Life in colour

Editorial

Rebecca Pool

Friday, May 4, 2018 - 14:30
Just one example of stunning colour SEM imagery from David Scharf; a Green Peach Aphid [Scharf]
 
Be it mosquitoes, neurons or microelectromechanical systems, David Scharf's colour SEM images changed how the world saw science, reports Rebecca Pool.
 
When David Scharf was only nine, he won a camera on the boardwalk of Asbury Park, New Jersey, and started taking pictures of his dog in his backyard.
 
By 12, he'd upgraded to a Kodak Brownie Hawkeye, discovered science, and combining his Brownie with a kids Gilbert microscope had produced his first micrograph.
 
Today that early image of potassium ferrocyanide crystals hangs alongside his many SEM images that have featured on the covers of Science, National Geographic, Nature and more.
 
Self-taught and self-employed, Scharf's micrographs of insects, arachnids, plants, cells, even World Trade Center dust, have stunned the world, inspired many and shone a dazzling light on microscopy.
 
David Scharf's ground-breaking SEM imagery has featured in National Geographic, Science, IMAX and more.
 
Scharf first came across electron microscopy in 1973, while heading up the Vacuum Physics Laboratory at computer system manufacturer, Burroughs Corp, in Newberry Park, California.
 
Having studied Physics at Monmouth University, New Jersey, the young engineer was designing control systems for high-vacuum deposition but soon became fixated with the lab's ETEC scanning electron microscope.
 
"The ETEC had just come out, was very new, so on my off-hours I taught myself how to use it, " says Scharf. "I got it tuned electronically and photographically, knew how to get the critical focus on the film recorder camera, maximising the dynamic range and getting the maximum light from the cathode ray tube without saturation... and was coming out with some incredible pictures of insects and plants."
 
"[My boss] told me to never, ever put anything wet into it, or I'd ruin it," he laughs. "Of course, I wasn't afraid as I knew if I broke anything, I could fix it. I had no fear of damaging that instrument."
 
But as well as amassing ground-breaking imagery, Scharf was also inadvertently developing new ways to image unfixed, uncoated, live organisms that deviated from standard methods of fixing, dehydrating and applying conductive coatings to dead specimens.
 
Kidney Stone: calcium oxalate crystals [Scharf]
 
Scharf would first anesthetise live specimens with refrigeration or carbon dioxide and then mount his subjects onto a SEM stub using denatured alcohol and graphite. His ETEC's vacuum system had a quick pumpdown time - some 60 seconds - giving him enough time to capture his specimen before it dehydrated.
 
During imaging, he used a 5 kV rather than a 20 kV electron beam, so the natural conductivity of living specimens could be used to conduct away the charge of the electron beam. And he adjusted the instrument's electronics and electron detection systems for maximum sensitivity.
 
"Once I got one good picture I was hooked," he says. "To me, it was amazing that you could put something live in there and it wouldn't blow up like everyone said it would."
 
But amazing or not, Scharf soon realised that he was pretty much alone in his creation of insect and plant micrographs.
 
"I honestly didn't know that I was doing anything different until I started to look through journals, and just couldn't find any decent-looking SEM photographs, let alone images of living things," he says.
 
David Scharf works with his electron microscope in his laboratory, at home in Los Angeles, US.
 
So, as a complete unknown in the worlds of science and electron microscopy, Scharf took his black and white images of live organisms to myriad publications including Time and National Geographic, and almost instantly, got published. 
 
"The response was incredible and I was treated like royalty at the National Geographic Society," he recalls. "I got a double-page spread in News Week, McGraw-Hill published me on their Science and Technology Encyclopedia yearbook covers and National Geographic just happened to be working on a story on electron microscopes at around that time."
 
Soon, Scharf was exhibiting his SEM photography across the US and published his first book, 'Magnifications: Photography with the Scanning Electron Microscope'.
 
By this time, the engineer-now professional photographer had left Burroughs Corp to focus on producing images, using an ETEC at Scanning Electron Analysis Laboratories in Culver City.
 
"They were happy to have me there as I kept that ETEC fine-tuned," he quips.
 
Anopheles Mosquito imaged for National Geographic; this is one of Scharf's favourite images [Scharf]
 
Come the early 1980s, Scharf was, as he puts it: "making a living with publishing', accepting commissions from magazines, newspapers, journals and books far and wide. "People would pay me to investigate, say, a pictorial story, and I would take the subject matter and see what I could come up with," he says.
 
In 1982, his photographs appeared in the film, 'Blade Runner', becoming the first SEM images to feature in a motion picture.
 
And in 1984 Scharf set up his own lab and bought a used ETEC SEM which had scanning coils for real-time TV scanning. This allowed him to produce the first black and white videos of insects alive and moving in the instrument.
 
