Continue hoge-resolutie Microscopische waarneming van Replicatieve Aging in Budding gist

The overall goal of this procedure is to track single haplo yeast cells during their entire replicative lifespan. The first step is to make a microfluidic chip containing an array of micro pads. Next, the yeast cells are loaded into the microfluidic chip, and because the area underneath the micro pads has a height similar to the diameter of a yeast cell, the cells will settle there.

Subsequently, a continuous flow of fresh medium is applied over the trapped yeast cells, and the emerging daughter cells are washed away by the flow of medium brightfield and fluorescence. Microscopy is used to visualize changes in cell or organelle morphology, protein expression, and protein localization that may occur in aging yeast cells. The main advantage of this technique over the classical microdissection method is that it's less labor intensive and that it enables long-term continuous microscopic images of the entire lifespan of individual yeast cells.

Though this method can provide insight into replicative aging, it can also be used for studies requiring continuous microscopic observation over prolonged periods of time. Generally, individuals new to this method will struggle because care needs to be taken at particular steps of the production of the micro feic chip. In addition, loading of the cells into the micro feic chip may require some experience.

The silicon wafer mold is produced by soft lithography. For details of its production, please refer to the accompanying protocol. Text cut a piece of tough tag into thin strips of about 0.5 millimeters by three millimeters.

With a scalpel, place the strips carefully on the silicon wafer mold slightly on top of the channel structure between the inlet channels and the micro padre. Next, cover a glass petri dish with a double layer of aluminum foil and put the wafer in the Petri dish. The aluminum foil will prevent PDMS from running in between the wafer and the Petri dish during the production of the microfluidic chips.

To begin the procedure for making microfluidic chips, place an empty plastic cup on the balance and tear the balance. Pour 40 grams of PDMS base into the plastic cup. Add PDMS curing agent in a weight to weight ratio of one to 10, which in this case is about four milliliters.

Using the disposable plastic pipette mixed thoroughly for several minutes, it is important that the mixture is well mixed to ensure proper polymerization of the PDMS later. Pour the PDMS on top of the wafer in the glass Petri dish. The PDMS mixture in the Petri dish will contain a lot of bubbles to Degas.

The PDMS place, the Petri dish in a desiccate connected to a vacuum pump for about 30 minutes when all bubbles have been removed. Polymerize the PDMS by placing the Petri dish with the wafer on top of a hot plate at 120 degrees Celsius for one hour, followed by 65 degrees Celsius for another hour. After the two hours, remove the aluminum foil wafer and polymerized PDMS from the glass Petri dish.

Peel off the aluminum foil into PDMS from the back of the wafer. Then carefully lift the layer of PDMS from the top of the wafer. Place the PDMS layer upside down with the channels facing up a the bench and carefully cut out the single chip designs imprinted on the PDMS with the scalpel.

Try to retain about three millimeters of PDMS around the edges of the channels. The next step is to punch holes in the PDMS at the ends of the inlet outlet and side channel as illustrated in this diagram. To punch a hole, push a 20 gauge lure stub all the way through the PDMS in a straight manner.

Remove the column of PDMS in the lure stub before pulling the stub out. Again, this prevents the PDMS column from blocking the newly punched hole. Once all the holes have been punched, clean the surfaces of the chip from dust particles and residual PDMS by placing scotch tape on it and immediately removing the tape.

Similarly, clean the cover glass to which the chip will be attached. Place the chip and cover glass below the UV lamp of a benchtop UV ozone cleaner with the sides that need to be bonded together, facing the lamp exposed to UV light for six to eight minutes, and put the exposed surfaces on top of each other. Gently tap around the sides of the chip to promote attachment of the PDMS to the cover glass, and to remove any air bubbles.

Do not tap on top of the channel structure as this may cause the micro pads to get attached to the cover. Glass as well. Place the newly made microfluidic setup on a hot plate at 100 degrees Celsius for one hour.

