Don't Send Her Flowers, She Just Wants the Pollen

This summer, while working as an intern at University of California in San Francisco at one of the light microscopy imaging cores there, I received the largest bouquet of flowers I’ve ever gotten. It had been probably a month and a half since I left Rochester, New York to take the internship and my presence was beginning to be missed. I called my boyfriend and thanked him and proceeded to spend the rest of the afternoon pulling apart one of each type of flower to make slides of their pollen. I mentioned it to him later and I could tell he was shaking his head, “You are just unreal,” he said with a giggle.

Perhaps it sounds a little nerdy, and cruel (I only pulled apart about an eighth of the bouquet, I promise) but the images of the pollen at about 100x magnification were just as gorgeous as the flowers themselves. The pollen grains were far too small to be photographed using brightfield microscopes, which are just the typical lab bench instruments most people are familiar with that simply shine a light through an objective lens to magnify the image. Luckily, pollen grains are autofluorescent meaning they absorb one specific wavelength of light and reemit light at a longer wavelength. A few modifications to the lab bench microscope enable us to create images of the emitted light without background brightness obscuring the image.

The light source is sent through an excitation filter, which passes only a specific wavelength of light, which will excite the subject. The light then reaches a beam splitter, a dichroic mirror, which selectively passes a particular wavelength of light while reflecting another. The beam splitter will pass the excitation light through to the objective lens and to the subject. The beam splitter reflects the light emitted by the subject at a longer wavelength directing it through the emission filter, which only allows light at the longer, emission wavelength to pass to the sensor, usually simply a modified CCD camera. It is important that the excitation and emission filters are mutually exclusive, that they do not allow any of the same light to pass so that no stray light makes it to the detector (Figure 1).

Figure 1 Widefield fluorescent light path

Although pollen can be viewed simply by mounting it on a slide and placing it in a fluorescence imaging system, most subjects of interest to biological research are not autofluorescent and must be stained. Green fluorescent protein (GFP) is protein produced by jellyfish that makes them glow green. It has ushered in a new era in microscopy and research. The gene that produces GFP can be inserted into organisms to make certain structures fluoresce green when subjected to blue light. The fluorphore, which generates the light, may also be attached to an antibody that will in turn attach to a protein of interest in the subject. Since the discovery of GFP, many other fluorescent stains have been created based on the same principle, making it possible to stain a single subject for several structures. To image a sample with multiple stains, the excitation and emission filters as well as the beam splitter are rapidly changed out to match the wavelength requirements of the different stains, making a single picture of the light emitted by each stain. These images are then combined into a single image, like the cells in Figure 2.

Figure 2 Bovine pulmonary artery cells, triple stained

Certain subjects, particularly thick samples, begin to present issues for this type of widefield, or modified lab bench microscope system because the objectives used to create magnification have a limited depth of field. As magnification increases, smaller and smaller slices of the subject are in focus, more and more features of the sample become obscured by out of focus light coming from different depths that are above or below the focal plane, as seen in Figure 3. With 100x magnification, which is really necessary to see a pollen grain in detail, that pollen grain is far too thick to be entirely in focus.

Figure 3 Immature daphnia eye spot, out of focus light above the focal plane obscures in focus information

Computational deconvolution attempts to reassign the out of focus light to its point of origin. This is possible because the way in which light spreads from its origin is affected by a number of factors which can be roughly estimated by software. Even when experimental values for these factors, such as the quality of the objective or how much the mounting media bends the light, are acquired whenever data must be quantified for research studies the accuracy of the deconvolution algorithms may be called into question.

For this reason, many researchers choose to use confocal imaging systems to image subjects that are thicker than the objective’s depth of field. There are several different types of confocal systems that are widely used in biological research but they all operate on the same principal, preventing out-of-focus light from ever reaching the camera. An aperture is placed in front of the camera and any light that is not in focus at the current focal plane falls outside the aperture, resulting in an image that is made up of only light that is in focus. To image the whole subject a series of images must be taken, moving the focal plane from the bottom of the sample to the top. This results in a sequence of images which are referred to as optical sections that can be realigned in three dimensions. The series of images that my flowers died for, the realigned and stacked images, and the 3D reconstruction can be seen in Figure 4.

Perhaps spending hours a day staring down into microscope eye pieces has warped by view of the larger world: I see it for its pollen rather than its flowers, and for their bits and pieces of transparent invisible cells that we’ve learned to make glow bright, beautiful colors in order to learn about how it is they function to build that larger world.