Houston Methodist. Leading Medicine.
Houston Methodist. Leading Medicine

Program Resources

Program resources

 

Brief introduction to optical microscopy

This is a brief introduction to the concepts and principles behind the optical microscopy that you can do in ACTM. The facility has a broad range of microscopy imaging equipment, and this overview covers most of the imaging systems and techniques available.

Microscope configurations

It is worth pointing out that most types of microscopes are available in two orientations: upright and inverted. Other than the orientation of the components, the principles are the same.

Fluorescence

Fluorescence is very useful for staining specific molecules within your sample and is the basis of much of what is done in the facility. The core principle of fluorescence is that the fluorophore is excited with one wavelength of light and light of a different wavelength is emitted. Common fluorophores, such as green fluorescent protein (GFP), fluorescein or DyLight 488, are chemical fluorophores bound to antibodies or fluorescent proteins. By forming an image from the fluorescence emission you learn where your protein of interest is located.

Fluorescence Microscopy

Fluorescence microscopes generate wide-field images. This is the basis of what happens in a fluorescence microscope:

  • The fluorescence source (from the arc lamp) produces bright white light which is passed through a colored filter to produce the specific excitation light, in this case blue for GFP.
  • This is reflected by the dichroic mirror, goes through the objective and to the sample.
  • The sample fluoresces as described above and some of the emission light backscatter through the objective.
  • This is the important bit: the dichroic mirror transmits the longer wavelength light so the very bright excitation and the relatively weak fluorescence are separated.
  • The emission is normally passed through another filter and then can be either seen with the naked eye or captured with a camera.

Laser Scanning Confocal Microscopy

A confocal microscopy produces an image in quite a different way from a wide-field fluorescence scope. Rather than exciting the entire field at once, the light is focused into a very small spot and is scanned across the sample so that an image is built up. This may sound like quite a strange way of making an image, so what is the point?

The key part of the confocal is the pinhole. Notice that only the light from the focal plane passes through the pinhole to the detector. The light from just below the focal plane (follow the dotted red lines) is mainly blocked. The same is for the light from just above (follow the blue dotted lines). By blocking the light from outside the focal plane, confocals have good z-axis resolution and are great for imaging thick samples because the haze from out of focus objects is mostly eliminated.

Confocal microscopes are much more complex than widefield systems. They consist of a normal microscope with the confocal bit stuck on the side. This shows the basics of a system: Lasers are used for excitation. The laser beam comes into the system and is reflected by the dichroic. Next, two scanning mirrors move the beam in a raster (like writing on a page) across the sample. The fluorescence light passes back through the objective and is de-scanned (i.e. reflects off both scanning mirrors). The light then passes through the dichroic and pinhole to the PMT (photomultiplier tube) detector. There are really lots more lens and mirrors involved, and our systems all have 3 PMTs for 3 different colors.

Spinning Disk Confocals

Spinning disk confocals are sort of half way between a wide-field fluorescence scope and a scanning confocal. The confocal principle is the same: a spot of light is scanned across the sample while a pinhole blocks the out-of-focus light. The main difference from a point-scanning confocal is that in a spinning disk there are many spots of light such that the spots are "moved" by means of a rotating disk with holes rather than scanning mirrors. The light is collected on a CCD camera rather than a PMT. The entire image is collected at the same time (faster).

Spinning disk microscopes are good for imaging living samples because they are fast and have relatively low phototoxicity. For fixed samples, you are probably better off with a point-scanning confocal.

Total internal reflection fluorescence (TIRF)

Total internal reflection fluorescence is a technique where only a very thin section close to the coverslip is excited. TIRF makes it great for studying events in the cell membrane. The technique relies on refraction between the coverslip and the aqueous sample so only really works with living samples.

The excitation from a laser is sent off-center up a high-NA objective. The light hits the coverslip at an angle such that total internal reflection occurs and light passes through the coverslip and generates an evanescent wave. This layer of excitation might be only 100 nm thick, much less than the z-axis resolution of a confocal. Aside from the special mode of excitation, the rest of the imaging is the same as a normal fluorescence.