Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Podsumowanie

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

Streszczenie

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

Wprowadzenie

Recent decades have witnessed the explosive development of the field of biomedical photoacoustic imaging^{1,2,3,4,5,6,7,8}. Based on the energy conversion of light into sound, the emerging photoacoustic imaging can visualize biological samples at scales from organelles, cells, tissues, organs to small-animal whole body and can reveal its anatomical, functional, molecular, genetic, and metabolic information^{1,2,9,10,11,12}. Photoacoustic imaging has found unique applications in a range of biomedical fields, such as cell biology^13,14, vascular biology^15,16, neurology^17,18, oncology^19,20,21,22, dermatology²³, pharmacology²⁴, and hematology^25,26. Its application in ophthalmology, that is, photoacoustic ocular imaging, has attracted substantial interests from both scientists and clinicians and is currently under active investigation.

In contrast to routinely used ocular imaging technologies²⁷, such as fluorescein angiography (FA) and indocyanine green angiography (ICGA) (based on fluorescence contrast), optical coherence tomography (OCT) (based on optical scattering contrast), and its derivative OCT angiography (based on motion contrast of red blood cells), photoacoustic ocular imaging uses optical absorption as the contrast mechanism. This is different from conventional ocular imaging technologies and provides a unique tool for studying optical absorption properties of the eye, which are usually associated with the pathophysiological status of ocular tissue²⁸. To date, significant excellent work has been done in photoacoustic ocular imaging^{29,30,31,32,33,34,35,36,37}, but these studies focus on the posterior segment of the eyes of small animals, such as rats and mice. The pioneering studies well demonstrate the feasibility of photoacoustic imaging in ophthalmology but there is still a long way to go towards clinical translation of the technology since eyeball sizes of rats and mice are much smaller (less than one-third) than that of humans. Due to the propagation of ultrasound waves over a significantly longer distances, signal intensity and image quality may greatly suffer when the technique is used for imaging the posterior segment of larger eyes.

Towards this goal, we recently reported the noninvasive, label-free chorioretinal imaging in living rabbits using integrated photoacoustic microscopy (PAM) and spectral-domain OCT (SD-OCT)³⁸. The system has excellent performance and could visualize the retina and choroid of the eyes of larger animals based on endogenous absorption and scattering contrast of ocular tissue. Preliminary results in rabbits show that the PAM could noninvasively distinguish individual retinal and choroidal blood vessels using a laser exposure dose (~80 nJ) significantly below the American National Standards Institute (ANSI) safety limit (160 nJ) at 570 nm³⁹; and the OCT could clearly resolve different retinal layers, the choroid, and the sclera. It is the very first demonstration of posterior segment imaging of larger animals using PAM and might be a major step towards clinical translation of the technology considering that the eyeball size of rabbits (18.1 mm)⁴⁰ is almost 80% of the axial length of humans (23.9 mm).

In this work, we provide a detailed description of the dual-modality imaging system and experimental protocols used for noninvasive, label-free chorioretinal imaging in living rabbits and demonstrate the system performance through representative retinal and choroidal imaging results.

Protokół

Rabbits are a United States Department of Agriculture (USDA) covered species. Its use in biomedical research needs to follow strict regulations. All rabbit experiments were performed in accordance with the ARVO (The Association for Research in Vision and Ophthalmology) Statement for the Use of Animals in Ophthalmic and Vision Research, after approval of the laboratory animal protocol by the University Committee on Use and Care of Animals (UCUCA) of the University of Michigan (Protocol PRO00006486, PI Yannis Paulus).

