The overall goal of this procedure is to demonstrate in vivo optical resolution photoacoustic microscopy or, or Pam of microcirculation that is non-invasive and label free. This is accomplished by first anesthetizing the experimental animal. The second step of the procedure is to position the region of interest by translating the imaging head of the optical resolution photoacoustic microscope scanning parameters are then set and image acquisition is initiated.
Ultimately, the distributions of total oxygen concentration and oxygen saturation of hemoglobin are obtained through computation of the multi wavelength experimental data. Optical resolution photoacoustic microscopy is based on laser excitation and ultrasonic detection. A short post laser beam is focused into biological tissue.
Light absorption generates transient heating and subsequent ultrasonic emission, which is received to form an image. The unique Advantage of, or Pam, is it's the ground free detection and exquisite sensitivity to optical absorption. In contrast, existing reflection, not optical microscopy technologies are primarily sensitive to optical scattering or fluorescence Capable of providing in vavo label free high resolution imaging of optical absorption or RPM has found broad biomedical applications in neurology, ophthalmology, vascular biology, and dermatology.
Optical resolution photoacoustic microscopy or, or pamm consists of both optical and ultrasonic components. The optical irradiation source is a dye laser pumped by a solid state pulse laser. The output laser beam is attenuated by a neutral density filter and coupled into a single mode optical fiber for light delivery to allow mechanical scanning of the microscope imaging head rather than the living object to effectively couple the multimode laser beam into a single mode optical fiber.
It is first filtered by a 50 micrometer diameter pinhole. The pinhole is positioned slightly away from the focus of the condenser lens to match its diameter with the fundamental mode beam diameter. To create a nearly diffraction limited optical focus, the output of the single mode fiber is collated and fills the back aperture of a microscope.Objective.
The ultrasonic detection involves a homemade acoustic optical beam combiner connected to an ultrasonic transducer. The combiner aligns the optical irradiation and the acoustic detection coaxially. A spherical cavity is ground into the bottom of the combiner to produce an acoustic lens with a numerical aperture of 0.5 in water, which provides an acoustic focal diameter of 43 micrometers at the 50 megahertz central frequency.
For the first alignment, it helps to use pulse echo ultrasound to determine the position of the acoustic focal plane. The imaging head is adjusted vertically until the maximum pulse echo ultrasonic signal is observed. To maximize the detection sensitivity, the optical and acoustic foci are aligned.Confocal.
An optically absorbing target, such as a piece of black electrical tape is placed in the acoustic focal plane. Then the optical focal position is adjusted to maximize the photo acoustic signal. The or PAM system also includes electronic amplifiers and analog digital converter, a mechanical scanner and a scanner controller.
The digitized signal is transferred to a computer for image formation. The software converts the raw data into a sequence of cross-sectional bcan images. As the microscope head raster scans the object, the recorded images can then be displayed as individual cross-sectional images, 2D maximum amplitude projection images or 3D renderings.
A water tank is used to house the microscope imaging head. An imaging window is cut into a Petri dish and sealed with an ultrasonically and optically transparent polyethylene membrane. The photo acoustic wave generated from the object is acoustically coupled to the ultrasonic detector first by ultrasonic gel between the object and the polyethylene membrane, and then by deionized water in the Petri dish.
These inventions and applications of available optics lasers and the ultrasound transducer make up the or pam system. In the next segment, we will image the microvasculature of a living mouse with this system. If the animal being imaged is not a nude mouse, 24 hours before the experiment, it is ideal to dete.
So the irritation to the skin vasculature is recovered. Anesthetized the mouse with 3%ISO fluorine vaporized in medical grade oxygen. The typical flow rate is between one and 1.5 liters per minute.
Depending on the animal's body weight. Maintain the anesthesia with 1%ISO fluorine Throughout the experiment, perform a toe pinch to ensure that the mouse is fully anesthetized. Transfer the mouse to a stereotactic stage with a heating pad to maintain its body temperature at 37 degrees Celsius.
Now flatten the ear on the plastic plate. Then apply a layer of ultrasound gel on top of the ear. Avoid trapping air bubbles inside the gel.
Also apply ophthalmic ointment to the eyes to prevent drying or accidental laser damage. Avoid touching the eye directly with the applicator. Then place the ear under the imaging window and slowly raise the animal stage until the ultrasound gel contacts the bottom of the membrane.
Soft contact is required because pressing the ear against the membrane may affect blood flow in the ear. Next, take a few precautions with the animal to monitor its physiological status. Clamp a pulse oximeter to the mouse, leg or tail.
Now lower the imaging head until the acoustic lens is immersed in deionized water and remove bubbles trapped under the acoustic lens. Set the laser to the external trigger mode and start trial scanning. Adjust the Z position of the imaging head until the optical and acoustic dual foci are seated at the middle of the ear.
Now set the scanning parameters and start formal image acquisition. To measure total hemoglobin concentration. Set the wavelength to an ISO spastic point where oxyhemoglobin and deoxyhemoglobin have equal molar extinction coefficients.
To measure hemoglobin oxygen saturation, set the second wavelength to where the molar extinction coefficients of the oxyhemoglobin and deoxyhemoglobin have a pronounced difference. When finished, clean the mouse ear and place the mouse in an incubator. With the environmental temperature set at 37 degrees Celsius, when it wakes up, naturally return the mouse to the animal facility.
This maximum amplitude projection image shows the vascular anatomy of a living nude mouse ear imaged using or Pam at 570 nanometers. This projection image shows hemoglobin oxygen saturation in the same ear. It is calculated from the images acquired at an ISO spastic wavelength of 570 nanometers and a deoxyhemoglobin absorption dominant wavelength of 561 nanometers.
A closer look at the boxed region shows the resolution of oramm. A densely packed capillary bed is clearly resolved. In this video, we demonstrated the typical experimental protocol for ORPM, including the system configuration, system alignment in vivo experimental procedure and functional imaging scheme.
One of the most important aspects in the instrument design and operation is the optical acoustic confocal alignment, Which is crucial to imaging sensitivity, optical resolution photoacoustic microscopy in combination with deep penetrating acoustic resolution photoacoustic microscopy and photoacoustic computed tomography promises in vivo, functional and molecular imaging and multiple end scales, organ nails, cells, tissues, and up to organs can be imaged with the same contrast. Origin, such as imaging scalability holds the potential to revolutionize research in multi-scale systems, biology and translation. From microscopic lab discoveries to macroscopic clinical practice.
