Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Summary

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Abstract

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 µm with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 µm with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here, in vivo small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Introduction

High-resolution optical imaging modalities, such as optical coherence tomography, confocal microscopy, and multiphoton microscopy, have numerous benefits. However, the spatial resolution decreases significantly as the imaging depth increases. This is because of the diffuse nature of light transport in soft tissues^1,2. The integration of optical excitation and ultrasound detection provides a solution to overcome the challenge of high-resolution optical imaging in deep tissues. Photoacoustic microscopy (PAM) is one such modality that can provide deeper imaging than other optical imaging modalities. It has been s....

Protocol

All animal experiments were performed according to the approved regulations and guidelines of the Institutional Animal Care and Use Committee of Nanyang Technological University, Singapore (Animal Protocol Number ARF-SBS/NIE-A0263).

1. AR-OR-PAM System (Figure 1)

1. System configuration: AR-PAM
   1. Use a nanosecond tunable laser system consisting of a diode-pumped, solid-state Nd-YAG laser (532 nm) and a dye laser with a tunability range of 559-576 nm as the optical irradiation source. Set the laser wavelength to 570 nm using an external controller and the repetition rate of th....

Results

The schematic of the AR-OR-PAM system is shown in Figure 1. In this setup, all components were integrated and assembled in an optical cage setup. The use of a cage system makes the AR-OR-PAM scanning head compact and easily assembled, aligned, and integrated onto a single scanning stage.

Two-dimensional continuous raster scanning of the imaging head was used during image acquisition. The time-resolv.......

Discussion

In conclusion, a switchable AR and OR PAM system that can achieve both high-resolution imaging at lower imaging depths and lower-resolution imaging at higher imaging depths has been developed. The lateral resolution and imaging depth of the switchable system was determined. The advantages of this switchable PAM system include: (1) the high-resolution imaging using tight optical focusing; (2) the deep-tissue imaging using acoustic focusing; 3) the dark-field illumination for AR-PAM, which prevents strong PA signals from a.......

Materials

Name	Company	Catalog Number	Comments
Q-switched Nd:YAG laser	Edgewave	BX80-2-L	Pump laser
Credo-High Repetition Rate Dye Laser	Spectra physics	CREDO-DYE-N	Dye laser
Precision Linear Stage	Physik Instrumente	PLS 85	XY raster scanning stage
Translation stage	Physik Instrumente	VT 80	Confocal determine
Mounted Silicon photodiode	Thorlabs	SM05PD1A	Triggering/Pulse variation
Motorized continuous Rotational stage	Thorlabs	CR1/M-Z7	Diverting laser beam
Mounted Continuously Variable ND Filter	Thorlabs	NDC-50C-4M	Intensity variable
Fiber Patch Cable	Thorlabs	M29L01	Multimode fiber
Microscope objective	Newport	M-10X	Objective
XY translating mount	Thorlabs	CXY1	Translating mount
Plano convex lens	Thorlabs	LA1951	Collimating lens
Conical lens	Altechna	APX-2-B254	Ring shape beam
Translation stage	Thorlabs	CT1	Translating stage
Optical condenser	Home made
Ultrasonic transducer	Olympus-NDT	V214-BB-RM	50MHz transducer
Plano concave lens	Thorlabs	LC4573	Acoustic lens
Pulser/Receiver	Olympus-NDT	5073PR	Pulse echo amplifier
Mounted standard iris	Thorlabs	ID12/M	Beam shaping
Plano convex lens	Thorlabs	LA4327	Condenser lens
Mounted precision pinhole	Thorlabs	P50S	Spatial filtering
Single mode fiber patch cable	Thorlabs	P1-460B-FC-1	Single mode fiber
Fiber coupler	Newport	F-91-C1	Single mode coupling
Achromatic doublet lens	Edmund Optics	32-317	Achromatic doublet
Protected silver elliptical mirror	Thorlabs	PFE10-P01	Mirror
Right angle kinematic mirror mount	Thorlabs	KCB1	Mirror mount
Z-Axis Translation Mount	Thorlabs	SM1Z	z translator
Lens tube	Thorlabs	SM05L10
UV Fused Silica Right-Angle Prism	Thorlabs	PS615	Right angle prism
Rhomboid prism	Edmund Optics	47-214	Shear wave
Dimethylpolysiloxane	Sigma Aldrich	DMPS1M	Silicon oil
Amplifier	Mini Circuits	ZFL-500LN	Amplifier
16 bit high speed digitizer	Spectrum	M4i.4420	Data acquisition card
Oscilloscope	Agilent Technologies	DS06014A
Mice	InVivos Pte.Ltd	ICR	Animal model
Ultrasound gel	Progress/parker acquasonic gel	PA-GEL-CLEA-5000	Acoustic coupling
Water tank	Home made
Translation stage	Homemade		Switching AR-OR
Gold nanoparticles	Sigma Aldrich	742031	Lateral resolution
Sterile ocular ointment	Alcon	Duratears	Animal imaging
1951 USAF resolution test target	Edmund Optics	38257	Confocal alignment
Data acquisition software	National Instrument	Labview	Home made software using Labview
Image Processing software	Mathworks	Matlab	Home made program using Matlab