NetNotes
the phase contrast trick described above, but instead close the aperture stop and you will again be able to see the diffracted light separated from the non-diffracted light in the focal plane. Te problem with poorly diffracting objects is two-fold, 1) very little light gets diffracted leaving little light for interference, and 2) the phase shiſt of the diffracted light is very small. Te end result being that samples like adherent cells only cause a very small decrease in intensity, and since we perceive light on a logarithmic scale, this is very hard for us to see. Terefore, phase contrast fixes both of these issues. One way (which you can see by looking at the focal planes) is that it uses a cone of light as the illumination pattern (due to the annulus at the front focal plane), and this cone perfectly lines up with an annular neutral density filter (the phase plate) at the back focal plane. Tis ring illumination is a clever way to spatially separate the diffracted light from the undiffracted light at the focal plane, allowing us to attenuate the undiffracted light without impacting the diffracted light, balancing the amount of diffracted and undiffracted light. Why use a ring to do this instead of, say, an aperture stop? Te ring illumination simply preserves more of the illumination NA and therefore preserves more resolution. However, the phase plate doesn’t stop there, as its name suggests,
it also enhances the phase difference between the diffracted and undiffracted light (ideally such that
the highest diffractive orders
are phase shiſted to 180°) such that the sharpest edges get perfect destructive interference. Now at this point, you may be asking yourself, how do they know how much to attenuate the undiffracted light (i.e., how dark to make the annular ND filter) and how much of a phase shiſt to impose, as some samples may be more diffractive than others. And this is why there are actually many different phase contrast objectives with different phase plates. In life sciences, most of the time we’re only using phase contrast to check cell culture, so we wind up dealing with just one phase plate optimized for that task. Hope this helps, and I really do recommend checking it out on your own microscope. I’ve found with students, looking at the correlation between the focal plane and image plane with and without a sample can really inspire that “aha” moment. Ben Smith
benjamin.smith@
berkeley.edu
SEM Filament Vibration Microscopy Listserver Looking for feedback from the EM community. It was recently
suggested to me that what appears to be wave-like image distortion normally attributed to electromagnetic interference (EMI) could actually be induced by vibration of the tungsten filament that results from incorrect centering or height adjustment in relation to the Wehnelt. Has anyone looked into this or know if this is possible? Mike Toalson
miketoalson@gmail.com
EMI effects can be easily checked when you go to a low working
distance like 6 mm and set SYNC on scan at 50 or 60 Hz (whatever your power grid has). If effect decreases you have some EMI influence. Using a magnetic field cancellation system like a Spicer 22 will greatly help. Only if this is ruled out then it might be something as strange as filament vibration. Is it dependent on HV value? It might also be a problem with the scan amp or a power supply there. Stefan Diller
diller@stefan-diller.com
Noise is the usual subject for bickering between users complaining
about noise and equipment manufacturers unwilling or unable to resolve issues with an instrument, or convincingly demonstrate that the problem originates from the environment. Fortunately, there are a few tests you can run on the SEM to point in the direction of the root cause:
68 You can use another carbon-coated grid on top of the grid loaded
with magnetic nanoparticles. Ravi Takkar
ravi.thakkar369@
gmail.com Te EMF is here if you have a magnetic field and movement.
So insert the sample without the magnetic field and then use the
www.microscopy-today.com • 2020 November
1. As already suggested, change working distance. If the root cause is EMI picked up by the electron beam, then a larger WD will increase the noise. Te same will happen if the root cause is mechanical vibration of the source. But if the source of the noise is in mechanical vibrations picked up by the stage or body of the instrument, then the magnitude of the vibrations wouldn’t depend on working distance.
2. Change acceleration voltage by a lot, like 3kV to 30kV (if you can). If the source of noise is EMI picked up by the beam, then lower-energy electrons would be more susceptible to it, and the magnitude of the noise with the lower acceleration voltage would increase.
3. Go through the range of acceleration voltages. If the source of the noise is some kind of resonance or cyclic process taking place with the source, or oscillations in the high-voltage power supply, then you may see not only magnitude, but also the frequency of the noise change.
4. Tap gently on the SEM column with a screwdriver. You will see additional noise but look instead at how such tapping affects the original noise that was present. If tapping enhances the original noise, or maybe suppresses it for some time, then the likely cause is some kind of mechanical resonance in the column or with the source.
5. Get a stethoscope (or surface-pickup microphone) and listen to sounds of the column. If you hear something when the source is up, and disappearing when the source is off, then there is a possibility of the root cause being associated with the source operation (oscillations, resonance, etc.).
6. Connect the surface-pickup microphone to an audio input of a laptop and run the audio spectrum analyzer - frequency and changes may point to the root cause. For example, if frequency matches rotation of the turbo then that could be the original source of the noise.
7. Connect the spectrum analyzer to the imaging output of the SEM. Tere will be frequencies associated with line scanning but look for something related to 50/60Hz or 100/120Hz. Tis is indicative of poor filtration or a ground loop somewhere. Do this with the electron beam blanked, open, and scanning. Differences could pinpoint which part of the SEM circuitry is affected.
8. If a spectrum analyzer is way too exotic an animal, then try working out frequency of the noise from the periodicity of waves and how it changes with change of the scanning frequency.
9. Switching the frequency of the high-voltage power supply or intermodulation between switching frequencies of
10. Change imaging modes of the SEM. If noise is present in one of the modes and not in another, the root cause is in SEM electronics.
11. If everything else fails, get engineering help from someone with experience in noise troubleshooting. Valery V. Ray
vray@partbeamsystech.com
Loading Magnetic Nanoparticles into a TEM Microscopy Listserver I have a user that wants to do HRTEM on magnetite nanoparticles.
My concern is that the nanoparticles will be pulled away from the carbon- coated grid by the strong magnetic field of the TEM lens and contaminate the column. If anyone has a similar experience and can provide advice it will be much appreciated. Te sample is powder which I usually dilute with alcohol, then apply a drop of the suspension onto a carbon coated grid. Fei Long
fei.long@queensu.ca
its various modules could be the culprit, if decoupling or filtration is inadequate.
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