Isometric multimodal photoacoustic microscopy based on optically transparent micro-ring ultrasonic detection

Abstract

Photoacoustic microscopy (PAM) is an attractive imaging tool complementary to established optical microscopic modalities by providing additional molecular specificities through imaging optical absorption contrast. While the development of optical resolution photoacoustic microscopy (ORPAM) offers high lateral resolution, the acoustically-determined axial resolution is limited due to the constraint in ultrasonic detection bandwidth. ORPAM with isometric spatial resolution along both axial and lateral direction is yet to be developed. Although recently developed sophisticated optical illumination and reconstruction methods offer improved axial resolution in ORPAM, the image acquisition procedures are rather complicated, limiting their capabilities for high-speed imaging and being easily integrated with established optical microscopic modalities. Here we report an isometric ORPAM based on an optically transparent micro-ring resonator ultrasonic detector and a commercial inverted microscope platform. Owing to the superior spatial resolution and the ease of integrating our ORPAM with established microscopic modalities, single cell imaging with extrinsic fluorescence staining, intrinsic autofluorescence, and optical absorption can be achieved simultaneously. This technique holds promise to greatly improve the accessibility of PAM to the broader biomedical researchers.

Fig. 1

Schematic of the multimodality microscopy system. (a) An integrated microscopic system combining laser scanning confocal microscopy and PAM was realized by an optically transparent MRR ultrasonic detector. The inset illustrates a magnified view of the placement of the fiber coupled MRR ultrasonic detector and specimen. (b) Illustration of MRR detection of laser induced PA waves. (c) A representative transmission spectrum exhibits a pronounced resonance dip under the critical coupling condition (black circle) and its corresponding Lorenz fitting (red line). Inset is the numerical simulation of the electric field distribution, which shows the fundamental TM mode in the waveguide. Using a narrow-band laser source (blue line), the pressure induced resonance shift (dashed red line) can be measured as the amplitude modulation of the transmitted optical signal. (d) The time-resolved PA pulse signal measured by the MRR ultrasonic detector. (e) Its corresponding power spectrum shows an ultra-broadband frequency response.

Fig. 2

PAM imaging of single RBCs in a mouse blood smear. (a) Trans-illuminated optical microscopic image. (b) PA MAP image of individual RBCs. scale bars: 10 µm. (c) Magnified PAM projection image of a single RBC along the x-y plane; scale bar: 2 µm. (d) Cross-sectional image of the same RBC along the x-z plane. (e) 3D visualization of the RBC, a movie can be found from the supplementary material.

Fig. 3

Simultaneous PAM and fluorescence confocal imaging of a human RPE flat mount. (a) Projection PAM image of the RPE along the x-y plane. (b) Cross-sectional PAM image along the x-z plane. (c) 3D visualization of the RPE cells imaged by PAM. (d) Actin stained confocal imaging highlights the boundaries of the RPE cells. (e) Autofluorescence confocal image shows the distribution of lipofuscin. (f) Overlaid image of all the three modalities acquired simultaneously. Scale bars: 10 µm.