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M. J. Huttunen and A. Kiviniemi
Diffraction of light and the Abbe’s law limit the practical achievable lateral resolution of an optical microscope to around 200 nm, while the axial resolution is often
even coarser (∼600 nm) [4, 5]. This resolving power limits the possibilities to study
biological objects, which all exhibit molecular-scale features. For example, crucial
building blocks of biological matter, such as DNA, RNA, and proteins, are all only
a few nanometers in size. The Abbe’s limit for optical resolution is not, however,
a fundamental limit. As a consequence, several super-resolution techniques have
been developed during the past two decades providing resolution greatly beyond
the diffraction limit [6–11]. The vast majority of these super-resolution techniques
are based on fluorescent probes, and often even require specific photo-switchable
dyes [10]. For example, photo-activated localization microscopy (PALM) is based
on photoactivatable dyes [11]. On the other hand, structured illumination microscopy
(SIM) provides around 100 nm lateral resolution without requiring any specific
dyes [7, 12].
Despite the fact that fluorescent dyes are widely used in optical microscopy,
their use is not without problems. The labeling procedure maybe time consuming,
some of the dyes are expensive and all exhibit photobleaching making long-term
investigations difficult. Dyes may also disturb the studied system compromising the
interpretation of results. Therefore, there is a need for the development of label-free
super-resolution techniques, which are not hindered by the aforementioned problems
and could be more suitable for clinical research and applications.
Several diffraction-limited label-free techniques have been already developed and
are mostly based on autofluorescence of molecules or coherent scattering of light
from the studied object and include holographic approaches and speckle imaging [13–
15]. In addition, label-free imaging is possible by utilizing intrinsic nonlinear optical responses of objects, which also provide entirely new contrast mechanisms for
microscopy [16, 17]. However, the development of label-free super-resolution techniques has proved challenging, since most of the realized super-resolution schemes
are based on specific properties of fluorescent dyes. Perhaps due to this reason, many
of the proposed and demonstrated label-free super-resolution techniques are based
on the SIM scheme [18–23].
In this chapter, we discuss and review the recent progress on label-free superresolution microscopy. In particular, we focus on techniques making use of the
intrinsic nonlinear responses of objects, and which are based on the SIM scheme.
The chapter is organized as follows. First, we introduce the reader to the theory
behind conventional and laser-scanning SIM, and explain how the lateral resolution
is increased using these techniques. Second, we provide a short tutorial on nonlinear
optical microscopy, with the emphasis on the most commonly utilized nonlinear optical processes and in their practical implementation. Third, we review recent advances
in nonlinear label-free super-resolution microscopy and discuss their applications.
We end the chapter by highlighting some of the current challenges and discussing of
the possible future directions in nonlinear label-free super-resolution microscopy.
M. J. Huttunen and A. Kiviniemi
Diffraction of light and the Abbe’s law limit the practical achievable lateral resolution of an optical microscope to around 200 nm, while the axial resolution is often
even coarser (∼600 nm) [4, 5]. This resolving power limits the possibilities to study
biological objects, which all exhibit molecular-scale features. For example, crucial
building blocks of biological matter, such as DNA, RNA, and proteins, are all only
a few nanometers in size. The Abbe’s limit for optical resolution is not, however,
a fundamental limit. As a consequence, several super-resolution techniques have
been developed during the past two decades providing resolution greatly beyond
the diffraction limit [6–11]. The vast majority of these super-resolution techniques
are based on fluorescent probes, and often even require specific photo-switchable
dyes [10]. For example, photo-activated localization microscopy (PALM) is based
on photoactivatable dyes [11]. On the other hand, structured illumination microscopy
(SIM) provides around 100 nm lateral resolution without requiring any specific
dyes [7, 12].
Despite the fact that fluorescent dyes are widely used in optical microscopy,
their use is not without problems. The labeling procedure maybe time consuming,
some of the dyes are expensive and all exhibit photobleaching making long-term
investigations difficult. Dyes may also disturb the studied system compromising the
interpretation of results. Therefore, there is a need for the development of label-free
super-resolution techniques, which are not hindered by the aforementioned problems
and could be more suitable for clinical research and applications.
Several diffraction-limited label-free techniques have been already developed and
are mostly based on autofluorescence of molecules or coherent scattering of light
from the studied object and include holographic approaches and speckle imaging [13–
15]. In addition, label-free imaging is possible by utilizing intrinsic nonlinear optical responses of objects, which also provide entirely new contrast mechanisms for
microscopy [16, 17]. However, the development of label-free super-resolution techniques has proved challenging, since most of the realized super-resolution schemes
are based on specific properties of fluorescent dyes. Perhaps due to this reason, many
of the proposed and demonstrated label-free super-resolution techniques are based
on the SIM scheme [18–23].
In this chapter, we discuss and review the recent progress on label-free superresolution microscopy. In particular, we focus on techniques making use of the
intrinsic nonlinear responses of objects, and which are based on the SIM scheme.
The chapter is organized as follows. First, we introduce the reader to the theory
behind conventional and laser-scanning SIM, and explain how the lateral resolution
is increased using these techniques. Second, we provide a short tutorial on nonlinear
optical microscopy, with the emphasis on the most commonly utilized nonlinear optical processes and in their practical implementation. Third, we review recent advances
in nonlinear label-free super-resolution microscopy and discuss their applications.
We end the chapter by highlighting some of the current challenges and discussing of
the possible future directions in nonlinear label-free super-resolution microscopy.