6 Absorption-Based Far-Field Label-Free Super-Resolution …
149
ratus was exploited to perform simultaneous multiwavelength PT imaging, enabling
the detection of two different species in one measurement [101]. The deployment of
diode laser pumped optical parametric oscillator finally enables full coverage of the
visible to NIR spectral region in PTM, which significantly enhances the ability to
differentiate complex samples using PT spectromicroscopy technique [102].
The concern of laser-induced photodamage, especially thermal damage, also
exists in visible beam excited PTM. Unlike fluorescence-based techniques that are
vulnerable to photobleaching, the photodamage in visible beam excited PTM mainly
comes from local thermal damage. The pump lasers used in PTM are usually nanosecond lasers, while the heat dissipation rate in condensed matters falls in tens of
nanoseconds to microsecond scale [103]. Therefore, potential damage from local
heat accumulation must be considered when determining the appropriate sampling
rate for PT experiments. Some hypothetical and experimental works investigating the
damage threshold of various biological samples have been reported [61, 104]. However, the experimental results of cell damage thresholds show dramatic variations
among different cell lines and pump beam wavelengths [105]. Thus, more systematic studies on the photodamage in visible excited PTM are in need to facilitate
spreading.
6.2.5 Mid-IR Excited Photothermal Microscopy
Ever since the publication of Coblentz’s high-quality IR spectral database in 1905
[106], advances in IR spectroscopy and spectromicroscopy including Fourier transform IR (FTIR) spectroscopy [26], FTIR imaging [27] attenuated total reflectance
IR imaging [107], and focal plane arrays IR imaging [108] have all improved the
measurement of IR absorption from the aspects of higher sensitivity and spatial resolution. However, the fundamental limit of IR diffraction (~5 µm) was not defeated
by preceding approaches. Besides, the broad spectral window (typically 2–10 µm)
invokes the problem of measurement accuracy since the wavelength-dependent light
scattering could produce dramatic fluctuations in the IR signal intensity, which leads
to huge baseline artifacts [109]. Furthermore, water shows strong absorption in the
entire mid-IR region, which hinders the application of IR spectroscopic imaging to
investigate biomolecules in living cells or organisms in aqueous environment. These
problems were addressed collectively by the invention of mid-IR excited PTM. We
will focus on the hardware and practical operation aspects of this technique in detail
in this section.
Mid-IR excited PTM was demonstrated by Furstenberg et al. in 2012 [110], about
20 years later than the first demonstration of visible excited PTM. The pivotal difficulties of developing mid-IR based PTM are: (1) the lack of high-quality mid-IR laser
sources; and (2) complications to integrate mid-IR pump beam with visible probe
beam in the same optical system without introducing severe chromatic aberrations
and mid-IR power losses.
149
ratus was exploited to perform simultaneous multiwavelength PT imaging, enabling
the detection of two different species in one measurement [101]. The deployment of
diode laser pumped optical parametric oscillator finally enables full coverage of the
visible to NIR spectral region in PTM, which significantly enhances the ability to
differentiate complex samples using PT spectromicroscopy technique [102].
The concern of laser-induced photodamage, especially thermal damage, also
exists in visible beam excited PTM. Unlike fluorescence-based techniques that are
vulnerable to photobleaching, the photodamage in visible beam excited PTM mainly
comes from local thermal damage. The pump lasers used in PTM are usually nanosecond lasers, while the heat dissipation rate in condensed matters falls in tens of
nanoseconds to microsecond scale [103]. Therefore, potential damage from local
heat accumulation must be considered when determining the appropriate sampling
rate for PT experiments. Some hypothetical and experimental works investigating the
damage threshold of various biological samples have been reported [61, 104]. However, the experimental results of cell damage thresholds show dramatic variations
among different cell lines and pump beam wavelengths [105]. Thus, more systematic studies on the photodamage in visible excited PTM are in need to facilitate
spreading.
6.2.5 Mid-IR Excited Photothermal Microscopy
Ever since the publication of Coblentz’s high-quality IR spectral database in 1905
[106], advances in IR spectroscopy and spectromicroscopy including Fourier transform IR (FTIR) spectroscopy [26], FTIR imaging [27] attenuated total reflectance
IR imaging [107], and focal plane arrays IR imaging [108] have all improved the
measurement of IR absorption from the aspects of higher sensitivity and spatial resolution. However, the fundamental limit of IR diffraction (~5 µm) was not defeated
by preceding approaches. Besides, the broad spectral window (typically 2–10 µm)
invokes the problem of measurement accuracy since the wavelength-dependent light
scattering could produce dramatic fluctuations in the IR signal intensity, which leads
to huge baseline artifacts [109]. Furthermore, water shows strong absorption in the
entire mid-IR region, which hinders the application of IR spectroscopic imaging to
investigate biomolecules in living cells or organisms in aqueous environment. These
problems were addressed collectively by the invention of mid-IR excited PTM. We
will focus on the hardware and practical operation aspects of this technique in detail
in this section.
Mid-IR excited PTM was demonstrated by Furstenberg et al. in 2012 [110], about
20 years later than the first demonstration of visible excited PTM. The pivotal difficulties of developing mid-IR based PTM are: (1) the lack of high-quality mid-IR laser
sources; and (2) complications to integrate mid-IR pump beam with visible probe
beam in the same optical system without introducing severe chromatic aberrations
and mid-IR power losses.