You’re a senior postgraduate student, a post doc or a junior PI with little knowledge on microscopes and someone between Senior PI /Dean level approaches you with this: “We have now the funds to buy the fluorescence microscope someone once told me we need. Can you handle this please? Oh, by the way, the funds have to be used by the end of the year.” After deciding it was not a question, and swallowing some choice expletives while looking at the bosses receding back, you dimly recall seeing that Bitesizebio.com has a new Microscopy Channel.
BitesizeBio to the rescue!
Maybe some help can be found there? You rush to your computer and find Rolly’s articles about choosing microscopes (you’ll find Part 1 of Rolly’s Guide here and Part 2 here). Okay, so, there are all kinds of microscopes available. Why are we buying this fluorescence microscope in the first place? You start to wonder (once again) whether the boss knew anything about the question at hand (doubtful). Now is time to use your best resource: colleagues. Find out who wants to use the microscope, what they really need to do with it and are there some other things they might possibly want to do later on.
More cost efficiency
In the tender invitation separate these into requirements and options. You should also double check that there isn’t a suitable accessible microscope somewhere nearby. Sometimes upgrading an existing microscope with new light source, fluorescence filters, objectives and/or camera can be more cost efficient than buying a new one.
Be nice to the microscopy core facility
Also if there is a light microscopy core facility nearby, consider making the whole project their problem (in as nice a way as possible- you want to keep on the right side of these people!). It’s much more cost efficient to have a core facility handle the microscope than training a new graduate student to handle the microscope user support every couple of years. Trust me- these systems are way too complicated to run reliably without having someone to take care of them.
Read these great resources
Now comes the tricky part: you have to convert the user community’s wishes to a tender invitation. For that, it may be a good idea to study some more. Some great resources include;
Molecular Expressions Optical Microscope Primer- everything you need to know about fluorescence microscopy is here: http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorhome.html
Read also the 50 Most Frequently Asked Questions about Optical Microscopy by Mortimer Abramowitz http://micro.magnet.fsu.edu/primer/faq.html. For example, questions 27&29 help you to circumvent the people who want to have a 100x objective just because they are looking at small things.
Try before you buy
Find out who the local microscope sellers are, contact them and tell them a broad outline on what you’ll need and ask them to visit with a short proposition on how they would fill your needs. Ask them if there are other nearby institutes which may let you try out some of the models they have on offer.
You’ll need a good engineer
Ask around what is the level of service and application support in your area. These can vary widely with all the four major microscope manufacturers (Leica, Nikon, Olympus and Zeiss) depending on your geographical location. Having a good service technician available close by is very valuable. Don’t blindly trust what the companies tell you about this, in some cases there might be several service engineers close by, but in reality they are part of a local laboratory equipment dealer’s workforce and are superficially trained to also service microscopes.
The most important parts – the objectives
As a starting point, here’s my view on specifying a fluorescence microscope for imaging fixed cells and thin sections stained with up to four colors mounted on cover slips:
Objectives are the most important part in a microscope. Select a combination of as high NA, as low magnification and as good aberration correction level as possible.
5x air is a useful objective for getting an overview of the sample,
10x with an NA of at least 0.4 gives way more efficient detection of fluorescence.
20x objectives are not that useful for single cell work but multi- or glycerol immersion ones are very good for imaging sections.
While 40x high NA air objectives are handy for medium resolution single cell imaging, in multiuser environment it’s best to steer clear, someone will always find a way to get oil on them.
60x (or thereabouts) Plan Apochromat immersion objectives are the best for single cell imaging. The basic solution is to get an NA 1.4 oil immersion objective. Depending on your sample thickness and refractive index (RI) you should consider whether a higher NA oil immersion, glycerol immersion or silicon oil immersion objective would give you better results. The first one is optimal for very thin or high RI samples. The second is best for around 5-10 micrometer thick samples mounted in glycerol based mount and the 3rd is optimized for tissues mounted in low RI media. In case your sample is mounted in water based solution and is not thick tissue, the best option is a NA 1.2 coverslip corrected water immersion objective.
We hope you found this first part useful, but, wait! Don’t go on a spending spree just yet- you’ll need to read part two of this buyer’s guide which will be published here next week.
The resolution of any microscope is related to the numerical aperture of the lens and the wavelength of light used to form the image, and can be calculated using Abbe’s law (read more about Abbe’s law here). This, however, is the ideal situation – the best case scenario. In real life, resolution must be defined […]
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