Fluorescent microscope checklist

Use for discussion of various microscopy and imaging techniques, such as phase, fluorescence and confocal microscopy, electron microscopy, and various other imaging techniques.

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Fluorescent microscope checklist

Postby RISC » May 13 2014 4:37 pm

Can you please tell me what are the things to check when buying a fluorescent microscope?
Thank you all
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Re: Fluorescent microscope checklist

Postby r.rosati » May 14 2014 9:36 pm

Hello! Which kind of samples would you like to visualize, and which techniques would you like to apply?
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Re: Fluorescent microscope checklist

Postby RISC » Jun 16 2014 2:04 am

we use mainly slides so upright microscope, and we do fluorescent antibodies, and bright field images of course HE stain etc..
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Re: Fluorescent microscope checklist

Postby rjm1975 » Aug 19 2014 10:02 pm

Hi, an important consideration is what is your budget?

Getting good quality images in fluorescent microscopy is all about having high efficiency transmission and collection of light. The major manufacturers, e.g. Olympus, Nikon, Zeiss, Leica all have well-optimized designs and high quality optics for efficient transmission of light. Apart from that you want to choose the highest quality filters. The modern generation of sputtered filters from suppliers such as Chroma, Semrock, Omega have much higher transmission and very precise passbands, versus the older generation of filters even from the same companies. In fluorescence microscopy the objective serves dual purpose as both condenser and objective, and you want to choose ones that are optimized to minimize spherical and chromatic aberations - these will have a designation like "Plan Fluor" (good) or "Plan Apochromat" (best) (but NOT "Achromat" which is what you see on entry-level or student microscopes). Resolution is ultimately a function of numerical aperature (NA) of the lens, which is a measure of the light collection efficiency (or the angle over which the lens can collect light). Higher is better. A good 60X oil objective will have NA of ~1.4.

Light source - you will probably get the most brightness out of an old fashioned 100 watt mercury arc lamp, although these have relatively short life, relatively poor stability. For similar output but longer life and better stability, you can get metal halide lamps, some of which include various bells and whistles like built-in intensity control or built-in shutter. Mercury and metal halide lamps have "spiky" outputs with high intensity in certain spectral regions, which is great for some fluorochromes, not for others. Xenon arcs and LEDs are other options.

Camera - this is one area where it is possible to pay way more than you need to. It really depends on what you're doing: how bright are your samples, and related to that, how long exposures are you planning to do. You can pay anywhere from $1000 for a very respectable uncooled CCD camera with 12-bit depth, to $30,000 or more for an ultra deep-cooled back-thinned electron multiplying CCD, or ultra-fast scientific CMOS camera. Basically if you have bright samples and can get away with short exposures with intense light, you can get by with a cheaper camera. If you are trying to do live cell imaging with long exposures and so forth, you start wanting features like a cooled camera. Too much detail to go into here, but for many microscopy applications you do not want very high number of pixels - you may see cameras with 5 megapixels on a 1/2" sensor, this may be good for astronomy but is a waste for microscopy - the pixel size is smaller than the resolution limit of the microscope. A good high-end camera for microscopy may be 1.3 megapixels on a 1/2" format sensor, which translates to roughly 6.5 micron size pixels - this is a good pixel size for microscopy with a 60X or 100X oil objective with numerical aperature of ~1.3 or 1.4. The one time you benefit from more pixels is if the image sensor itself is bigger; you may see 5 megapixels on a 1" sensor or something, which translates to a larger field of view.

There's much more to consider, and other bells and whistles like automated focus, motorized stage, filter wheels, etc are all nice to have but a matter of budget. Another consideration that depends a lot on budget is imaging software, you can go anywhere from free (Micromanager) to >$10,000 for some commercial packages.
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