1. System configuration

1. Photoacoustic microscopy (PAM)
   1. Use an optical parametric oscillator (OPO) laser pumped by a diode-pumped solid-state laser as the light source of the PAM. Select proper technical specifications, such as pulse repetition rate 1 kHz, pulse duration 3 - 6 ns, and tunable wavelength range 405 - 2600 nm.
   2. Reflect the emanating beam from the laser at 570 nm by two mirrors (M1 and M2), then pass it through a half-wave plate attenuator mounted on a motorized rotation stage, and finally focus, filter, and collimate it by a beam collimator (Figure 1). Optimize the design of the beam collimator. An example configuration of the beam collimator includes a focusing lens L1 (focal length 250 mm), a pinhole (diameter 50 µm), and a collimating lens L2 (focal length 30 mm).
   3. Divide the collimated beam by a beam splitter (BS1) with a split ratio of 90/10 (reflection/transmission). Record the transmitted portion by a photodiode for pulse-to-pulse laser energy monitoring. Successively deflect the reflected portion by a mirror (M3) and a dichroic mirror (DM) and raster-scan it using a two-dimensional galvanometer. The galvanometer is a shared component with the spectral domain (SD)-OCT system (described below).
   4. Deliver the scanned beam through a telescope composed of a scan lens (focal length 36 mm) and an ophthalmic lens (OL, focal length 10 mm) and finally focus it on the fundus by the rabbit eye optics.
   5. Select a needle-shaped ultrasonic transducer with proper technical specifications, for example, center frequency 27 MHz, two-way −6 dB bandwidth 60%. Place it in contact with the conjunctiva off the central visual axis to capture excited photoacoustic signal.
   6. Amplify the signal using an ultrasonic amplifier (for example, gain 57 dB), filter it by a low-pass filter (for example, cutoff frequency 32 MHz), and digitize it by a high-speed digitizer at a sampling rate of 200 MS/s.
   7. Place a power meter above the rabbit eye and measure the laser pulse energy on the rabbit cornea to keep it below the ANSI safety limit 160 nJ at 570 nm³⁸. Block the beam to avoid laser overexposure using a laser shutter controlled from Matlab through a synchronization electronics.
   8. Synchronize the laser, the galvanometer, and the digitizer through a data acquisition (DAQ) board. Program the software for system control and data acquisition in Matlab.
2. Spectral-domain optical coherence tomography (SD-OCT)
   1. Adapt the SD-OCT system based on a commercially available system by adding an ophthalmic lens (OL) after the scan lens (SL) and a piece of dispersion compensation glass (DCG) in the reference arm (Figure 1). The modification enables that the OCT system can image the posterior segment of the rabbit eye.
   2. Use a zooming housing tube to adjust the length of the reference arm to ensure its match with the optical path length of the sample arm. Use an iris to control the intensity of retro-reflected reference light to ensure its match with back-scattered light intensity from the rabbit fundus to achieve maximum image contrast.
   3. Employ a charge-coupled device (CCD) camera encapsulated in the scan head for real-time visualization of rabbit fundus with an illumination light emitting diode (LED) as an external illumination source.

2. System alignment

1. Initialize the position of the galvanometer and align the OCT system by adjusting the screws of the fiber collimator mount and the beamsplitter cube.
   NOTE: The step-by-step procedures are available in the manual of the commercially acquired OCT system and will not be covered here. This step is mainly to ensure correct alignments of the fiber collimator, the reference arm, and the scan lens to maximize the performance of the OCT system.
2. Adjust the x, y, and z position of the pinhole around the focus of the focusing lens to spatially filter out the laser beam and maximally transmit laser energy. Check the heights of the laser beam before and after the pinhole using a height measurement tool to ensure that they are the same.
3. Adjust the tilt, decenter, and z position of the collimating lens L2 to collimate the filtered beam. Ensure that the beam has approximately the same size and height when observed in the near field and the far field.
4. Co-axially combine the PAM laser beam and the OCT light beam by tuning the tilts of the mirror and the DM. After this step, the PAM laser and OCT light should be fully coincident and scanning regions on the rabbit fundus are the same.
5. Adjust the tilt and decenter of the OL lens to correctly align it in the optical path. Once done, the dual-modality system is ready for imaging.
   NOTE: One can use the auto-collimation method to achieve this, i.e., checking the light back-reflected by the OL lens surface to make sure that it goes back along the same way as the incident light.

