Hi, I'm Shamal, a postdoctoral fellow with TUR Kika at National Center for Biological Sciences India. In this video I'm going to show you how to fabricate a bio their micro device, using soft graphy techniques In a regular lab environment. I'm going to use these devices for high resolution, cellular and subcellular imaging of Genetic model organisms.
The current protocol will allow researchers to perform this time lapse imaging in anesthetic free intact organisms. So let's get started. We have developed simple microphonic device to perform anesthetic free in vivo imaging of genetic model organisms such as sea elegance, intact oph and zebra fish lab.
In this study, we demonstrate a membrane based clear mobilization device fabricated using of and soft molding of PDMS. In order to immobilize single organism, we locate individual animal under a stereo microscope and pick the animal using a small drop of buffer solution using a micropipet, insert the individual animal into the flow channel and immobilize it under the PTMS membrane. Using a pressurized nitrogen gas connection through a three-way ball.
Design the micro fredic structures using cleaning software. Clean two centimeter by two centimeter silicon wafers in 20%KH for one minute and rinse di water. Blow dry the pieces with nitrogen gas and dehydrate at 1 22 Celsius for four hours.
Spinco hexa metal on silicon pieces at 500 RPM for five seconds and 3000 RPM for 30 seconds. Spinco SU 8 2 0 2 5 on silicon wafers at 500 RPM for five seconds and 2000 RPM for 30 seconds. Soft bake the coated silicon pieces at 65 degrees Celsius for one minute and 95 degrees Celsius for 10 minutes.
Expose the baked SU eight coated wafers to 200 watt UV lamp through the photo mask for design one and design two for a flow layer and control layer respectively. Post bake the wafers at 65 degrees Celsius for one minute and 95 degrees Celsius for 10 minutes. Develop the pieces using SUA developer solution for 20 minutes.
Code the pieces with tri chloro per fluoro CET seline vapor in a desiccate for two hours. Mix the poly dyl xox base and curing agent in 10 is to one ratio by weight. Degas the mixture to remove all air bubbles from during mixing.
Pour a five millimeter thick PDMS mixture over silicon wafers with SC weight pattern of design. Two gently to avoid formation of air bubbles. Spin coat and uniform layer of PDMS on silicon wafers with design one for the flow layer at 500 RPM for five seconds and thousand 500 RPM for 30 seconds.
Bake both pieces of design one and design two containing PDMS at 50 degrees Celsius for six hours. Cut out the PDMS piece from silicon substrate using a sub blade containing SCH pattern two. Punch a small hole on top of the reservoir connecting the main trap in the PDMS mold.
Using a highest puncture or 18 gauge needle. Expose the bottom surface of the control layer and the top surface of the flow layer to 18 wat air plasma for two minutes. Place the two plasma treated surfaces together with gentle pressure and bake them at 50 degrees Celsius for two hours.
Cut out the bonded devices from the silicon substrate with SUH design one and punch access holes at the inlet and outlet reservers of the flow channel. Expose the bottom PD on of the device and a clean glass cover. Sleep to 18 volt air plasma for two minutes.
Place the two plasma treated surfaces with gentle pressure and bake at 50 degree surface for two hours. Store the device in a clean environment for future usage After the fabrication of the devices, I'm going to show you how to use PDMS membrane to immobilize c elegance into the device. Connect one end of a microflex tube to a pressurized nitrogen gas supply regulator and other end of the main trap reservoir through an 18 gauge needle.
A three-way stop cock used in the middle of the tube connection allows application or release of the pressure on the membrane. Fill the tube and connecting to the PDMS device with a 10 centimeter column of distilled water before connecting it to the reserv of the main trap. Turn the valve of the nitrogen supply to read protein PSI and monitor the membrane of the main trap deflecting towards the flow channel at low magnification of an inverted microscope.
So release the three way stopcock world and put the membrane in the resting position. Fill the flow channel with buffer for 10 minutes. Before an experiment, locate a single sea elegance using a lone magnification studio microscope.
Pick a single animal using a small volume of buffer solution into a micro tip. Push the single one through the flow channel inlet. Adjust the pipetting pressure to position the individual animal under the main trap.
Increase the pressure of the PDMS membrane slowly to position the animal against the channel boundary and immobilize it with seven to 14 PSI pressure. Use an inverted microscope to desired setting for high resolution right fill or fluorescence imaging. Acquire single or timed up fluorescence images at predefined frame rate real.
Release the trap pressure and monitor the locomotory behavior of the animal. For five minute at low magnification, flush the animal and insert a fresh animal. To repeat DevOp steps, use the cut micropipet tip to pick single oph larva and insert it into the larger size micro ferric device.
Push the single larva through the flow channel inlet. Increase the pressure of the PDMS membrane slowly and immobilize it with seven ESI pressure. Use an inverted microscope for fluorescence imaging at low magnification, acquire time lapse fluorescence images for transport imaging.
Release the trap pressure and monitor the locomotive behavior of the larva at low magnification. Use the same device principle to immobilize and acquire time lapse images of 30 hour post fertilized zero fish. Val heart.
After watching this video, you should be able to fabricate similar micro devices in your own laboratory and use them successfully for immobilizing organisms for high resolution amazing experiments. Thanks for watching the video and good luck for your experiments.
