain is presented. Photoacoustic waves and modulated fluorescence are generated in chromophores by using a modulated diode laser. The photoacoustic waves, recorded with a hydrophone, and the fluorescence signals, acquired with an avalanche photodiode, are simultaneously measured using a lock-in technique. Two possibilities to optimize the signal-to-noise ratio are discussed. The first method is based on the optimization of the excitation waveform and it is argued why square-wave excitation is best. The second way to enhance the SNR is to optimize the modulation frequency. For modulation periods that are much shorter than the relaxation times of the excited chromophores, the photoacoustic signal scales linearly with the modulation frequency. We come to the conclusion that frequency-domain photoacoustic microscopy performed with modulation frequencies in the range of 100 MHz can compete with time-domain photoacoustic microscopy regarding the signal-to-noise ratio. The theoretical predictions are confirmed by experimental results. Additionally, images of stained and unstained biological samples are presented in order to demonstrate the capabilities of the multimodal imaging system.
In this paper a multimodal optical resolution microscope in frequency domain is introduced. Fluorescence and photoacoustic signals are generated simultaneously using an amplitude modulated diode laser. The resulting signals are recorded by a lock-in amplifier. By scanning the sample, two-dimensional plots of the fluorescence and photoacoustic signals can be generated. In this paper, the signal-to-noise ratios of amplitude modulated signals are compared to those expected for pulsed excitation under the assumption that the maximum permissible exposure limits are fulfilled. Furthermore, it is described how to determine the excited state life-times from the measured frequency responses of the fluorescence or photoacoustic signals, respectively.