fluorescent (FL) protein in the 1960s, which allowed to “highlight” the structures
inside living cell, the achievement which heralded a new era in cell biology. In the
1990s, it was shown that certain optical nonlinear and single-molecule FL detection
properties allow achieving extraordinary high, nanometer-scale resolutions in
methods such as stimulated emission depletion (STED) and localization microscopies. Due to very strong contrast mechanism and extreme sensitivity up to a
single-molecule level, these techniques became a method of choice in biomedical
studies. The development of super-resolved FL microscopy was recognized by the
2014 Nobel Prize in Chemistry awarded jointly to the pioneers of these methods,
Eric Betzig, Stefan W. Hell, and William E. Moerner. Although the resolutions
available in these methods go far beyond the classical diffraction limit, these
methods also have drawbacks. The speed of FL imaging can be rather slow because
of the relatively weak signals. The FL intensity dims over time because the
fluorophore is being degraded by light (photobleaching). In addition, FL labeling
may induce undesirable effects like phototoxicity. Besides, labeling itself may be
difficult for some specimens.
The label-free microscopy (LFM), which does not require sample staining, is a
desirable option and it is in a high demand for even wider range of applications than
FL microscopy. However, the development of LFM methods took longer time
compared to a rapid progress of FL microscopy since the LFM mechanisms rely on
subtler light-scattering processes in nanoscale objects, resulting in lower effective
image contrasts. The pursuit of super-resolution in LFM is also somewhat complicated since we cannot rely on nonlinear and localization properties offered by
individual dye molecules. It should be also noted that the resolution quantification
can be a controversial issue in the case of LFM performed using coherent or partly
coherent illumination schemes compared to more straightforward resolution
quantification in the case of incoherent FL imaging.
The turning point in the LFM development can be dated to the 1950s when the
phase-contrast microscopy (PCM) was introduced by Frits Zernike (1953 Nobel
Prize in Physics). It was followed by the development of a large family of interferometric detection techniques such as differential interference contrast (DIC),
reflection, and Mirau interference microscopy, as well as more modern digital
holographic microscopy (DHM). Although being diffraction-limited, these methods
demonstrated the best spatiotemporal resolution among regular microscopy techniques for various biomedical samples. On a somewhat separate note, the development of electron microscopy with unsurpassed subnanometer spatial resolution in
the 1960s was an extraordinarily important achievement; however, the light
microscopy has remained the most widely used method for biomedical imaging. As
it was already stated, a powerful impact on the development of LFM techniques
was produced by the invention of NSOM in the 1980s, which can offer a
nanometer-scale resolution, but by the expense of being relatively slow
point-by-point scanning technology. One more important development was structured illumination microscopy (SIM) introduced around the year 2000 providing a
twofold increase in resolution compared to the diffraction limit. A new momentum
for LFM imaging was produced in the past two decades with the advent of
vi
Preface
inside living cell, the achievement which heralded a new era in cell biology. In the
1990s, it was shown that certain optical nonlinear and single-molecule FL detection
properties allow achieving extraordinary high, nanometer-scale resolutions in
methods such as stimulated emission depletion (STED) and localization microscopies. Due to very strong contrast mechanism and extreme sensitivity up to a
single-molecule level, these techniques became a method of choice in biomedical
studies. The development of super-resolved FL microscopy was recognized by the
2014 Nobel Prize in Chemistry awarded jointly to the pioneers of these methods,
Eric Betzig, Stefan W. Hell, and William E. Moerner. Although the resolutions
available in these methods go far beyond the classical diffraction limit, these
methods also have drawbacks. The speed of FL imaging can be rather slow because
of the relatively weak signals. The FL intensity dims over time because the
fluorophore is being degraded by light (photobleaching). In addition, FL labeling
may induce undesirable effects like phototoxicity. Besides, labeling itself may be
difficult for some specimens.
The label-free microscopy (LFM), which does not require sample staining, is a
desirable option and it is in a high demand for even wider range of applications than
FL microscopy. However, the development of LFM methods took longer time
compared to a rapid progress of FL microscopy since the LFM mechanisms rely on
subtler light-scattering processes in nanoscale objects, resulting in lower effective
image contrasts. The pursuit of super-resolution in LFM is also somewhat complicated since we cannot rely on nonlinear and localization properties offered by
individual dye molecules. It should be also noted that the resolution quantification
can be a controversial issue in the case of LFM performed using coherent or partly
coherent illumination schemes compared to more straightforward resolution
quantification in the case of incoherent FL imaging.
The turning point in the LFM development can be dated to the 1950s when the
phase-contrast microscopy (PCM) was introduced by Frits Zernike (1953 Nobel
Prize in Physics). It was followed by the development of a large family of interferometric detection techniques such as differential interference contrast (DIC),
reflection, and Mirau interference microscopy, as well as more modern digital
holographic microscopy (DHM). Although being diffraction-limited, these methods
demonstrated the best spatiotemporal resolution among regular microscopy techniques for various biomedical samples. On a somewhat separate note, the development of electron microscopy with unsurpassed subnanometer spatial resolution in
the 1960s was an extraordinarily important achievement; however, the light
microscopy has remained the most widely used method for biomedical imaging. As
it was already stated, a powerful impact on the development of LFM techniques
was produced by the invention of NSOM in the 1980s, which can offer a
nanometer-scale resolution, but by the expense of being relatively slow
point-by-point scanning technology. One more important development was structured illumination microscopy (SIM) introduced around the year 2000 providing a
twofold increase in resolution compared to the diffraction limit. A new momentum
for LFM imaging was produced in the past two decades with the advent of
vi
Preface