3. Rabbit preparation

1. Take a New Zealand White rabbit from the animal facility and record individual information, such as body weight and animal number.
2. Mount rabbit platforms, including the body support and the head support on the optical table below the imaging system. Put a water-circulating heating blanket on the body support and set the temperature of the circulating water to 38 °C to help keep the body temperature of the rabbit warm for the duration of the experiment and recovery.
3. Record the pre-procedure vitals, including overall animal state, mucous membrane color, heart rate, respiratory rate, and rectal body temperature. Anesthetize the rabbit with a mixture of ketamine (40 mg/kg) and xylazine (5 mg/kg) through intramuscular (IM) injection and record the usage of ketamine (Schedule III controlled substance). Confirm the anesthesia level by checking its heart rate, respiratory rate, and overall state.
4. Dilate the rabbit pupils using one drop each of Tropicamide 1% ophthalmic and phenylephrine hydrochloride 2.5% ophthalmic.
5. Use a speculum to hold the eyelids out of the way and apply a drop of eye lubricant to moisten the cornea. Instill a drop of topical Tetracaine 0.5% in the eye before the imaging procedure.
   NOTE: For longer procedures or procedures with possible discomfort to the animal, give the rabbit a subcutaneous injection of meloxicam before the experiment to ensure animal comfort.

4. SD-OCT imaging

1. Transfer the rabbit to the imaging platform of a clinical fundus camera and take 50 degree fundus, red free, and autofluorescence images before the OCT imaging session. This helps check optical transparency of the eye and recognize fundus vessel morphology and landmarks, such as the optic nerve and medullary ray retinal vasculature.
2. Transfer the rabbit to the platform of the OCT system and adjust its posture to roughly position one of the eyes under the OL. Illuminate the eye using the LED light.
   Note: To facilitate the clinical translation of the technique, the rabbit eyes are not stabilized using any other methods and the rabbit head is just put on the head support without any fixation.
3. Open the OCT software and first check the CCD camera image of the fundus. Finely adjust the height and angle of the head support if necessary to ensure region of interests (ROIs), such as choroidal vessels and retinal vessels, are within the field of view (FOV) of the camera.
   Note: If the rabbit is under a good anesthesia level, one sequential imaging session can last as long as 10 min without the need of re-adjusting the rabbit head.
4. Draw a straight line to represent the OCT B-scan of interest and start scanning. Adjust the reference arm length to visualize the OCT image and optimize the dispersion compensation factor in the OCT software to get the sharpest images.
   Note: When adjusting the reference arm length, two mirrored OCT images will appear one after the other. The correct image could be distinguished based on prior knowledge of the fundus anatomy.
5. Set data acquisition parameters, such as number of pixels and averages, and save images.
6. Observe the respiratory rate and heart rate of the rabbit to estimate the anesthesia level and animal comfort. For longer sessions, a one third dose of supplemental ketamine or inhaled isoflurane could be considered with endotracheal intubation, a V-gel, or a face mask to maintain the plane of anesthesia when necessary.
7. Rinse the rabbit cornea with eyewash every 2 min during the experiment to prevent corneal surface dehydration and corneal superficial punctate epithelial keratopathy. Monitor and record animal vitals every 15 min.

