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TwoMP Autofocus Failure: Causes and Troubleshooting
What are the common reasons why the TwoMP fails to find focus in droplet-dilution mode, and how can I prevent this?
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Refractive Index affected by salts
When I look at samples with elevated salt concentrations, like MgCl2 for example, I see what looks like TV static in the background. I see from a quick search that the salt is likely affecting the landing behavior and the refractive index contrast.
I'm curious if anyone has done a study of different salts at different concentrations that I could look to for guidance when doing sample prep? I don't want to burn through the consumables on something like that.
Thanks in advance for any help or insights!
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Austin Graves
Sep 16
Hi Mike,
Is your buffer just MgCl2 or does is it added to PBS? We have observed background noise coming from magnesium but this often occurs when the MgCl2 buffer is prepared in PBS.
At the moment we do not have a document that outlines the possible effects of different buffer components but I will certainly carry that feedback back to the team. Thanks!
Austin G.
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How necessary is a vibration table?
We originally purchased our OneMP with a vibration table (Everstill K-400) and have always used the table during measurements. Over the past couple weeks, though, I’ve tested making measurements with the table off and have noticed no difference in the recordings. In fact, I was able to obtain an accurate reading of a 40-50 kDa sample quite well (an exceedingly clean glass and PBS blank helped). Does the apparent lack of impact of the table on measurement accuracy simply mean that the bench where our device rests is solid enough to not warrant a table?
I should also note that, when the table is on, I always notice that even light taps to the work surface are observable in the blue trace in the radial profile monitor, making me suspect the table’s antivibration efficacy. But I later understood this to be normal as the table is intended only to…
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Amy Chau
Aug 27
Hi Chase,
If turning off the anti-vibration table doesn't affect the focus position fluctuation, it means your bench is very solid! You're correct that the anti-vibration table only counteracts building vibrations or low frequencies from nearby equipment. We still wouldn't recommend leaning against the bench, or tapping the table as these actions introduce vibrations that can't be isolated by the anti-vibration table.
Amy
Sr. FAS
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Are gaps in the focus ring really a problem?
At the initial training for our OneMP, I was told that gaps in the focus ring due to air bubbles in the objective oil were detrimental and should be removed prior to a measurement by lifting then resettling the coverslip. I recently made a measurement with gaps in the ring and it appeared to be fine. Can you explain how gaps in the focus ring affect measuring accuracy?
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Amy Chau
Aug 27
Hi Chase,
Bubbles have different refractive index than oil, which causes light to bend differently than intended. This results in less light reaching to the particles of interest and reducing the amount of scattering from the particles. Since we capture the interference between particle scattering and background reflection, less scattering from particle caused by oil would reduce the interference, thus lower contrast and mass.
If it's a small gap in the autofocus ring, it's likely not a huge mass shift. If the gap gets wider (more bubble), you will likely see a bigger shift. A simple test you can do is to measure the sample before and after introducing bubble to the oil. Move some oil to a microcentrifuge tube and shake it to add bubbles.
If you noticed your measurements tend to create gaps in the autofocus ring, you can heat the oil at 60 C for 2-8 hours and then cool to room temperature.
Amy
Sr. FAS
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What increases signal on apparently clean glass?
Over the years working with self-cleaned coverslips, I’ve noticed that when finding focus on a PBS blank, I can obtain a baseline signal value as low as 0.05 and up to over 0.10 on the same slide, even though the FOV in all wells appears to be over clean glass in the native view. This occurs in the absence of any obvious artifact, such as diagonal lines as from well leakage. In the corresponding ratiometric view, the FOV simply appears noisier at the higher signal values. As I am using the same PBS for all wells, I have to believe glass itself is causing the elevated signal, even in the absence of an obvious contaminant. Can you explain how the glass itself, in the absence of unwanted particles in solution or on the glass surface, can elevate background signal? What about the glass might be doing this?
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Benjamin CAPPE
Aug 31
Dear Chase,
I’m Benjamin Cappe, Product Manager at Refeyn.
Thanks for the question.
What you're seeing is most likely due to subtle variations at the glass-liquid interface rather than visible contamination. Mass photometry is highly sensitive, and even when a field of view appears clean in the native image, microscopic differences in glass surface properties, batch-to-batch variation, residual contamination, surface roughness, or other locally adsorbed material can increase the background signal and make the ratiometric image appear noisier.
When using self-cleaned coverslips from third parties, it can be difficult to pinpoint the exact source, as factors such as glass batch variability, cleaning procedures, handling, buffers, and plasticware can all contribute to background noise.
This is one reason we developed MassGlass™ UC. These slides are specifically selected by our team, cleaned, and quality-controlled to provide a more consistent glass surface for mass photometry measurements, thereby reducing variability between experiments.
