Mass Photometry

Question 1

What is mass photometry

Answer 1

Measures binding to a surface

refractive index at the surface changes when molcule bind to it

Question 2

Explain the incident light scattered light and reflected light for mass photometry

Answer 2

The incedent light comes from the bottom to the top

When molecules bind near the surface, they scatter the light

The incedent light is reflected from that surface as reflected light

Question 3

In a mass photometry experiment what does the resulting image look like

Answer 3

Shows molecules that bound to the surface as darker

The more mass, the more contrast (more mass not more size) meaning a tetramer shows more than the same sized dimer

Question 4

What type of technique is mass photometry

Answer 4

A single molecule technique

Shows individual binding of single molcules and collects each binding event the separated them based on their contrast (more contrast more mass)

Question 5

Explain the scatter plot for a mass photometry

Answer 5

Shows the peaks from higher to lower contrast and how many particles are making those peaks

The number of particles (concentration) goes down overtime because more protiens have come out of solution and already bound to the coverslip

This is why less particle show overtime because already bound

Question 6

Explain the mass photometry setup

Answer 6

Has the glass coverslip at the top with the molecule on the coverslip causing scattering and reflection

The laser shines light past the WFL to the The partial reflector (PR) and lets the light go up past the BFP of the obejective lens to hit the coverslip

The reflected light goes down and it attenuated by a mask on the PR and it goes past another lens (L) and collected by a camera , the mask removes the direct reflected light to only collect the high constant scattered light

The camera has to refresh fast enough to take videos of the binding events

Question 7

How what are the problems with measuring in mass photometry and how can the signal be improved

Answer 7

Need to differentiate between incedent light that interacts with the object and the background light

Also the contrast is very small for a single molecule and we really need to suppress the noise to actually measure a signal

so we increase the contrast by measuring the reflection (measuringin diff direction the incedent light) rather than the transmission (directly across incedent light)

Need to detect a high 10^8 photons to get a signal to noise good signal to noise ratio, but digital cameras detect only 10^5 (so there needs to be a better way

Question 8

What is special about molecules near the coverslip of the machine

Answer 8

They radiate the most photons into the machine at wide angles due to scattering, this is why the scattering give the most contrast

Question 9

Explain the mask in mass photometry

Answer 9

Masks the angles of light that are sent down by the reflected light to reduce the amount of reflected light sent to the camera

The scattered light goes at wider angles and so that would be collected

Basically separated the scattered light from the reflected light to increase the constrast since scattered light gives more contrast

Question 10

What are the steps to the mass photometry experiment

Answer 10

Prepare instrument and materials

Aquire data

Analyze data

Question 11

How do you Prepare instrument and materials for mass photometry

Answer 11

Warm up the instrument

Cleans the coverslips with water,ethanol etc.)

Find the good side of the coverslip

Filter the protiens solutions or centrifuge them

Get small volume of protiens and find the concentrations

Question 12

How do you acquire data from mass photometry

Answer 12

Add immersion oil to the microscope then the coverslip and you small volume of buffer

Load the protiens sample

Set the acquisition time to set how long each video frame of the landing event would be

Monitor the landing events,you want to have the landing events separated in space, not on top of each other

Question 13

Why is the concentration of sample so low in mass photometry

Answer 13

you want to have the landing events separated in space, not on top of each other, so low concentration with do this

Question 14

How do you analyze the data from mass photometry

Answer 14

Adjust the sensitivity to pick out each of the landing events

Look through the images to see if any need to thrown out due to artifacts

To remove noisy images you can add spatial and temporal masks (like something weird happens at a specific time so you just cut the time off)

Convert the contrast values to molecular weights using standards every series of measurements

Plot the data with histograms and adjust the histogram bin width (to show the peaks well in the histogram)

Fit the histogram to the Gaussian functions

Question 15

Why do we fit the histograms with a Gaussian function

Answer 15

Because the histograms look noisy

Also to find the amplitude, width, and where the peaks are potisioned

Question 16

How can you find the mass of the particles based on their constant

Answer 16

Use the molecular ladder standard curve

Y=MX+B

MW=m (contrast) + B

On sheet

Question 17

Whag are the limitations of mass photometry

Answer 17

There is a small MW range that is detects, because molecule has to be small enough to have scattering

Have to use a narrow range of protien concentrations because of high have overlapping landing events

• This is why is hard to measure Kd, since to make those measurement you need high concentrations

-Also lose a lot of sample from its binding to lookers tips because concentration is already small

Has no detection specificity so any impurites in the sample that scatter light are also measured

M

Question 18

Advantages of mass photometry

Answer 18

Single molecule techniques

On sheet

Question 19

Explain the antigen antibody complex example for mass photometry

Answer 19

Bivalent antibody so it can bind two antigens at once

If just free antigen, only see one peak

If antigen