Chapter 7
Label-Free Pump–Probe Nanoscopy
Paolo Bianchini, Giulia Zanini and Alberto Diaspro
Abstract In the last few decades fluorescence microscopy has been the most widely
used microscopy technique and much effort has been put into the development of
advanced super-resolution fluorescence microscopy techniques to circumvent the
diffraction limit. Despite their well-established benefits, these techniques have to
rely on the photo-physical properties of fluorescent molecules to obtain the desired
contrast and spatial resolution. The labeling procedure may cause unwanted alterations in the sample. With the advent of ultrashort-pulsed laser sources, it became
possible to better explore novel non-fluorescent-based contrast mechanisms that rely
solely on intrinsic properties of the molecules of interest and which led to the development of label-free microscopy approaches. In this chapter, the imaging capabilities
of absorption-based pump–probe microscopy are presented. This technique explores
the ultrafast dynamic properties of the sample with high spatial and temporal resolution, as well as high sensitivity and chemical specificity. Two pulses, a pump and
a probe, with a proper spatial and temporal overlap are used. The pump is absorbed,
inducing a measurable change in the sample carrier population, which is then monitored by a delayed probe pulse. The development of new label-free approaches
also represents a key challenge for the exploration of super-resolution approaches in
non-fluorescence-based methods.
7.1 Nonlinear Optical Interactions
Familiar optical phenomena, like reflection, refraction, and absorption, which allow
us to see the colors or to focus the light by a lens, are a consequence of the interaction
of light with matter. They can be explained taking into account the wave-like nature
of light and assigning a set of optical parameters to the material. In normal conditions
these parameters are constant (linear optics), while under strong illumination they
become functions of the light intensity (nonlinear optics).
P. Bianchini (B) · G. Zanini · A. Diaspro
Istituto Italiano di Tecnologia, via Morego 30, 16163 Genoa, Italy
e-mail: paolo.bianchini@iit.it
© Springer Nature Switzerland AG 2019
V. Astratov (ed.), Label-Free Super-Resolution Microscopy,
Biological and Medical Physics, Biomedical Engineering,
https://doi.org/10.1007/978-3-030-21722-8_7
171
Label-Free Pump–Probe Nanoscopy
Paolo Bianchini, Giulia Zanini and Alberto Diaspro
Abstract In the last few decades fluorescence microscopy has been the most widely
used microscopy technique and much effort has been put into the development of
advanced super-resolution fluorescence microscopy techniques to circumvent the
diffraction limit. Despite their well-established benefits, these techniques have to
rely on the photo-physical properties of fluorescent molecules to obtain the desired
contrast and spatial resolution. The labeling procedure may cause unwanted alterations in the sample. With the advent of ultrashort-pulsed laser sources, it became
possible to better explore novel non-fluorescent-based contrast mechanisms that rely
solely on intrinsic properties of the molecules of interest and which led to the development of label-free microscopy approaches. In this chapter, the imaging capabilities
of absorption-based pump–probe microscopy are presented. This technique explores
the ultrafast dynamic properties of the sample with high spatial and temporal resolution, as well as high sensitivity and chemical specificity. Two pulses, a pump and
a probe, with a proper spatial and temporal overlap are used. The pump is absorbed,
inducing a measurable change in the sample carrier population, which is then monitored by a delayed probe pulse. The development of new label-free approaches
also represents a key challenge for the exploration of super-resolution approaches in
non-fluorescence-based methods.
7.1 Nonlinear Optical Interactions
Familiar optical phenomena, like reflection, refraction, and absorption, which allow
us to see the colors or to focus the light by a lens, are a consequence of the interaction
of light with matter. They can be explained taking into account the wave-like nature
of light and assigning a set of optical parameters to the material. In normal conditions
these parameters are constant (linear optics), while under strong illumination they
become functions of the light intensity (nonlinear optics).
P. Bianchini (B) · G. Zanini · A. Diaspro
Istituto Italiano di Tecnologia, via Morego 30, 16163 Genoa, Italy
e-mail: paolo.bianchini@iit.it
© Springer Nature Switzerland AG 2019
V. Astratov (ed.), Label-Free Super-Resolution Microscopy,
Biological and Medical Physics, Biomedical Engineering,
https://doi.org/10.1007/978-3-030-21722-8_7
171