We have Nikon Ti microscope, the diffraction limit is about 200nm, the images of 200nm channel is blurry at the boundaries, how can I improve it, can updating hardware or software solve this problem?
As we know that the lateral resolution depends on the numerical aperture (NA). NA is a constant for the microscope objective you use. Some microscope objective have a NA of 1.3. This value could be increased using Immersion oil and thus NA = n.NA, where n is the refractive index of the immersion liquid. I hope this helps!
First of all, if 200 nm resolution is the theoretical value, you will never obtain such a resolution in real imaging. Deconvolution may help to improve the quality of images, however it can not substitute for an appropriate objective or well prepared sample. If you want better resolution, go for high quality immersion objective (the immersion liquid - and hence the objective - should match the medium of your sample, i.e. for imaging cells in medium, PBS or whatever, use water immersion, for fixed samples use immersion oil etc.). You should prepare your sample or rather choose the best objective and immersion so that refractive indexes of immersion liquid and medium are as close as possible.
From my experience the immersion oil is very important for those commercial microscope setups. You should only use the Nikon immersion oil that is dedicated for this application, since the internal optics are adjusted to it!
To resolve down to 100nm, a simple optical microscope will not cut it (you will be lucky to be getting 250nm). Since you are not trying to image a fluorophore, you will need to use a true super-resolution imaging technique such as structured illumination microscopy.
Most microscope manufactures are offering turn-key solutions, (including Nikon based on the Eclipse Ti). Unfortunately, It is not just a matter of adding a single attachment to your Ti, but you may want to talk to your rep if you are interested in the upgrade. I will caution that even though structured illumination can approach low-100nm spatial resolution, this is under optimal conditions. YMMV.
Resolution is normally defined as the distance you can resolve two points apart. This is different to detection which can be significantly less, for example you may detect a microbe of less than 200nm but not separate two same microbes which are 200nm micron apart and they will appear as an oval. Another example is Oil Immersion Darkfield Microscopy, where sub 200nm micron particles can be detected but not separated. A good introduction to the principles of microscopy is: Introduction to Optical Microscopy by Jerome Mertz 2010, ISBN: 978-09815194-8-7
Hope this helps.
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