Refeyn Mass Photometer
This technique is based on iSCAT that uses the scattering and interferometry of light against single molecules (biomoleucules) that are correlated with the size of said molecule via a standard curve.
Manufacturers site: Refeyn - Measuring molecules with light
Version 1 - 12.09.22
Maintain Room temp to have the most consistent measurements without recalibration. Check the Temperature meters. The room should be around 20-25C with little fluctuations.
Calibrate every 1 hour for best results
This covers data acquisition
Warm UP
1) Turn on Power Supply (aka Laurelin) the switch is at the back.
2) Let warm up for 30-45 minutes
3) Turn on Vibration stabilizer (push the button for isolation, a blue light should come on)
4) Turn on the software Acquire MP and Discover MP. This is the data acquisition software. When the software is on, the device will turn on and reset the stage.
Preclean (Ask if unsure)
1) Remove dust from the work area.
2) Clean lens with properly folded lens paper (DO NOT USE KIMWIPES!!! WHILE CLEANING DO NOT TOUCH THE OBJECTIVE WITH ANYTHING BUT LENS PAPER)
Prepare a slide
1) Remove plastic on one side of a 6-well gasket
2) Place in center of the Refeyn slide holder
3) Remove plastic on other side of the gasket.
4) Place a precleaned, Refeyn slide on top the gasket and align.
5) Press gently on the interstitial spaces to let the gasket stick to the slide.
Oil Objective (Ask if unsure)
1) Swirl the wand inside the oil.
2) Remove the wand and let the first drop fall
3) Turn the wand horizontal. The oil should not form a drop.
4) Hover above over the center of the objective. DO NOT TOUCH OBJECTIVE
5) Slowly rotate the wand where now a corner is pointing towards the center of the objective.
6) Let the oil droplet form and let it drop.
Alignment and Focussing and Acquisition
1) Flip the prepared gasketted slide on to the objective.
2) Apply the two magnets to the top left and bottom right corners to hold into place.
3) Using the 'Lateral Control' settings, bottom right of screen. Move the laser light close to the center of the first drop at 50X. The machine will make a slight pitched sound. If it is TOO highly pitched, STOP.
4) Add buffer, 18 uL, into your first gasket.
5) Turn to 'Native' view.
6) Go to 'Focus Control', also bottom right. Then 'Droplet dilution'
7) Check for dust
8) Go to 'Ratiometric' view, top left.
9) Add 2 uL of 100 nM (starting recommended concentration) of target protein, to the 18 uL buffer. Mix with pipette gently. Dont touch anything other than droplet.
10) Press record. It will record a movie for 60seconds.
11) Open the movie in the Discover MP software.
12) Further samples 2-6 on the same slide, repeat Steps 3-10.
13) After all the gaskets have been used up, one MUST remove excess oil from the objective (ask if unsure), then start anew from 'Prepare a Slide' (above)
Clean up
1) When you are done for the day, cleaning up is critical. This removes all parts and oil from the objective.
2) Using a piece of LENS paper, slide it from below the stage and gently with wicking action, remove excess oil.
3) Using Lens paper, fold a piece of lens paper, preferably with tweezers, into the appropriate size. Dip the lens paper into isopropanol. Shake off excess.
4) In circles, starting from the center, gently clean the objective until you reach the edge
5) Repeat 3)
6) Once again, starting in circles move outwards. This time, in places where there appears to have oil, apply more pressure on the lens tissue to remove oil. DO NOT TOUCH ANYTHING WITH TWEEZERS DIRECTLY. DO NOT GO BACKWARDS TOWARDS CENTER.
7) You may may have to repeat this one more time (2-3x with isopropanol in total)
8) Let Isopropanol dry, Close the cover to protect from dust.
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