If you'd like to discuss your specific observations in more detail, feel free to contact our support team at support@refeyn.com. We'd be happy to review the data and help troubleshoot.
Best regards,
Benjamin Cappe, Product Manager
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Is subtracting negative mass events from the positive histogram a valid baseline correction in mass photometry?
Hi everyone,
I was recently suggested to perform baseline correction by subtracting the negative mass events from the positive histogram (or using the negative side to correct the positive side) before interpreting the protein peaks.
Is this an accepted approach in mass photometry data analysis, or should the negative mass events simply be considered background while interpreting only the positive protein peaks?
Has this type of baseline subtraction ever been used or validated in any published MP study? If so, could you please point me to the relevant reference?
I'd appreciate any guidance on the recommended best practice. Thanks!
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Bhaskar Barnwal
Aug 04
Dear Sathya
Thank you for your question. My name is Bhaskar Barnwal and I am the senior field application scientist for Refeyn based out of Singapore.
As the molecules land on the glass coverslip (binding event), it scatters light, creating a contrast—or dark spot (negative contrast)—on the imaging sensor. The intensity of this contrast directly correlates with the mass of the particle: the darker the spot, the higher the particle’s mass. Conversely, when a previously bound molecule detaches and diffuses back into the solution (unbinding event), it appears as a white spot (positive contrast) that is registered as a negative mass on the -X axis. Most of the proteins will bind well to the coverslip and you will see fewer unbinding events in comparison to the binding events unless you run samples like apoferritin which show significant unbinding. To answer your question, in short, we do not do any kind of blank or baseline correction by subtracting negative mass events from positive side. The histogram data (mass vs counts) is directly taken from DiscoverMP software and can be presented or published.
Before running your protein samples, it is always recommended to run your buffer/additives to ensure there is little to no noise due to your buffers. Here, the unbinding data helps us interpret the noise from the buffer and additives. When you run a buffer/additive then you might see the mirror-image peak – with equivalent, ‘negative’ apparent mass and the same height – as you see in picture below in case of PBS (low-count, low-mass noise). If you get higher noise due to the additives then you can run your samples in it provided the mass of your protein sample is higher and is not affected by the noise as you can see in picture below (mirror-image noise due to buffer and a protein peak at 160 kDa). If there is any interference due to additive, you need to dilute your sample in buffer without additive to suppress the noise.
We have a very nice application note on detergents which will give you more information about mirror-image peaks and noise due to different detergents. If you don’t mind, you can provide email id so I can forward you that for your reference and can guide you better on your experiments. You can always reach out to me at bhaskar.barnwal@refeyn.com. Please let us know if we can help you further. Thanks!
Regards
Bhaskar
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Modification of imaging conditions
Hi!
I am trying to reproduce the imaging conditions found in the following paper: Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics
Briefly: "The following settings have proven to work for MSPT on a commercial mass photometer (see Table of Materials): FOV of 128 pixels x 35 pixels, a frame rate of 1 kHz resulting in roughly 200 frames per second after subsequent 5-fold frame averaging, and an exposure time of 0.95 ms." ... "The movie duration can be set in advance and depends on the type of experiment. In most cases, an acquisition time between 5 min and 7 min is recommended."
We are using a OneMP with the latest software and I am having some difficulty modifying the frame rate/exposure time/acquisition time such that in one recording they are equivalent to those mentioned (ie. frame rate: 1 kHz; exposure time 0.95 ms; acquisition time 5-7 min). Is this…
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Team Refeyn
Mar 05
Hi Cecilia,
Mohammed is our support person for software.
You can modify the Photometer Settings from the Settings menu, at the top left of AcquireMP; here you have the field of view, frame rate and frame averaging – check the user manual for further details. You might need to activate Advanced Mode first, from the Preferences menu. If you are trying to reproduce the results of the paper you mentioned, I would also recommend you unselect Compress Movies from the Preferences menu, to enable custom movie analysis with your own scripts. As for the recording time, you can change the number of seconds from the text box right below the movie, in AcquireMP’s main window.
The settings described should be achievable with your setup, but do not hesitate to contact our support@refeyn.com service if needed.
Kind regards,
Refeyn Team
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Mass Photometry on IDPs
Hello, I am starting a new project with a protein that has extensive intrinsically unstructured regions. What are the experiences of the community with this? I am searching for good references. I know it should be possible, but I am struggling to find literature references where authors did this. Thank you.
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Amy Chau
Feb 27
HI Annette,
You can use the Feature Publication search (Resources: Publications - Refeyn) to look up keywords on proteins with IDR. Here are some preliminary search from using the tool:
Happy to help if you have more questions
Amy Chau
Sr. Field Applications Scientist
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Hi Ying,
There are a couple of possible reasons why this could be happening but we would need more information to be able to diagnose it. Please reach out to support@refeyn.com with your issue and we can connect you with your local representative who can help figure out what's going on.