December 6, 2009 | David F. Coppedge

Microscopy’s Golden Age Is At Hand

Like test pilots breaking the sound barrier, microscope makers are breaking a light barrier some said was physically impossible: the diffraction limit.  Within the next 5 to 10 years, we may see more and more images of phenomena at the molecular scale – not with electron microscopes, but with light microscopes in real time.  What amazing vistas will come into focus?
    Nature posted a short Technology Feature on microscopy.  The lead article1 describes how the diffraction limit was believed for well over a century to be unbreakable.  Ernst Abbe, a German physicist, had said in 1873 that the wavelength of light posed a fundamental limit on the resolution of optical instruments.  “For many years it was a source of frustration for biologists that the internal components of a cell were practically invisible to them.” wrote Kelly Rae Chi in her article, “Microscopy: Ever-increasing resolution.”2  “Researchers believed that the wavelength of light determined a fundamental limit to the resolution of optical microscopes.”
    Never say “never” to inventors.  Chi described several “diffraction-busting technologies” that are breaking the barrier without killing any test pilots.  We have Hell to pay for breaching the limit: Stefan Hell, that is, a German physicist who first suggested that the diffraction limit could be beaten. 

Hell, while a postdoc at the University of Turku in Finland in the 1990s, thought that, with the right lasers, he could activate a fluorescent spot and then shrink it by superimposing a larger, hollow beam of light to deplete all the light emission except for that at the centre of the spot.  He called the technique stimulated emission depletion (STED) microscopy.  Although many physicists were initially sceptical of Hell’s ideas, by 2000 he had used STED to produce the first nanoscale fluorescence images.
    Super-resolution microscopy has blossomed since, allowing researchers to see cellular processes unfolding at nanometre scales.  “This is something that the field has desired since people first started looking through light microscopes,” says Jan Liphardt, a biophysicist at the Lawrence Berkeley National Laboratory in California.

Other methods include compiling images of billions of fluorescent proteins, photo-activated localization microscopy (PALM), and “stochastic optical reconstruction microscopy (STORM), which uses photoswitchable probes to temporally separate the overlapping images of individual molecules and so boost resolution to ten times better than the diffraction limit,” and fluorescence PALM (fPALM), which “involves looking at thousands of fluorophores at once, and localizing on small numbers at a time.”  Then there’s iPALM (interferometry PALM) that creates its images in 3-D.  Another method, 3-D SIM, creates images by bar-coding samples with light patterns and creates images of the underlying structure by analyzing the Moire fringes produced.  Chi writes that “the field is just warming up.”
    In her second article, Chi quoted W. E. Moerner (Stanford) sharing his excitement: “There’s a huge explosion of interest and progress.  That makes it very exciting to watch and to participate in.”  These technologies are just beginning to produce products from leading manufacturers like Carl Zeiss, Leica, and Nikon.  “Super-resolution technology allows researchers to see details that are difficult or impossible to image with conventional light microscopes – at resolutions of 100 nanometres or better.
    What can the public expect to see over the shoulders of these pioneers?  Already these techniques have been used to watch proteins in action.  3D STORM has been used to image microtubules in kidney cells as well as whole cells.  And “Hell’s group, early in 2008, used the STED method to show the movement of synaptic vesicles inside living neurons at video rate.” 

“To people like me who were trained in physics or optics in the 1990s, it’s just unbelievable that one can image below the resolution of light,” says Bernardo Sabatini, a neurobiologist at Harvard Medical School in Boston, Massachusetts.  “The major revolution for the next 5 or 10 years is getting these advances to answer biological questions.”

The sky’s the limit as these new test pilots are on a roll maneuver.  Using combination strategies, “The pioneers of super-resolution microscopy are continuing to improve their methods with better sample preparation, a few strategically placed pieces of hardware and more sophisticated algorithms.”  And these techniques are now being applied to live imaging of cells.  That was always a drawback of electron microscopes – the samples had to be killed and coated before turning on the electron beam.  Already a SIM test has allowed scientists to “see proteins moving along individual microtubules within living cells…at 100 nm resolution.”  And it’s going to get faster – cameras shooting 1000 frames per second are already available.  Add additional colors to the probes, and real-time 3-D visualization of the molecular machines in living cells is a distinct possibility.
    Chi quoted a biologist who compared this revolution to another optical triumph: “We’re starting to get pictures out of the mouse brain that rival anything from the Hubble Space Telescope, and we’re just getting started,” Stephen Smith (Stanford) said.  Chi ended,

The promise of super-resolution microscopy – thought for so long to be little more than a dream – is starting to become a reality.  Researchers have taken different approaches and are using tools and techniques borrowed from physics, chemistry and computing technology to bring the nanoscopic world to our macroscopic eyes.  Although commercialization is progressing, there is still plenty of room for the do-it-yourself biologist to modify and improve their systems, and produce images of stunning complexity that will rival anything else in science.

Leeuwenhoek’s best microscope had a top resolution of 1.4 microns (micrometers).3  These new instruments are exceeding 100 nanometers – 100 times more detail.  Some of the systems are already succeeding in getting images down in the 10-20 nm range.  A bacterial flagellum motor is about 60nm in diameter and its tail is 500nm long.  ATP synthase is about 10×20 nm.  Till now, micro-imaging techniques have seen these structures “through a glass, darkly.”4  The super-resolution microscope revolution is approaching the frontier of bringing these molecular machines into clear focus.

1.  Technology Feature, “Microscopy: Breaking the light barrier,” Nature 462, 676 (3 December 2009) | doi:10.1038/462676a.
2.  Kelly Rae Chi, “Microscopy: Ever-increasing resolution,” Nature 462, 675-678 (3 December 2009) | doi:10.1038/462675a.
3.  See our biography of Leeuwenhoek in full at this site.  It was published in Christian History & Biography (Issue 76, 2002).
4.  I Corinthians 13:12, King James Version.

Wow; this is a wonderful application of scientific knowledge producing more scientific knowledge – not the Darwinian storytelling kind, mind you, but real observational knowledge.  You know, this could do to the Darwin Party what high-resolution ultrasound is doing to the abortion industry – showing real-time evidence that challenges its assumptions.  Abortionists have long treated the human fetus as a lump of tissue.  3-D live ultrasound is showing more and more moms and dads that there is a real, live baby there in the womb – a human being with emotions, feelings and a personality all its own.  A picture is worth a thousand claims by Planned Parenthood that abortion is just a “medical procedure” for “women’s health”.  World Magazine echoed this idea in its article on “Daniel of the Year” Stephen Meyer: “As ultrasound machines have undercut abortion, so information revolutions have led more scientists to embrace ID” (intelligent design).
    Terrific animations of cellular machines exist (as in Unlocking the Mystery of Life and the movie Expelled), but they tend to be highly stylized models.  The impact of the real thing could be as great as a Hubble image compared to a painting.  So what will the Darwinists do when real-time, 3-D images of actual cellular machines hit the big screen?  Oh, they will continue to claim this is all the product of chance mutation and natural selection.  Facts have never gotten in the way of their ideology.  But people will trust their eyes, not the Darwinian spin doctors.  The Darwin Party hacks will have to face reality, or will be cast in the role of the academics who refused to look through Galileo’s telescope.  Bring it on.

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