The overall goal of the following experiment is to acquire a micron scale, three dimensional images of the oxygen concentration in the immediate environment of live cells by electron spin resonance or ESR microscopy. The protocol will be demonstrated on cyanobacteria, which are common photosynthetic cells. This is achieved by first preparing a sample of cyanobacteria cells inside the ESR microscopy sample holder.
Embedded in stable organic radical solution. The sample holder is placed inside the imaging probe, which goes into the static electromagnet of the ESR microscope system. Now, several three dimensional ESR micro images of the organic radical solution are acquired by the ESR microscope.
This is carried out for several variants of pulse sequences and often under various external conditions such as under light and dark conditions. Next, the raw data images are processed by the computer in order to extract the three-dimensional spatially resolved relaxation times of the radical. These relaxation times provide a direct measure to the oxygen concentration through pre-existing calibration leading to full three-dimensional oxygen maps of the measured samples under various external conditions.
Results are obtained that clearly show the lack of oxygen production by the cyanobacteria when no light is applied. Furthermore, it is shown that under light irradiation, significant oxygen gradient and oxygen heterogeneity are created near the cyanobacteria cells. Hi, I am al from the laboratory of BLA in the S Faculty of Chemistry at the Technion, the Israel Institute of Technology in Haifa.
Today we will show you a procedure for mapping oxygen concentrations near life cells in three dimensional manner at high special resolution by the means of fierce arm microscopy. We use this procedure in our laboratory to study a oxygen consumption rate and metabolic activity of cells. So let's get started.
Electron spin resonance or ESR is a spectroscopic technique in which electromagnetic radiation at a specific frequency is absorbed by paramagnetic species having unpaired electron spin placed under an external static magnetic field. It is a powerful method for studying the environment of paramagnetic molecules in live species, including the concentrations of oxygen and reactive oxygen species. Here a typical oxygen concentration image of a tumor bearing mouse is shown for heterogeneous samples.
The ESR spectral information can be obtained in a spatially resolved manner through the use of magnetic field gradients. A stable, free, organic radical was injected into the mouse and its ESR characteristics provide the oxygen concentration at its environment. The ESR based results are superimposed on an MRI anatomical image.
Up until now. Such ESR imaging techniques of live specimens were only carried out with relatively large samples at millimeter scale. Resolution ESR microscopy involves the measurements of millimeter and sub millimeter size samples, providing three dimensional ESR images with a resolution approaching one micron.
Here are two examples of high resolution micron scale ESR, images of generated sample with NNC 60 powder and lithium fellow cyanide paramagnetic crystals. An ESR microscope is similar to a conventional ESR spectrometer, having a magnet for generating the static field, a microwave system for spin excitation and signal detection, a probe for holding the sample and a computerized console to control the acquisition process and data handling. Other components of the system include magnetic field gradient drivers, which are part of the electronic system and gradient coils that are located in the imaging probe.
The gradient coils also have an aperture to allow for light irradiation of the sample. A controlled temperature moist air is applied to the sample to ensure minimal dehydration during the experiment. The main unique features of ESR microscopy are the small size of the probe and the gradient coils, the higher spin sensitivity of the system and the high strength of the magnetic field gradient drivers Prior to the start of this procedure, prepare a cyanobacteria suspension at a concentration of 40 milligrams per milliliter.
To begin ESR micro imaging sample preparation, take a few squares of absorbent paper at a size of approximately 400 by 400 microns using fine tweezers. Then insert them into an einor tube. Subsequently filled the tube with 1.2 milliliters of the cyanobacteria suspension.
Then centrifuge the suspension for two minutes at 6, 000 RPM in a micro centrifuge following centrifugation, completely remove the snat except for approximately 50 microliters, which is left to avoid cyanobacteria dehydration. This process results in the saturation of the absorbent paper with the cyanobacteria cells. Next, prepare a specially prepared cup like glass sample holder, extract a few fibers from the paper using fine tweezers, and place them on the bottom of the sample holder.
Following that, add three millimolar of tridel and BG 11 solution, which is prepared as described in the written protocol by the eight of a syringe. Then seal the sample holder with UV curable glue by applying the glue between the edges of the glass sample holder and its matching cover glass piece, leaving a small air outlet open. Finally, cure the glue with UV light for two seconds.
To begin the imaging experiment, turn on the ESR micro imaging system and insert the sample into the resonator that goes inside the imaging probe. Now using the computer control software, set the system on tune mode and find the resonance microwave frequency of the probe, which will be used for the ESR measurements. Following that, set the static magnetic field on the value that matches the applied microwave frequency.
Set the timing parameters for the pulse sequence and observe the ESR signal to make sure that the system functions well and the sample is well prepared. Then set the imaging parameters such as the number of pixels, the strength of the gradients, and the length of the gradient pulses to their required values. Following setup, collect three 3D ESR images by a Han Echo Imaging pulse sequence with interpulse separation tau values of 500 600 and 700 nanoseconds.
At this point, turn the light projection at the sample on if required for the experimental conditions throughout the acquisition, the data is automatically saved. Following data acquisition process, the raw data files via MATLAB software script. Using the software generate images of the trill radical concentration and the relaxation time.
T two map then translate these images to an oxygen concentration image via a preexisting calibration curve that links the oxygen concentration to the relaxation time. Here T two zero is the spin, spin, relaxation time of the probe under anoxic conditions, depending on the probes concentration C and its diffusion coefficient D.Typical raw data images from several three dimensional ESR imaging experiments performed on cyanobacteria are shown. These images were acquired with different tau values under either dark or light conditions.
Under dark conditions, the results are very similar except for decreased intensity for the larger T values due to spin, spin relaxation time T two. Conversely, under light irradiation, the image pattern changes due to different relaxation times throughout the sample. This data is then processed to obtain an amplitude image from this image.
It is evident that the cyanobacteria is located mainly on the right side of the sample holder. T two images are translated to oxygen concentration values via a pre-existing calibration curve that links the oxygen concentration to the relaxation time. The results are shown for cyanobacteria in the presence and absence of light.
It is clear that oxygen production is initiated with light and causes significant increase in the solution oxygen concentration. This leads to a decrease in the radical T two, mainly in the voxels near the absorbent paper fibers with the cyanobacteria, We have just shown you how to prepare sample containing life cells with stable free radical for mapping oxygen concentration by E ES R microscopy. When doing this procedure, it is important to remember to keep it as short as possible to assure S variability and to avoid sample de duration.
So that's it. Thank you for watching us, and good luck with your experiment.
