American Scientist

My perspective on science turned inside out, literally, during my student days, while I was taking a microscopy course at Rochester Institute of Technology (RIT). While learning about the history and techniques of microscopy, we were challenged to find samples that would reflect the project we were working on. One time, I was digging around in my bag for something new to observe when I found an old Polaroid I had taken. I decided to cut it open to see what was inside. Under magnification, I discovered an unexpected array of chemical colors and shapes. There was an entire landscape of images within the image itself—a completely new view of a common object I’d never given much thought to before.

I had always been drawn to photography but had no interest in conventional portraiture. I had found my calling: photomicrography. Over the years I have come to concentrate specifically on the biological side of microphotography, finding ways to capture elusive or overlooked aspects of the living world. Sometimes I work solely as an imaging technician, helping researchers make their micrographs as clear as possible so that the images have the most scientific value. Other times I make more use of my artistic background, experimenting with field of view and other parameters. But I always enjoy trying to blend in my artistry as much as I can.

Composing microscopic pictures poses a joyous challenge, especially when the subjects are alive. Simply preparing a slide takes a lot of practice; so does determining the optimal illumination and framing. The images shown here were created mainly for their aesthetic value, but the colorful, sharp processing needed to produce a pleasing image also draws out biological details that might otherwise be lost or overlooked. Above all, I love revealing and sharing aspects of the microscopic realm that not many people have seen before.

One of my favorite tools for my photomicrography is the confocal microscope, which uses lasers to illuminate a specimen. Researchers will often stain the specimen with light-emitting molecules that bind to specific cellular structures or other objects of interest. These molecules glow, or fluoresce, when stimulated by specific wavelengths produced by a laser beam. But certain biomolecules will light up even without a stain, a process called autofluorescence.

A challenge with fluorescence is that it tends to produce a lot of out-of-focus light from different parts of the samples. Confocal microscopes get around that problem using a point-scanning technique: They collect light through a tiny pinhole that moves across the specimen, building up a clear, high-resolution image across multiple depths. I also work with a widefield microscope, which uses a light source instead of lasers to illuminate the subject to provide a quick, broad view.

My current process typically begins by getting a big-picture view of something that captures my curiosity, such as a sample of water from the ocean. I’ll examine it under a microscope with the lowest magnification and widest field of view, most often a stereo microscope, to zero in on a specific target. Then I’ll go to whichever microscope is available—confocal or widefield—to see if the target fluoresces. If my specimen is large, like the chameleon embryo, I would want to use the widefield or stereo microscope. If I’m working on the confocal microscope, I will start out at low magnification, typically 10×, to hunt around for an interesting target to photograph, before going to 20× or 65× to isolate a feature and to capture it at high magnification.

As I begin making the image, there are many parameters to play with, including scan speed, frame average, frame size, laser power, gain, and image size. Because of the microscope’s limited depth of field, often I have to do what is called a z-stack. The z refers to the depth direction on the specimen. A z-stack will take images at multiple z-positions, which can then be combined to create a single image made up of many overlapping layers, all of them in focus.

I will typically test out many different combinations of parameters and z-stack sizes before I find a composition that works for me. Some images take less than a minute to complete; others take hours and test my patience. Samples that give off a relatively faint fluorescent signal require longer scan times. They may also require combining multiple scans to create a smoother image.

Once I have raw images in hand, I edit them to adjust contrast, highlights, darken the background, or remove specks that distract from the main object. At this point I also start to experiment with color. In fluorescence microscopy, the color given off by the specimen has no connection to what your eye would see if it were directly illuminated by white light. Using the image-processing software, I may assign colors that match the color of the fluoresced light or I might choose something completely different, depending on what is most meaningful or beautiful.

When it comes to selecting the subjects for my images, I’m a scientific omnivore. I’ll go for anything I’m excited to see under the microscope, including ordinary objects around me: makeup, premade slides, or water from my fish tank. (The latter of these inspired a children’s book I wrote, The Hidden World of the Aquarium.) Because most of my images rely on fluorescence, finding a specimen that provides a good fluorescent signal is key. Many living things autofluoresce in response to red laser light. Plants often respond to green as well as red.

Embryos are some of my favorite subjects. The chameleon embryo was especially exciting for me because it was a specimen I had prepared from start to finish: eviscerating it, washing it, dissolving the soft tissue, and staining the cartilage. For the resulting image, I applied two fluorescent stains and also exploited autofluorescence. I had to take multiple pictures with different fields of view to take in the whole creature. It was amazing to see the result: The skeletal structure of the embryo stands out in bright red, almost like an x-ray, but with the surrounding tissue still visible in great detail.

As soon as I imaged one embryo, I wanted to do them all. The turtle is another favorite of mine. It’s easy to forget that these reptiles have a full set of bones inside their shells; one clearly can see the ribs and other skeletal details inside the shell in this photograph.

My microphoto of recently fertilized sea-urchin eggs, created for a developmental biology class at RIT, allowed me to apply my techniques on a more realistic scientific scale, working entirely with fluorescent stains in this case. The fluorescence highlights three key structures: the blue chromosomes duplicating and expanding; the red lines of the mitotic spindle, which is the structure that orders and segregates the chromosomes during cell division; and the green of the cell membranes.

I hope that other people look at these photos and feel as amazed as I do. Science is a source of insight, but it’s also a tool for beautiful artistry. It was daunting and confusing at first, but I found a way to combine the two. You can, too.