2 Interferometric Scattering (iSCAT) Microscopy and Related Techniques
29
where the early success of staining techniques had quashed the necessity to search
for more elaborate means to improve contrast. Another interferometric modality that
has since become popular is Differential Interference Contrast (DIC) microscopy,
in which regions of differing optical paths in the sample, such as those occurring at
edges, are shown with enhanced contrast through the interference of parallel sheared
beams. The spatial displacement and angular deviation of the beams, on the order of
half the airy disk diameter, is achieved through a special prism introduced by Georges
Normarksi (1919–1997) which bears his name [13]. Interestingly, the conceptual
roots of DIC microscopy can be traced to the interferometer developed by Jules
Jamin (1818–1886) in 1856 [14], and the first interference microscope from Jacobus
Laurens Sirks (1837–1905) in 1893 [15]. Another important related invention was
that of holography as proposed by Dennis Gabor (1900–1979) in 1947 [16], an
accomplishment that would lead to Gabor being awarded the Physics Nobel Prize
in 1971. As a new technique, Holography was mainly employed in macroscopic
imaging, one of the first holographic microscopes was reported in 1966 [17], enabled
by the commercial availability of the laser which was invented several years prior.
By the era of the late 1950s, interferometric microscopy was in its heyday, with
a bewildering array of interferometric microscopes demonstrated, several of which
found commercial release. A comprehensive review of these microscopes is provided
in [18]. Popular categories of design for these interferometric microscopes include
[19, 20]: (1) the beam-shearing type, (2) a two-arm design, and (3) the dual focus type
in which the reference wave is focused to a different plane than that of the specimen.
Unfortunately, interferometric microscopies witnessed a general decline after the
1950s, save for their application in surface profilometry, e.g., in precision engineering, and later for inspection in the microelectronics industry, where reflection-based
Normarksi DIC and the Mirau interferometer objective remain workhorses to this
day [21]. The decline of interferometry as the principle imaging technique in the life
sciences was the result of a series of innovations and developments in fluorescence
microscopy [22, 23], such as the invention of immunolabeling in 1950, which led to a
meteoric rise in fluorescence imaging, aided by the subsequent invention of confocal
scanning microscopy in 1955 by Marvin Minsky (1927–2016) [24].
One interferometric technique that would thrive throughout the 1960–70s was
Interferometric Reflection Microscopy (IRM). This simple technique, which in fact
does not require an explicit interferometer, relies on the sample coverslip to provide the reference beam. The rise of IRM is widely accredited to Adam Curtis
(1934–2017), who investigated and estimated the nanometric separation between
glass and cell at sites of adhesion in living cultured fibroblasts [25]. The interferometric nature of the image provided nanometer-level information on the cell–
substrate distances—a resolution unmatched by fluorescent methodologies of the
time, enabling researchers using IRM to pioneer investigations into cell adhesion
[26–30] as well as associated cytoskeletal components [31]. Johan Sebastiaan Ploem
(born 1927) would later introduce instrumental refinements to better the sensitivity
and performance of the technique under the alternative name Reflection Contrast
Microscopy (RCM) [26]. Ultimately the failure to interpret quantitatively the interferometric content of the images, i.e., accounting for contributions from variations
29
where the early success of staining techniques had quashed the necessity to search
for more elaborate means to improve contrast. Another interferometric modality that
has since become popular is Differential Interference Contrast (DIC) microscopy,
in which regions of differing optical paths in the sample, such as those occurring at
edges, are shown with enhanced contrast through the interference of parallel sheared
beams. The spatial displacement and angular deviation of the beams, on the order of
half the airy disk diameter, is achieved through a special prism introduced by Georges
Normarksi (1919–1997) which bears his name [13]. Interestingly, the conceptual
roots of DIC microscopy can be traced to the interferometer developed by Jules
Jamin (1818–1886) in 1856 [14], and the first interference microscope from Jacobus
Laurens Sirks (1837–1905) in 1893 [15]. Another important related invention was
that of holography as proposed by Dennis Gabor (1900–1979) in 1947 [16], an
accomplishment that would lead to Gabor being awarded the Physics Nobel Prize
in 1971. As a new technique, Holography was mainly employed in macroscopic
imaging, one of the first holographic microscopes was reported in 1966 [17], enabled
by the commercial availability of the laser which was invented several years prior.
By the era of the late 1950s, interferometric microscopy was in its heyday, with
a bewildering array of interferometric microscopes demonstrated, several of which
found commercial release. A comprehensive review of these microscopes is provided
in [18]. Popular categories of design for these interferometric microscopes include
[19, 20]: (1) the beam-shearing type, (2) a two-arm design, and (3) the dual focus type
in which the reference wave is focused to a different plane than that of the specimen.
Unfortunately, interferometric microscopies witnessed a general decline after the
1950s, save for their application in surface profilometry, e.g., in precision engineering, and later for inspection in the microelectronics industry, where reflection-based
Normarksi DIC and the Mirau interferometer objective remain workhorses to this
day [21]. The decline of interferometry as the principle imaging technique in the life
sciences was the result of a series of innovations and developments in fluorescence
microscopy [22, 23], such as the invention of immunolabeling in 1950, which led to a
meteoric rise in fluorescence imaging, aided by the subsequent invention of confocal
scanning microscopy in 1955 by Marvin Minsky (1927–2016) [24].
One interferometric technique that would thrive throughout the 1960–70s was
Interferometric Reflection Microscopy (IRM). This simple technique, which in fact
does not require an explicit interferometer, relies on the sample coverslip to provide the reference beam. The rise of IRM is widely accredited to Adam Curtis
(1934–2017), who investigated and estimated the nanometric separation between
glass and cell at sites of adhesion in living cultured fibroblasts [25]. The interferometric nature of the image provided nanometer-level information on the cell–
substrate distances—a resolution unmatched by fluorescent methodologies of the
time, enabling researchers using IRM to pioneer investigations into cell adhesion
[26–30] as well as associated cytoskeletal components [31]. Johan Sebastiaan Ploem
(born 1927) would later introduce instrumental refinements to better the sensitivity
and performance of the technique under the alternative name Reflection Contrast
Microscopy (RCM) [26]. Ultimately the failure to interpret quantitatively the interferometric content of the images, i.e., accounting for contributions from variations