NetNotes
sounds promising when you get similar labeling pattern as in previous light microscopy data. In this case, you might be able to fine-tune the fixation step and permeabilization if the latter was used. In my experience, this approach worked better for the Flower, a synaptic vesicle protein in the neuromuscular junction (NMJ) which we were working on. We tried several fixation combinations but found that overnight 4% paraformaldehyde fixation achieved sufficient labeling. Although ultrastructure quality was not the same as routine TEM, the organelles were still recognizable. We were a bit lucky because the NMJs lie close to the muscle surface and go only as deep as 5–8 microns into the tissue. Also, we worked on fillet-dissected third instar Drosophila larvae and did not need 50 micron thick Vibratome sections. Even so, we still needed permeabilization steps but not as drastic as used in regular immunofluorescence. Aſter trying different detergents and concentrations, we added 0.01% Tween 20 in the blocking step. Te nice thing about using FluoroNanogold is we can examine the fluorescence labeling before proceeding to next steps. In addition, you can post fix with 3% Glut and use lower concentration of osmium tetroxide prior to dehydration and embedding so the sample quality should be more enhanced. I am not an expert in Immunocytochemistry like Drs Paul Webster and Jan Leunissen and others out there but one thing I learned as I was getting into this is that there is no single labeling approach. Pre-embed method is not suitable for all samples but would likely work on cell monolayers/ suspensions, Vibratome sections and samples where antigen is easily accessible. We try to fit the best immuno-EM method to your samples which we all know usually takes time. I hope you have plenty of time to optimize the method for your samples. I will send a PDF of the Flower paper so that you can check the micrographs. A detailed pre-embed method with Fluoronanogold is on the supplemental material. Claire M. Haueter
chaueter@bcm.edu Tue Jan 25
Light Microscope: stuck objective What tricks/tools do people suggest for unscrewing objectives
from the turret when you cannot loosen them by hand? David Knecht
david.knecht@
uconn.edu Wed Feb 16 Buy a strap wrench. If the rubber slips on the objective barrel,
use channel pliers to grab the rubber strap and turn. Don’t squeeze excessively. Focus on turning with the pliers. You may be able to use leather gloves with the pliers, but the teeth of the wrench could penetrate and cause gouges in the outer case. A small sheet of thick rubber may also work, but strap wrenches are cheap and store easily. Gregg Sobocinski
greggps@umich.edu Wed Feb 16 I want to thank you all for your suggestions and report back.
I put WD-40 on the threads with a plastic Q-tip, waited a while, then used a small strap wrench (Harbor Freight Tools) and I was able to get them all loose. David Knecht
david.knecht@
uconn.edu Sat Feb 19
TIRF:
laser alignment question We have the Nikon TIRF system and have three laser lines going
into the TIRF arm via a single fiber. When we project through the 100× objective through the sample onto the wall, we see that the lines go through the sample at different angles. (You can see a picture of the projection at approx 45 degrees at
http://www.flickr.com/photos/ mcammer/5359189090/.) It is also noticeable in the TIRF images that the field depth is different for each wavelength. Is this unavoidable due to the different wavelengths or is it possible to align the optics better so these spots would be more coincident? Michael Cammer michael.
cammer@med.nyu.edu Sun Jan 16
64
Tat is a beautiful photograph of the unavoidable relationship
between the angle of incidence and the wavelength of light used to illuminate the sample. It stems from the fact that the relative index of refraction of the glass-water interface is slightly different for each wavelength. To ensure that the penetration depth is nearly equal for all laser lines, you would have to offset each laser beam to a different specific radial position on the back focal plane of the objective—and these relative spacings would change as well if you wanted a different penetration depth. You can work out what these spacings need to be by examining the basic equation for the TIRF penetration depth (you have to assume you know the wavelength dependent index of refraction of the sample which ranges from 1.33 to 1.38) and you’ll find that they are on the order of a few tens of micrometers depending on the specific penetration depth you desire (see
http://micro.magnet.
fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html). Tere’s a nice little ImageJ plug-in that can be used:
http://rsbweb.nih.gov/ij/ plugins/tirf/
index.html. John Oreopoulos john.oreopoulos@utoronto. ca Sun Jan 16 Tanks for the reply. Reading it and the referenced websites
jogged my memory. A few years ago we were having problems with the first commercial Olympus TIRF system because we could not get consistent evanescent waves with the one angle adjustment with the laser lines we had from 405 to 568 nm that were delivered via a single fiber (it was worse when we later added a 633 or 638 nm laser). I suggested we pump each laser in through a separate path that could be angled independently. We did not build it, but I think Olympus now sells a TIRF system that does this. Another issue is that when I first heard about TIRF maybe 15 years ago, it was introduced as a ring illumination at the outer edge of the back aperture, not as a single point or crescent at the periphery on only one side. A ring, or at least a series of points around the periphery, seems like a better way to provide a uniform field due to aberrations from coherent light in the imperfect optics. Any thought on this? Michael Cammer michael.
cammer@med.nyu.edu Sun Jan 16 Yes, you are right. See e.g. this paper about this very topic: Fiolka,
R., Belyaev, Y., Ewers, H., & Stemmer, A. (2008). Even illumination in total internal reflection fluorescence microscopy using laser light. Microscopy Research and Technique, 71(1), 45–50. doi:10.1002/jemt. 20527. Janne Hyoetylae
janne.hyoetylae@stud.unibas.ch Tue Jan 18
EM:
radiation risk I am thinking about the EM radiation for an expectant mother.
Are there any policies regarding the use of SEM and TEM by pregnant employees? Tank you very much! Xiaolan Wu xiaolan.wu@
dartmouth.edu Tue Feb 15 In our Facility (located in Austria) we had the pleasure to deal
with this situation a year ago. What we found out was that with the maximum X-ray emission of our 100 and 300 kV TEMs as measured in the acceptance test (approx. 200 nSv/h), it takes an expecting mother under intensive, but realistic working conditions more than 2 years to reach the maximum dose allowed here by law. To confirm this, we had both microscopes re-measured by an external, certified expert. It turned out that close to our 100 kV machine, the radiation level is even below environmental radiation, as the column shields more than it emits. Subsequently, we received an official safety clearance from the local work inspectorate for both scopes. We also had our pregnant colleagues carry live dosimeters with them, which we checked daily, to make sure the conditions at the microscopes did not change. Guenter Resch
guenter.resch@
imba.oeaw.ac.at Wed Feb 16
www.microscopy-today.com • 2011 May
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