Beyond black and white
But while Scharf's initial success had stemmed from black and white imagery, this was soon to change.
 
In 1980, the editor of Time magazine had asked the up and coming photographer for a colour image of a fruit fly. Scharf replied, 'there is no colour in these images' and was told, 'just experiment; we'll give you the money to buy whatever you need'.
 
Scharf bought some maskers, toners, made 11 by 14 inch black and white prints, and eventually coloured his first image of a Mediterranean fruit fly. 
 
"It took three to four days and you had to wear a respirator while painting the maskers on. It was just excruciating," he says. "But you know, it was pretty cool and after that was published nobody really wanted black and white images anymore."
 
David Scharf: a pioneer of colour SEM.
 
Scharf went on to use Photoshop to colourise his images, but his initial tinkerings with the world of colour had also sowed the seeds for an invention that would revolutionise his SEM image production.
 
"I had been exasperated with having to spend three days to make a colour image and thought there's got to be another way to do this, so I looked back in my laboratory notebook from the 1970s and decided it must be possible to use multiple detectors to do this," he says.
 
Scharf was right. In 1994, he was awarded a US Patent for his 'Multi-detector Color Synthesizing Scanning Electron Microscope', with worldwide patents following.
 
Scharf had put together the system using parts from an ETEC system, kindly donated by Jean-Pierre Slakmon of Canada-based scientific instrument supplier, Soquelec.
 
The system uses multiple secondary electron detectors which share the electron emission from the sample.
 
Each detector’s signal is encoded with an arbitrary colour using a signal mixing system. The red, green and blue signals from the mixing system are sent to an analogue to digital converter to facilitate acquisition in digital format.
 
"In the beginning this technology was actually ahead of the available imaging technology, so I could only produce poor-quality images at 400 pixels or so," highlights Scharf. "But then 4Pi Analysis developed a serial analogue-digital system so I was then able to get 2048 by 1536 pixel images."
 
Human embryonic stem cell [Scharf]
 
Eventually, thanks to a donation from Nion co-founder, Ondrej Krivanek (then with Gatan), Scharf was able to use Gatan's DigiScan beam control and imaging processing system to accelerate colour image production further. And with system in tow, he won a contract for a new IMAX 3D film, 'The Hidden Dimension'. 
 
At this point, Scharf also switched from his beloved ETEC to a Cambridge Leo 440 SEM, financed by IMAX. As he puts it: "This stuff had never been done but the Leo with its LaB6 source was much better... and [the instrument] had this bend beaming option for 3D imaging."
 
So Scharf added his extra detectors and colour system to his latest SEM, and collaborating with Gatan software designers, John Hunt and Jacob Wilbrink, Scharf, used computer-controlled stop-motion techniques to create colour stop-motion imaging of his specimens traveling through microspace.
 
Scharf's breakthrough SEM movies were a first in the world of SEM and cinema, and from here he went onto create stop-motion colour high definition TV video sequences for US broadcaster PBS's prime-time documentary on microbiology, 'Intimate Strangers: Unseen Life on Earth'.
 
SEM video sequences for the National Geographic TV documentary on parasites, 'Body Snatchers', followed, with Scharf winning an EMMY for his contributions. He has been exhibiting, lecturing and winning awards, ever since.
 
Intuitive art
For Scharf, a good image means getting composition, lighting, dynamic range, sharpness and focus exactly how he wants it.
 
"All of these things put together have an emotional impact and it's hard to put a science to it as there's so many elements," he says. "You just kind of have to feel it after a while... try it, see how it feels and see how it looks."
 
The advent of digital photography has slashed the time taken for the photographer to create an image. But as he says: "Sometimes you still don't get it right first time and then you just have to trash that picture."
 
According to Scharf, his images of the Anopheles mosquito - which he captured for National Geographic - are amongst his favourite photographs.
 
"This was the most difficult image of a live insect that I ever had to do as the little guy was very delicate and just didn't last long in a vacuum," he says. "Many [specimens] just shriveled up while I was trying to photograph them, but the final results just turned out beautiful."
 
MEMS electrode array [Scharf]
 
Looking forward, Scharf would now like to collaborate with cancer researchers to capture the whole array of different cancers as well as revisit bacteria. In each instance, he would need to swap his 'now obsolete SEMs for a higher resolution field-emission SEM. 
 
"These new microscopes have upper secondary and upper back-scatter detectors, for example, so it would be interesting to experiment with a combination of different detectors and see the effect on colour," Scharf highlights.
 
Without a doubt, the end-results would be mind-boggling.
 
"I have always been able to exploit equipment to limits beyond what the manufacturers thought they were capable of," he adds. "FE-SEM microscopes take such astounding and beautiful images, and I just know with something like this I would be able to take my images so much further. It is such a privilege to be doing this work."
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