Microscopy   Leave a comment

Microscopes have been around for about 500 years- they are one of the oldest scientific instruments that are still in regular use. Obviously, the microscopes we use today greatly outperform the microscopes used by Hooke and van Leeuwenhoek, but you may be surprised to learn that the world’s best microscopes from the 1960s work as well as state-of-the-art microscopes made today.

In fact, there has been only two major advancements in optical microscopy since the 1930s, one of which is technical in nature (infinity corrected objectives), while the other has truly revolutionized biology- fluorescence imaging. The use of fluorescence techniques has directly led to a huge number of specialized imaging techniques, including confocal, TIRF, FRET, STORM, FLIM, and two-photon.

Here is a picture of our main research microscope:

It’s a fully motorized Leica microscope with a Prior LumenPro fluorescence source, an intensified camera and a digital video camera. We also enclose the microscope in an incubator to keep the cells warm and wet.

Imaging our cells presents a challenge: because they are grown on suspended filters, we are restricted in what techniques will work- differential interference contrast, for example, will not work. Fluorescence imaging allows us to image the cells at high resolution, and furthermore to image the parts of the cell we want to image. For live-cell imaging, we use cells transfected with GFP-fusion proteins and Fura-2 to visualize the amount of Calcium present in the cytosol. Otherwise, we kill the cells and use standard techniques to selectively stain proteins of interest.

These are the lenses we have available:
10x/0.30 HCX PL-S APO: one of the primary lenses we use. An excellent objective lens, made especially useful due to the large exit pupil diameter: the optical throughput of this lens is impressive.
40x/0.80 HCX APO L W U-V-I: a ‘dipping’ objective. This objective lens is used for live-cell imaging, where the lens is designed to be dipped directly into the culture media, without the use of a coverslip.
63x/0.90 HCX APO L W U-V-I: a higher magnification dipping objective, one of the primary lenses used
63x/1.47 HCX PL APO OIL CORR TIRF: an oil immersion lens, highest numerical aperture available. This lens has the highest resolution we own, and gives spectacular DIC images.
100x/0.95 HCX PL APO: a dry objective, no coverslip correction. We can use this as a dipping objective.

The fluorescent filter cubes we have are:

Sedat Quad Filter
Texas Red
Tweezer cube

And we also have a deSenarmont compensator for color DIC imaging.

Here’s a picture of another microscope we use:

This is a Zeiss Ultraphot III and was rescued along with a full set of lenses and attachments. We use this one for epi-illumination and macro imaging. This microscope was originally specified for use in a metallurgical laboratory, and so the lenses are all designed to work without a coverslip. This microscope can perform brightfield, darkfield, phase contrast, and DIC imaging all in reflected mode. To be sure, we have the parts needed to operate this microscope in trans-illumination mode, but we have yet another scope, so this one is a dedicated reflected-field scope. The objectives we have are:

Inkof Epiplan 4/0.1 Pol
Inkof Epiplan 8/0.2 Pol
Inkof Epiplan 16/0.35 Pol
Inkof Epiplan 40/0.85 Pol
Inkof Epiplan 80/0.95 Pol
Epiplan HD 16/0.35

and in addition, the microscope came with a near-full set of Luminar lenses. Zeiss Luminars are the premier macrophotographic lenses ever made, producing images without peer (nearly all of the macro images we produce use these lenses). These are:

Luminar 16 mm f 2.5 with iris
Luminar 25 mm f3.5 with iris
Luminar 63 mm f4.5 with iris
Luminar 100 mm f6.3 with iris, with dovetail for Luminar Head
Luminar 2.5 – 5.0: 1, for use with macro stage 47 25 61
Epi-Luminar 20 mm, M 40 X 0.75 mm thread for macro-illuminator 47 25 75
Epi-Luminar 25 mm, NA 0.14, M 40 X 0.75 mm thread for macro-illuminator
Epi-Luminar 40 mm, NA 0.11, M 40 X 0.75 mm thread for macro-illuminator
Epi-Luminar 63 mm , M 40 X 0.75 mm thread for macro-illuminator 47 25 76

For routine cell culture activities, we use this:

A Nikon TMS.

Finally, we also have a Nikon SMZ-6 stereozoom microscope:

And a Zeiss ACM microscope:

The stereo microscope is used a lot for fine assembly and inspection work, while the ACM is able to provide limited fluorescence capabilities. One nice aspect of the ACM is the ability to accomodate large objects by raising the objective lens via the rack and pinion mechanism.

Posted August 27, 2010 by resnicklab

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