Apr 22, 2020
Nanosight LM10
- 1Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Protocol Citation: Joshua A Welsh, Bryce Killingsworth, Jason Savage, Tim Traynor, Jennifer Jones 2020. Nanosight LM10. protocols.io https://dx.doi.org/10.17504/protocols.io.be58jg9w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 15, 2020
Last Modified: April 22, 2020
Protocol Integer ID: 35744
Keywords: extracellular vesicles, EVs, exosomes, nanoparticle tracking analysis, NTA, nanosight, malvern, LM10,
Abstract
This is a protocol for performing nanoparticle tracking analysis measurements on the Translational Nanobiology Sections Nanosight LM10 (405 nm) module.
Guidelines
The optical flat of the instrument is very sensitive, make sure to only wipe it with lens paper and dry using compressed air
Materials
MATERIALS
DPBSInvitrogen - Thermo FisherCatalog #14190
Lens Paper, 4L x 6 in. W (10.1 x 15.2cm); Sheets per book: 50Thermo FisherCatalog #11996
Covidien Monojectâ„¢ Rigid Pack 12mL SyringesFisher ScientificCatalog #22-652-090
Air-Titeâ„¢ All-Plastic Norm-Jectâ„¢ 1 mL SyringesFisher ScientificCatalog #14-817-25
Micro-90® Concentrated Alkaline Cleaning SolutionInternational Products Corporation (IPCOL)Catalog #M-9050-12
Safety warnings
Do not turn the laser on when the metal plate covering the optical flat is removed
Before start
Gather PBS, an empty waste container, 12 mL syringes, and 2 mL low protein-binding Eppendorf tubes
Equipment Start Up
Equipment Start Up
15m
15m
Ensure that the black power strip is turned on. (Specific to Translational Nanobiology Section's lab)
Turn on the computer on the shelf, login using the password written above screen (Specific to Translational Nanobiology Section's lab)
Turn on the Nanosight microscope, you should hear the camera fan turn on
Remove the storage lens paper which is positioned between the stainless steel cover and the optical flat of the LM10 module
Inspect the optical flat of the module and window in the steel cover to see if there are any obvious smudges which require immediate cleaning
If immediate cleaning is necessary:
Examine translucent window component of steel cover to see if there are any particles or smudges. The outside part of the window shouldn't need to be lceaned of smudges but may have dust and link which can be blown off using a can of compressed air
The inside of the window can be cleaned by blowing off the dust and pouring a small amount of 1% MICRO 0- into the cap of a 50 mL conical and dipping the end of a folded piece of lens paper into it. Gently clean the surface, and repeat this same procedure using a new piece of lens paper dipped into DPBS. Dry the window with a piece of lens paper and set the steel cover aside.
The optical flat can be cleaned with 1% MICRO 90. Pour it into the cap of a 50 mL conical and dip a piece of folded lens paper into it. Wipe the optical flat, focusing on the area of the optical flat that contains a translucent rectangle. PRess firmly perpendicularly to the edge of the optical flat and parallely, not in circles.
Quickly place the steel cover back onto the module and screw it in, tightening the screws in a diagonal pattern and ensuring not to over-tighten
Quickly load 10 mL of DPBS into a 12 mL syringe
Flush the LM10 module with DPBS by inserting the syringe into the white plastic port on the side of the module. One side of the stainless steel has the entry port, while the other side has the exit port. The exit port is directly adjacent to another dark metal port while the entry port is the only port on its side. Hold the module as vertically as possible and slowly inject DPBS, ensuring that no bubbles form on the viewing window
Once liquid can be seen filling up the fluid exit port, flip the Nanosight module so that the syringe is standing straight up and slowly press on the plunger until all of the DPBS has passed through the module and into a waste container
If bubbles are formed, DO NOT REVERSE THE SYRINGE to remove the DPBS, but rather push it all the way through
Once the syringe is empty, return the Nanosight module to its original vertical position
Draw out the remaining DPBS that remains int he viewing window of the Nanosight slowly, ensuring that minimum fluid is left behind
Discard the DPBS in the syringe in a waster container
Draw up another 6 mL of DPBS into the syringe. If there were bubbles present in the initial flush, push the entire volume through and add another 6 mL of DPBS to the syringe
Flush the NanoSight with ~5 mL of DPBS, leaving less than 1 mL remaining in the syringe
Slide the LM10 module into the grooved slot on the microscope table
Make sure the 20X lens is in use on the microscope
Open up the most recent version of the NanoSight software (3.4 as of January 2020)
Turn the camera on inside the NTA software by clicking "Start camera"
Adjust the settings to those optimal for the instrument. These settings are subjective depending upon instrument parameters such as the wavelength and module alignment, along with the sample.
