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November 22, 2016 at 11:33 am #3598
I am trying to use the enhanced darkfield to observe Au nanoparticles with a mean size of 7 nm.
I am putting a drop of a solution of Au in water with a concentration of 0.32 mg/L of top a coverslip and I covered with a glass slide.
Do you have idea if this is the recommended concentration and the correct sample preparation?
I observed some bright spots, unfortunately I also observed bright spots when I observe only the coverslip and the glass slide, so the bright spots are dust.
Thanks in advance!
AlexandreNovember 22, 2016 at 2:41 pm #3599
I wouldn’t expect to be able to see sub-10 nm AuNPs. However you should certainly be able to detect the larger AuNPs in the population or aggregates.
It’s always hard to say what the appropriate concentration should be, especially with NPs so small, but you may try to increase the concentration.
A couple of suggestions to try first:
1. Make sure your coverslip is on top when viewing under the microscope.
2. Check all areas under the coverslip. Sometimes the AuNPs cluster at one side.
Hope this helps,
ElyseNovember 23, 2016 at 11:55 am #3600
Thanks for the help!
The largest NPs in the population should have 11 nm of diameter. But maybe I can observe aggregates.
I am trying to use the enhanced darkfield (in transmission mode).
Does it requires a special type of filter placed in the fluorescent cube torrent?
How can I check if the alignment between the condenser and the objective is correct?
Thanks in advance!
AlexandreNovember 23, 2016 at 12:24 pm #3601
Hello this is Jamie Uertz. How are you?
I would suggest checking out this video on our website to ensure your darkfiled alignment is correct.
After watching let us know if this helped and if you have any more questions!
JamieNovember 23, 2016 at 12:24 pm #3602
Sorry I forgot to attach the link!
http://www.cytoviva.com/products/microscopy-2/high-resolution-illuminator/December 3, 2016 at 8:42 am #3608
After trying to observe Au NPs, on top of a glass slide, using the enhanced dark field in transmission mode, I was able to record a spectra with that probably correspond to the scattering of the Au NPs. But I also measured other scattering that probably corresponds to dust or defects of the slide that also scattered the light. Is there some tool of the Cytoviva software that can perform a correction that would “subtract” this two scattering spectra?
Thanks in advance!
AlexDecember 4, 2016 at 11:06 am #3609
I would suggest the easiest fix for this issue is to use Ultra Clean glass slides. These can be acquired by the company Schott -Nexterion. We recommend Glass B. Here is the link:
This will ensure that anything on your slides is your sample and not dust, scratches, etc.
If you need ordering information we can help you with this!
Let us know.
JamieFebruary 10, 2017 at 8:58 am #3731
I recorded the spectra of Au NPs, using the enhanced dark field in transmission mode.
The spectra is similar to the one in the article I am atacching.
Can/should I perform some type of correction in order to take in account the lamp profile?
Thanks in advance!
Attachments:February 10, 2017 at 2:13 pm #3734
There is a Lamp Correction routine built into ENVI. It can be accessed from the main toolbar–> CytoViva Analysis–> Calibration and Correction–> Normalize for Lamp Spectrum.
Lamp correction/normalization isn’t always necessary, especially for plasmonic NPs, but it can be useful in pulling out features that are otherwise obscured by lamp. The spectra in the app note you attached were not corrected.
There is a pre-processing step involved, which requires that you first take a recording of the lamp spectrum. This needs to be done every so often (e.g., once a month for daily use) since the light bulb and light guides change over time. Please see the steps for acquiring a lamp recording below:
1. Sample slides are usually pretty dirty, but if you can find a clean area, you can use it to take the lamp scan. Otherwise, I would get a clean slide and mark on it with a permanent marker. This will give you something to focus on.
2. Once you have something to focus on, use 100x and adjust until particles, mark, or whatever is in focus.
3. Move to a clean area.
4. Open up the iris on the 100x objective. If you look through the eyepieces it will be very bright.
5. On HSI controls, adjust exposure to 0.005 seconds or so. The same rules for setting exposure apply.
6. I usually name the sample “100x lamp DATE”.
7. Take a quick or 21 line scan.
8. Take a ROI across the width of the lamp scan. It’s easier to choose a rectangle shape to draw with (under ROI_Type).
9. Hit Stats. Once this opens, right click the plotPlot Key.
10. Right click the plot–>Options–>New Window Blank.
11. Drag and drop the Mean into the new window from the plot key.
12. In the main toolbar–>Basic Tools–>Spectral Math–>Restore–>Desktop–>Click normalizing.exp–>Highlight–>Click OK
13. Highlight the Mean in the next window that pops up–>OK
14. In the window with the old spectrum and the new spectrum–>Right click–>Plot Key
15. Right click the first Mean–>Remove
16. Right click the plot area–>Reset Range
17. Save this as a spectral library. I would name it “100x NORM lamp DATE.”
Now you can proceed with the actual lamp correction of the image.
Lamp correction/normalization often introduces noise on the ends of the spectrum that will need to be eliminated before analysis. Let us know if you need more information on spectral subsetting.
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