28
R. W. Taylor and V. Sandoghdar
the overall brightness [3]. In conventional bright-field microscopy this means that if
small, thin or otherwise weakly interacting samples cannot cast a shadow of sufficient
darkness then, to our eyes, it cannot be seen. An apt example of our limited contrast
sensitivity is that of the spider web [4]; viewed against the bright sky the web is
practically invisible, but when seen illuminated by the same sunlight against the
darker surface of the ground, the web is now readily observed. In other words, we
can circumvent the limitations of our visual sensitivity by diminishing the brightness
of the background against which we wish to detect a shadow. This situation is also
familiar to the astronomer, as favorable conditions for observing the faint light of
distant stars occurs when the surrounding sky is as dark as possible.
In microscopic imaging, the deliberate removal of background illumination is
known as dark-field imaging, with the first recorded instance of its implementation
dating back as early as 1830 to Joseph Jackson Lister (1786–1869) [5]. Despite its
effectiveness and advocates, dark-field microscopy initially failed to gain appreciation in the wider circles of microscopy, and was seen somewhat of a novelty. The
technique remained largely overlooked [6] until the realization of the potential for
dark-field to observe minute specimens that were otherwise invisible in bright-field.
Austrian Chemist Richard Adolf Zsigmondy (1865–1929) would beautifully demonstrate this principle in 1902. Zsigmondy, along with Henry Siedentopf (1872–1940),
presented the technique for the observation of nanometer-sized gold particles by eye.
Consisting of an orthogonal objective and condenser, the “ultramicroscope”—as it
was so named, allowed the observation of light scattered by the nanoparticles in the
absence of a background [7]. This technique facilitated a more sensitive detection
microscopy, which would enable, for example, the breakthroughs into identifying
the microoranisms responsible for syphilis. In recognition of his achievements, Zsigmondy would be awarded the Chemistry Nobel Prize in 1925, the first of many Nobel
Prizes to be awarded for advances in optical microscopy. It should be noted, however, that dark-field imaging becomes increasingly challenging for smaller objects,
especially those in the presence of larger ones, as noise and incomplete removal of
the background obscures detectability.
Where dark-field imaging seeks to better detection sensitivity through complete
removal of the background, a more powerful approach to higher imaging contrast is to
selectively shift the phase of the background illumination with respect to the incident
light that interacts with the sample. This realization would be made in the decade
following Zsigmondy’s Nobel Prize by Frits Zernike (1888–1966), who developed
such wisdom from the study of aberrations and coherence in diffraction gratings and
telescopic optics [8–10]. Phase Contrast Microscopy (PCM) [10–12], as it became
to be known, involved the addition of a phase plate to the illumination path in the
microscope with the effect of enhancing the bright-field contrast of specimens with
refractive index similar to that of the surrounding media. Phase Contrast Microscopy
represented a monumental advancement in microscopy, garnering Zernike the 1953
Physics Nobel Prize, with the successes brought about within the research of cancer
being cited as one of the many advances enabled by PCM.
Arguably, Zernike’s greater impact was to bring interferometric principles to the
fore of contemporary microscopy, specifically into biologically-orientated imaging,
R. W. Taylor and V. Sandoghdar
the overall brightness [3]. In conventional bright-field microscopy this means that if
small, thin or otherwise weakly interacting samples cannot cast a shadow of sufficient
darkness then, to our eyes, it cannot be seen. An apt example of our limited contrast
sensitivity is that of the spider web [4]; viewed against the bright sky the web is
practically invisible, but when seen illuminated by the same sunlight against the
darker surface of the ground, the web is now readily observed. In other words, we
can circumvent the limitations of our visual sensitivity by diminishing the brightness
of the background against which we wish to detect a shadow. This situation is also
familiar to the astronomer, as favorable conditions for observing the faint light of
distant stars occurs when the surrounding sky is as dark as possible.
In microscopic imaging, the deliberate removal of background illumination is
known as dark-field imaging, with the first recorded instance of its implementation
dating back as early as 1830 to Joseph Jackson Lister (1786–1869) [5]. Despite its
effectiveness and advocates, dark-field microscopy initially failed to gain appreciation in the wider circles of microscopy, and was seen somewhat of a novelty. The
technique remained largely overlooked [6] until the realization of the potential for
dark-field to observe minute specimens that were otherwise invisible in bright-field.
Austrian Chemist Richard Adolf Zsigmondy (1865–1929) would beautifully demonstrate this principle in 1902. Zsigmondy, along with Henry Siedentopf (1872–1940),
presented the technique for the observation of nanometer-sized gold particles by eye.
Consisting of an orthogonal objective and condenser, the “ultramicroscope”—as it
was so named, allowed the observation of light scattered by the nanoparticles in the
absence of a background [7]. This technique facilitated a more sensitive detection
microscopy, which would enable, for example, the breakthroughs into identifying
the microoranisms responsible for syphilis. In recognition of his achievements, Zsigmondy would be awarded the Chemistry Nobel Prize in 1925, the first of many Nobel
Prizes to be awarded for advances in optical microscopy. It should be noted, however, that dark-field imaging becomes increasingly challenging for smaller objects,
especially those in the presence of larger ones, as noise and incomplete removal of
the background obscures detectability.
Where dark-field imaging seeks to better detection sensitivity through complete
removal of the background, a more powerful approach to higher imaging contrast is to
selectively shift the phase of the background illumination with respect to the incident
light that interacts with the sample. This realization would be made in the decade
following Zsigmondy’s Nobel Prize by Frits Zernike (1888–1966), who developed
such wisdom from the study of aberrations and coherence in diffraction gratings and
telescopic optics [8–10]. Phase Contrast Microscopy (PCM) [10–12], as it became
to be known, involved the addition of a phase plate to the illumination path in the
microscope with the effect of enhancing the bright-field contrast of specimens with
refractive index similar to that of the surrounding media. Phase Contrast Microscopy
represented a monumental advancement in microscopy, garnering Zernike the 1953
Physics Nobel Prize, with the successes brought about within the research of cancer
being cited as one of the many advances enabled by PCM.
Arguably, Zernike’s greater impact was to bring interferometric principles to the
fore of contemporary microscopy, specifically into biologically-orientated imaging,