5. PAM imaging

1. Wear appropriate laser safety goggles and turn on the OPO laser.
2. Start the PAM control software, tune the laser wavelength to one of the absorption peaks of the targeted chromophores (e.g., 570 nm for hemoglobin), initialize the position of the galvanometer, and monitor laser energy before the rabbit cornea to ensure that it is below the ANSI safety limit.
3. Mount the ultrasonic transducer on a three-dimensional (3D) translation stage and position the transducer tip in contact with the rabbit conjunctiva pointing to the fundus. Use a drop of eye lubricant to better couple the transducer tip and rabbit conjunctiva.
4. Turn on the LED illumination light and visualize the rabbit fundus through the Matlab software.
5. Set the scanning ROI (either retinal vessels or choroidal vessels), including the center and the physical size. Open the laser shutter and start B-scan of the beam. By roughly align the transducer, one should be able to see detected photoacoustic signal on the oscilloscope. If not, slightly adjust the eye position to scan a different region of the cornea or switch to the other eye and repeat the above processes.
6. Observe the detected photoacoustic signal on the oscilloscope and finely tune the transducer position to maximize signal intensity along the entire B-scan.
   Note: Due to limited beam width, ultrasonic transducer usually has a small FOV⁴¹. This step determines the background modulation of final PAM images. Misalignment will lead to PAM images with heterogeneous background and degrade image quality greatly.
7. Set data acquisition parameters. This includes the number of pixels (e.g., 256×256 pixels), sampling rate (e.g., 200 MS/s), and delay time. Start data acquisition. The Matlab software will automatically open the shutter to pass the laser beam when started and close the shutter to block the beam when finished to avoid laser overexposure.
   Note: Limited by the pulse repetition rate (1 kHz) of the laser, it takes about 1 min to finish the data acquisition of an image with 256 × 256 pixels.
8. Process the raw data and visualize the PAM image in two dimensions (2D) through maximum intensity projection (MIP)¹³ or in 3D through volumetric rendering³⁸.
9. Unmount the ultrasonic transducer, rinse the tip using deionized water, and put it back to the storage case.
10. Transfer the rabbit to the fundus camera and re-examine the fundus. This step helps to check whether there are any morphological changes of the fundus after the imaging session.
11. Rinse the rabbit cornea with eyewash every two min during the experiment to prevent corneal surface dehydration and keratopathy. Monitor and record animal vitals every 15 min.
    Note: The PAM, OCT, and fundus imaging sessions take approximately 1 h.

6. Post imaging

1. After fundus re-examination using the fundus camera, disconnect the V-gel if connected. Rinse the eye using eyewash, apply flurbiprofen ophthalmic and neomycin and polymyxin B sulfates and dexamethasone ophthalmic ointment, and close the eye.
2. Transfer the rabbit with the water-circulating blanket to a recover chamber. Shield the box from light and wait until the rabbit naturally wakes up. During this period, monitor animal vitals every 15 min and keep the record and return a copy to the animal facility for recordkeeping.
3. Once the rabbit wakes up and is active, alert and walking normally, transport it back to the animal facility. If an acute experiment is planned, euthanize the animal using euthanasia solution (e.g., Beuthanasia, 0.22 mL/kg, intravenous injection in the marginal ear vein) and dispose of the carcass.
4. Turn off the software and the laser. Clean the optical bench.

Wyniki

The dual-modality imaging system and experimental protocol have been successfully tested in the authors' laboratory using four New Zealand White rabbits. The following showcases some representative results.

Figure 1 shows the schematic of the PAM and SD-OCT dual-modality imaging system. It is composed of the following modules: photoacoustic light source, variable laser attenuator, beam coll...

Dyskusje

An intact and regular tear film is essential for high-quality fundus images. An irregular and deteriorated tear films can significantly degrade image quality⁴². To preserve the integrity of the tear film and prevent corneal superficial punctate keratopathy, it is critical to lubricate the cornea using eyewash very frequently, approximately every two min. If there are any concerns regarding the opacity of the eye, use a slit lamp and fluorescein strips to check the cornea conditions.

