Hi, I am Sava Saje from Photo Migration Imaging Laboratory of David Bo in Martino Center for Biomedical Imaging at Massachusetts General Hospital. And I'm a boss also from the David Bow Laboratory And I'm from the same lab. Today we are going to show you two procedures of measuring oxygen tension in cortical microvasculature in small rodents based on oxygen dependent quenching of phosphorescence.
We use these techniques in our laboratory to study oxygen delivery and consumption under normal and pathological brain conditions. So let's get started. First, prepare several pieces of cover slip glasses surrounded by optically absorptive masks made from black electric tape.
The masks should have openings approximately 3.5 millimeters in diameter. These cover slips are used for sealing the cranial windows while the absorptive mask blocks phosphorescent signal from any dye that has leaked from the cranial windows edges. Now prepare the sealed cranial window on the peral bone.
The details of the preparation can be found in papers by Kleinfeld et al and Masini etal referenced in this publication. In this preparation, the Dura matter is removed. Make sure that the mask covers all edges of the cranial window.
Now using a dental drill, make an additional one millimeter bur hole in the frontal bone. This hole will be used later to induce cortical spreading depression. Be careful not to damage the cortical vasculature in any step.
The imaging setup consists of a quantel brilliant pulsed ind YAG laser at 532 nanometers and 10 hertz in which a small portion of the pulse energy is coupled to a multimode fiber and delivered to the sample. The energy of each pulse is also sampled with a photo diode. A CCD camera imager.
QE from law vision is mounted on a vertical Z stage. We use an Olympus four X objective with a 29 millimeter working distance. A high pass filter with a 650 nanometer cutoff wavelength and a 100 millimeter focal length tube lens are mounted in front of the camera with the animal in place.
Adjust the position of the objective. Now set the laser to minimal pulse energy and turn it on. Measure the beam energy with an optical pulse energy meter, adjust the Q switch.
Delay time such that the pulse energy delivered to the sample does not exceed 10 millijoules per centimeter squared. Next, ensure that all physiological parameters of the animal are within normal physiological range. Then dissolve a weight appropriate amount of the oxygen sensitive phosphorescence probe.
Oxy four R three, developed by Libi Dev et all in one milliliter of saline to achieve a concentration of 40 micromolar in blood. Slowly inject the solution into the femoral vein. Now inject approximately one microliter of a one molar solution of potassium chloride through the bur hole in the frontal bone to induce cortical spreading depression and start the data acquisition software.
Imaging of phosphorescence should last around 10 minutes. During the experiment, phosphorescence images are displayed in real time on the screen. After imaging, take another blood sample to confirm that the animal's blood gases are still within normal physiological range.
Here are a few representative results from wide field CCD imaging of PO O2 during cortical spreading depression. The left side of this figure displays a wide field of view image of oxygen pressure before the arrival of A CSD wave. The right side shows the temporal evolution of the average oxygen pressure during CSD propagation within the region of interest marked on the left.
This movie shows the temporal evolution of the oxygen pressure in the whole cranial window. During propagation of the CSD wave, the scale bar indicates oxygen pressure in millimeters of mercury for two photon imaging of oxygen tension and cortical micro vasculature of a mouse. Prepare the cranial window as described in Jo of article number six 80 and insert the cannula into the femoral artery.
For imaging, we use an Olympus 20 x infrared coated water immersion objective, dissolve a weight appropriate amount of the oxygen sensitive phosphorescence probe, PTPC 3 43, developed by Kova et al in 0.2 mils of saline and filter it through a two micron filter. After ensuring that the animal's physiological parameters are within normal physiological range, very slowly inject the PTP C3 43 solution into the femoral artery. The main components of the experimental setup are a femtosecond laser set to 840 nanometers, an electro optic modulator, galvanometer scanners, Olympus scan, and tube lenses, and a four channel detector with PMTs.
One detection channel contains both A PMT and a discriminator. Photon counts are collected with a high speed digital board. All aspects of the experiment are controlled by software written in lab view, and c.
Begin the experiment by setting the desired time intervals for dye excitation and emission collection At each pixel, first acquire a survey scan, which is a slow two dimensional raster scan of phosphorescence intensity decay that displays the vasculature structure at the current depth. Using the survey scan image, select intravascular locations to measure PO two. During measurements, the software redirects the excitation laser to the selected locations by changing the position of the galvanometer scanning mirrors.
Set the desired amount of averaging measurement interval and experiment duration and start the measurement. PO two measurements are acquired at the selected locations at the specified measurement interval for the duration of the experiment. After collecting data at various cortical depths, inject dextrin conjugated fluorescent dye into vasculature for imaging.
The micro vasculature structure. Obtain a stack of structural images of the vasculature by performing the two photon imaging of fit c fluorescence using a green channel in the four channel detector. Here we present a 3D projection of the imaged microvasculature stack.
The shades of gray represent a volumetric vessel mask created based on the structural image. The measured PO two values were color coded. The scale bar is 200 micrometers.
We have just shown you two procedures for measuring oxygen tension in cortical microvasculature, in small rodents based on oxygen dependent crunching of phosphorescent. When performing these procedures, it's important to have very clean brain surgeries and also to wear safety goggles when operating the lasers. So that's it.
Thank you for watching and good luck with your experiments.