A guide for the 405 nm LM10 module (Specific to Translational Nanobiology Section's lab)
Capture -
Screen Gain: 1
Camera Level: 14
Process -
Screen Gain: 1
Detection Threshold: 4
Advanced -
Uncheck all "AUTO" boxes
Min Track Length: 8
Plug the power source into the side of the LM10 module
Turn on the laser by flipping the silver switch on the right side of the LM10 module
Use the microscope focusing knobs/stage mover to ensure that you are in the correct viewing window.
Make sure that the "thumb-print" is centered in the microscope window. The thumb will look like a large, purple oval that takes up the majority of the screen
Once centered using the thumb-print, move the screen to the right until you see a large, vertical line. The viewing frame for sample analysis should be as close to this line as possible without having the line being in the actual viewing window (position the viewing frame immediately to the right of the line)
If the line is not vertical, the camera module on top of the microscope may need to be slightly rotated
With DPBS loaded, ensure that there are no particles remaining in the NanoSight from the previous samples
If there are particles, turn off the laser and disconnect the power source. Get a new aliquot of PBS and a new syringe (at the user's discretion) and repeat the flushing process
If there are large, highly fluorescent smudges, or significant amounts of white backgroup noise, turn off the laser and disconnect the power source. Clean the optical flat and viewing window and flush the module as described in steps 6-15.
If the NanoSight is clear, turn off the laser and pull the remaining PBS out with the syringe, ensuring to go slow and leave behind as little fluid as possible
Sample Analysis
Sample Analysis
10m
10m
Dilute samples as needed in a 2 mL low protein-binding Eppendorf tube
Vortex samples thoroughly
Draw up sample in 1 mL syringe, pulling down the syringe plunger to remove any bubbles
Load sample into the LM10 module through the fluid entry port
Turn on the laser
Ensure the NanoSight is in the correct viewing window, adjust as necessary
Ensure that the sample is in the correct viewing window and properly focused
Ensure the sample is not too dilute or concentrated
It is too dilute when there are too few (<10) particles on screen at any given time
It is too concentrated when there is significant fluorescent background or particles cannot be distinguished from one another
To adjust settings, press:
SOP -
1. Standard Measurement
2. Number of Captures: 3
3. Capture Duration(s): 30
4. Current temperature: Measure using thermometer on table
To begin recording, press "Create and run script." Enter the sample name and the dilution amount, then hit "OK" on the windows that pop up
When the fan turns off, the instrument is recording. Ensure that nothing bumps, vibrates, or moves the table while the recording is taking place and refrain from leaning over the table.
After each 30-second capture, you will be prompted to advance the sample and press "OK." Do this by carefully pressing the plunger in until you see the particles on the screen have moved.
At the end of the 3 captures, allow the software to automatically analyze the data
As soon as the data analysis begins, turn the laser off
When data analysis is complete, hit "Export" on the box that pops up
If you need to measure another sample:
Flush the device with at least 2x12 mL syringes of DPBS (they need not be entirely full, technique is more important than quantity of DPBS flushed through)
Check the viewing device to ensure that the viewing frame is particle free and does not have smudges or excessive background noise
IMPORTANT NOTE: if there are particles remaining, the module was flushed insufficiently and will need to be opened and cleaned as described in steps 6-15
Device Shutdown
Device Shutdown
15m
15m
After you've finished with all of your samples, the device must be opened and cleaned as described in step 6 with the following changes:
After wiping with MICRO 90, obtain another piece of lens paper and gently wet it with pure water, then wipe down the optical flat. Use another piece of dry lens paper to wipe off the water.
ALL COMPONENTS of the module which may have come into contact with DPBS must be wiped gently with a Kim wipe or a lens paper wetted with pure water, then dried gently with compressed air. This includes the sides of the module, especially the corners and edges where DPBS can pool and crystalize
Water should NOT be poured or pipetted onto the LM10 module as it could enter the electronic components and cause damage. Only use a wetted piece of lens paper if you need to apply fluid to the module
Place a piece of folded, dry lens paper between the optical flat and steel cover plate
Ensure that all power sources are turned off
File Export
File Export
5m
5m
To transfer data to a flash drive, click:
1. This PC
2. J'D (H:)
3. Nanosight files
4. Nanovideos
5. Date of recording
(Specic to 'Translational Nanobiology Section's lab'