Materiały

Name	Company	Catalog Number	Comments
Dual-modality imaging system
OPO laser	Ekspla (Vilnius, Lithuania)	NT-242
Beam attenuator	Thorlabs, Inc. (Newton, NJ, USA)	AHWP10M-600
Motorized rotation stage	Thorlabs, Inc. (Newton, NJ, USA)	PRM1/MZ8
Motorized rotation stage controller	Thorlabs, Inc. (Newton, NJ, USA)	TDC001
Focusing lens	Thorlabs, Inc. (Newton, NJ, USA)	AC254-250-B
Pinhole	Thorlabs, Inc. (Newton, NJ, USA)	P50S
Collimating lens	Thorlabs, Inc. (Newton, NJ, USA)	AC127-030-B
Photodiode	Thorlabs, Inc. (Newton, NJ, USA)	PDA36A
Laser shutter	Vincent Associates Inc. (Toronto, Canada)	LS6S2T0
Laser shutter driver	Vincent Associates Inc. (Toronto, Canada)	VCM-D1
Dichroic mirror	Semrock, Inc. (Rochester, NY, USA)	Di03-R785-t3-25×36
Scan lens	Thorlabs, Inc. (Newton, NJ, USA)	OCT-LK3-BB
Ophthalmic lens	Thorlabs, Inc. (Newton, NJ, USA)	AC080-010-B-ML
Ultrasonic transducer	Optosonic Inc. (Arcadia, CA, USA)	Custom
Amplifier	L3 Narda-MITEQ (Hauppauge, NY, USA)	AU-1647
Band-pass filter	Mini-Circuits (Brooklyn, NY, USA)	BLP-30+
Digitizer	DynamicSignals LLC (Lockport, IL, USA)	PX1500-4
Synchronization electronics	National Instruments Corporation (Austin, TX, USA)	USB-6353
OCT module	Thorlabs, Inc. (Newton, NJ, USA)	Ganymede-II-HR
Dispersion compensation glass	Thorlabs, Inc. (Newton, NJ, USA)	LSM03DC
Illumination LED light	Thorlabs, Inc. (Newton, NJ, USA)	MCWHF2
Power meter	Thorlabs, Inc. (Newton, NJ, USA)	S121C
Power meter interface	Thorlabs, Inc. (Newton, NJ, USA)	PM100USB
Height measurement tool	Thorlabs, Inc. (Newton, NJ, USA)	BHM1
Fundus camera	Topcon Corporation (Tokyo, Japan)	TRC 50EX
Matlab	MathWorks (Natick, MA, USA)	2017a
Oscilloscope	Teledyne LeCroy (Chestnut Ridge, NY, USA)	WaveJet 354T
Animal experiment
Water-circulating blanket	Stryker Corporation (Kalamazoo, MI, USA)	TP-700
Ketamine hydrochloride injection	Par pharmaceutical, Inc. (Woodcliff Lake, NJ, USA)	NDC code 42023-115-10
Xylazine hydrochloride	VetOne (Boise, ID, USA)	NDC code 13985-704-10
Tropicamide ophthalmic	Akorn Pharmaceuticals Inc. (Lake Forest, IL, USA)	NDC code 17478-102-12
Phenylephrine hydrochloride ophthalmic	Paragon BioTeck, Inc. (Portland, OR, USA)	NDC code 42702-102-15
Eye lubricant	Hub Pharmaceuticals LLC (Rancho Cucamonga, CA, USA)	NDC code 17238-610-15
Eyewash	Altaire Pharmaceuticals, Inc. (Aquebogue, NY, USA)	NDC code 59390-175-18
Tetracaine hydrochloride ophthalmic solution	Bausch & Lomb, Inc. (Rochester, NY, USA)	NDC code 24208-920-64
Flurbiprofen sodium ophthalmic solution	Bausch & Lomb, Inc. (Rochester, NY, USA)	NDC code 24208-314-25
Neomycin and Polymyxin B Sulfates and Dexamethasone Ophthalmic Ointment	Bausch & Lomb, Inc. (Rochester, NY, USA)	NDC code 24208-795-35
Meloxicam injection	Henry Schein Inc. (Queens, NY, USA)	NDC code 11695-6925-1