Afterwards, check the bonding of the cover glass to the PDMS chip by slightly lifting up the edges of the PDMS chip. If this is not possible, the bonding is successful and the chip is ready for use. To prepare the microfluidic chip for cell loading, place the chip in the metal holder with silicone gel between the glass of the chip and the metal parts of the holder.

To create a waterproof seal, screw the nuts gently to avoid breaking the glass. Connect thin tubes to the side channel and the outlet channel of the chip. The use of tweezers makes it easier to insert the tubing in the punched holes.

Fill a 50 milliliter lure lock syringe with culture medium. Remove the air from the syringe and connect it sequentially to a syringe. Filter a 20 gauge lure stub, a short, thick tube and a thin tube.

Place the syringe in the syringe pump and fast forward the pump until the thin tube is completely filled with medium. Push the thin tube connected to the syringe into the inlet channel of the and let the medium flow through the chip at a rate of 10 microliters per minute. Collect the medium leaving the chip in a Petri dish.

The medium will run out via the side channel. Because of the resistance difference between the large tough tag made side channel and the outlet channel. Place the chip on the microscope stage and set the focus of the microscope on the pads.

To begin this procedure, connect to five milliliter lure tip syringe to a 20 gauge lure stub and a thick tube. Load the syringe with approximately one milliliter of the cell suspension to be loaded into the chip. The preferred cell count for loading is between one and five times 10 of the six cells per milliliter.

Decrease the flow rate of the syringe pump to 0.5 microliters per minute. The most difficult part of this procedure is the loading of the cells into the microme chip. This usually requires some practice.

Connect the syringe containing the cell suspension to the tube of the outlet channel. Load the cells by pressing on the plunger of the syringe. Gently observe the cells coming in and settling underneath the micro pads via the ocular or the computer screen.

Maintain pressure on the plunger until sufficient cells settle underneath the pads. The optimum load is one to three cells per pad. Disconnect the syringe used for cell loading and flush the side channel for a couple of minutes at higher flow rates to remove air bubbles and or cells that did not yet exit the chip.

Decrease the flow of the syringe pump to 0.5 microliters per minute again, and close the side channel off by connecting it to a thick tube containing a catheter plug at its end. Increase the flow of the syringe pump to a final flow rate of one to five microliters per minute. Flow rates may vary depending on the medium and yeast strain.

Start a movie with the microscope and camera setting suitable for the goal of the experiment. This is an example time-lapse movie of a single wild type E cell growing on YPD. Medium images were taken every 10 minutes and the scale bar just shown represents five micrometers.

The cell produces a total of 30 buds before it dies. Replicative lifespan can be determined by counting the number of buds produced by a single mother cell. This data is transformed into a lifespan curve by plotting the percentage of viable cells against the number of buds produced or the number of generations.

This figure shows an example of lifespan curves obtained for a wild type yeast strain and two mutant strains. Deletion of the SER two gene results in a shorter lifespan while deletion of the FOB one gene results in a longer lifespan. Mitochondrial morphology as a function of age can be studied using the microfluidic device.

An aging experiment was performed with BY 7 42 wild type yeast cells expressing ILV 3G FP, which is targeted to the mitochondria. In this figure, A representative example of age associated changes in mitochondrial morphology in a single cell is indicated by the white arrow. All images are scaled identically and the scale bar represents five micrometers.

The second time lapse movie is of a single cell expressing ILV 3G FP from the experiment where mitochondrial morphology as a function of age was observed. Images were taken every 30 minutes and the scale bar represents five micrometers. This final figure shows the mitochondrial morphologies observed in the same set of cells at different replicative ages before producing their first bud after 10 buds and prior to death.

An example image of each morphology class is included tubular, fragmented tubular, and spots and large spots. The morphology changes seen in mid aged cells resemble those reported previously By continuously monitoring cells as they divide. This method can help answer key questions in yeast replicative aging, such as why does a yeast cell age, what phenotypic changes are taking place, and how do these influence the replicative lifespan of the yeast?

After watching this video, you should know how to create your own micro chips and study replicative aging in cells using basically any fluorescent microscope.