The goal of this experiment is to demonstrate the feasibility to photoactivate enzyme using near infrared irradiation. Caged-protein kinase A is immobilized on this surface of upconversion nanoparticles micro-injected into cells and kinase induced response in living cells is observed after near infrared irradiation. Traditional photoactivation require phototoxic UV light and inevitably, will co-induce undesirable chemical reaction and similar response, however, in our friendly options, like near infrared irradiation cannot trigger reaction due to its low energy.
The upconversion nanoparticle can effectively upconvert bio-friendly low energy near IR photon into high energy UV photon to trigger a chemical reaction near the particle surface without direct UV irradiation. Kinase plays a key role in oncogenesis, this technique can help us examine more signal transduction information with much lower interfering noise from UV phototoxicity and its side reaction. Generally, individuals new to this field will encounter difficulties in modifying instruments and optical setup for upconversion application.
To begin, install a 4x objective lens next to the cuvette holder of the fluorospectrometer, so that the laser can be focused in the middle of the cuvette. Place a tunable 980 nanometer laser diode 90 degrees against the objective lens. Install a full reflectance mirror and the light path intersection of the objective lens and laser.
Don't forget to place an anodized metal plate to block the excitation window of the spectrometer to protect the interior parts. Then place a cuvette of silica coated upconversion nanoparticle solution in the holder. A bright upconversion beam can be visualized when the laser turns on.
Switch the spectroscopy to luminescence mode, with the lowest PMT voltage during the alignments. Adjust the mirror and laser stages so that the beam is through the middle of the cuvette for the PMT to pick up the strongest signal. After installing a dichroic mirror and an excitation filter, install a 980 nanometer laser diode in the lower tier light path.
Add a collimator in between the laser and the mirror to help diffuse and enlarge the NIR irradiating area in the focal plane. Then use an upconversion nanoparticle solution to visualize the near infrared beam. Adjust the position of laser stage to center the near infrared light beam to finish the alignment.
Perform caged-PKA synthesis and demobilize the caged-PKA/UCNP, to generate caged-PKA/UCNP as described in the text protocol. To measure the activity of both constructs before UV photolysis, measure the decrease in NADH absorbents in the assay mixture over time after the addition of two microliters of PKA solution to the assay mixture. Repeat the measurement for PKA/UCNP.
Next, perform UV photolysis at 200 milliwatts per square centimeter of caged-PKA and caged-PKA/UCNP, in an ice water bath, to make sure PKA can be activated. PKA activity is determined by a pyruvate kinase lactate dehydrogenase enzyme coupled assay. With a decrease in NADH absorbents, is measured over time after the addition of PKA solution.
Incubate the cells in 10%fetal bovine serum containing L15 medium during the microinjection period for stable pH control outside the incubator. Configure the needle puller for a cone shaped tip with a diameter of around 1.5 microns for later tip opening. For opening the tip, install the needle on the microinjector move the needle tip in contact with the edge of a slide and follow with a mild knob on the microscope stage.
This vibration force is sufficient to slightly break the tip and enlarge the opening. Examine the needle opening to ensure that it has an external diameter from 1.3 to 1.5 microns which can be confirmed by microscope images using a 40x objective lens. Next, set the injection pressure from 50 to 75 hectopascals with a pressurized interval of 200 to 300 milliseconds and set the constant compensation pressure at 15 to 30 hectopascals to prevent medium backflow into the needle.
Load the caged-PKA/UCNP complex with TAMRA dye at the back position of the needle. The loaded needle is then set up on one axis hydraulic micromanipulator, with an angle from 30 degrees to 45 degrees. Check the needle with microscope to make sure no bubble generated after sample loading.
After immersing the needle into the medium, switch to fluorescence mode, to confirm a burst release of dye when injection pressure is applied and a slow release of dye when compensation pressure was constantly applied. Then switch to phase contrasted bright field mode, to start cell microinjection. The membrane shockwave can be observed in successful injections.
Wash the cells twice and check the injected cells for co-injected TAMRA fluorescence to locate successfully microinjected cells. Cell with UCNP admission but without a co-injected dye signal, indicate the cell membrane was ruptured during injection and should be excluded from further experiments. Irradiate the cells with a 980 nanometer laser diode in the lower tier light path focused by a 10x objective lens for 15 minutes.
Allow for a five minute dark interval after every five minutes of irradiation to avoid heating. If a shorter activation time is needed, such as seconds or even subseconds, simply switch to a 40x objective lens for higher focus photon density. Following photolysis and cell recovery, fix the cells in one milliliter or 4%paraformaldehyde in PBS for 20 minutes, before further alexa 594 followed within staining.
Finally, visualize the NIR induced stress fiber disintegration. After NIR irradiation, cells microinjected with caged-PKA/UCNP as indicated by white arrows, show upconversion emission. Stress fiber disintegration caused by a successful PKA NIR photoactivation, exposes more f actin and consequently results in strong green staining near UCNP.
A negative control where the cells are microinjected but remain in the dark, show no disintegration of stress fibers. The level of f actin exposure after NIR photoactivation can be stained with alexa 594 followed within and quantified by image J.Intensity curves of f actin staining along the red arrow were plotted, providing quantitative information of stress fiber disintegration which can be correlated to NIR induced PKA activity. Once familiar with microinjection, this technique can be done within one day, including preparation and the quality control of caged-PKA/UCNP.
With our instructions for our instrument modifications and the experiments, the desired reaction can be triggered by near IR, in highly spatial temporal resolution like, traditional UV for titration methods, but without the phototoxicity, it's not contagious. After watching this video, hope you have a good understanding of how to deliver enzyme nanoparticle complex into a cell by microinjection and photoactivate the enzyme, using bell compatible near IR light. This technique paved the way for researchers in the field of chemical biology, who need to use light to trigger desired enzymatic reaction in living cells for spatial temporal resolution when the cell cannot tolerate UV phototoxicity.
Following this procedure, other bio affectors can be associated to upconversion nanoparticle, to answer additional questions that need spatial temporal photoactivation. If the bio affector works extracellularly, microinjection is not even needed. The near IR photoactivation platform can also provide more penetration depth in tissue and can therefore be applied in small animals to trigger desired reactions at the given time and the location.
