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C. Li and J.-X. Cheng
sensitivity. The limit of detection of visible excited PTM was first determined to be
at femtogram level [95]. This limit was soon pushed to single nanoparticle detection
[81], statistically sub-molecule detection [96], and single-molecule imaging [82].
Note that the impact of solvents in sample cells on SNR has already been noticed
and investigated. Since the PT signal is proportional to the refractive change rate as a
function of the change in temperature (∂n/∂ T ), this value determines the signal level
and SNR when other parameters are the same. It was demonstrated experimentally
that performing PT detections in glycerol results in five times improvement of the
SNR than in water [74]. Furthermore, recent experiments indicated that critical Xeon
medium will enhance the SNR by over 100 times compared to glycerol [97].
Visible excited PTM endows several advantages over other label-free microscopy
techniques. First, for many molecules, electronic state absorption cross-sections are
much larger (>10
9 times) than vibrational state absorption or Raman scattering processes, resulting in extremely sensitive detection of various species [98]. For instance,
heme proteins are known to have extremely fast internal conversion rate and short
excited state lifetime (<50 fs) of Soret band such that almost all absorbed photon
energy is converted to heat [99, 100]. Therefore, PT effect provides ideal contrast for
the detection of these molecules at fairly low concentrations (100 µM hemoglobin
solution) [98]. Besides, gold nanoparticles and nanorods are a group of nano-objects
that often being studied using visible excited PTM since the plasmonic resonances
at 550–800 nm increases local electromagnetic fields and enhances the PT signals
allowing nanoparticles as small as 1.4 nm to be detected [80].
Another advantage is that optics are well-designed and readily operated for visible
beams compared to mid-IR sources that require special materials for coatings and
substrates. There are no specific sample preparation requirements for visible excited
PTM compared to fluorescence microscopy or IR microscopy. Liquid sample cells
or sandwiched glass slides are usually used for transparent samples at forward detection mode. Although some experiments were conducted in glycerol or other organic
solvents in order to enhance the SNR, the practical steps are nothing more complicated than preparing samples suspended in water. For opaque samples, backward
detection mode allows visible excited PT imaging of only the surface of the samples
due to dramatic power attenuation as both pump and probe beams propagate deep
into the samples. But the operation is relatively easy in this case. Such advantage significantly simplifies the customized development, broader applications, and routine
maintenance of the PT microscopes.
Third, laser engineering has come a long way in developing visible laser sources
with higher power, lower noise, and broader tunable range, which all benefits the
overall performances of PTM with visible pump beams. At first, the pump and probe
beams power in samples are limited to 2 and 0.1 mW, respectively [96]. The application of high-power lasers increases the power dissipated to samples to 10 times
for each beam, which leads to 100 times PT signal and 10 times SNR, given the
quadratic relation of laser power and signal level in PT process [74]. Before tunable
lasers were implemented, PTM is excited by monochromatic visible pump beams
including He–Ne laser, Ar–ion laser, and 532 nm diode laser, through which only
one type of molecules can be determined at a time. Then, dual pump beams appa-
C. Li and J.-X. Cheng
sensitivity. The limit of detection of visible excited PTM was first determined to be
at femtogram level [95]. This limit was soon pushed to single nanoparticle detection
[81], statistically sub-molecule detection [96], and single-molecule imaging [82].
Note that the impact of solvents in sample cells on SNR has already been noticed
and investigated. Since the PT signal is proportional to the refractive change rate as a
function of the change in temperature (∂n/∂ T ), this value determines the signal level
and SNR when other parameters are the same. It was demonstrated experimentally
that performing PT detections in glycerol results in five times improvement of the
SNR than in water [74]. Furthermore, recent experiments indicated that critical Xeon
medium will enhance the SNR by over 100 times compared to glycerol [97].
Visible excited PTM endows several advantages over other label-free microscopy
techniques. First, for many molecules, electronic state absorption cross-sections are
much larger (>10
9 times) than vibrational state absorption or Raman scattering processes, resulting in extremely sensitive detection of various species [98]. For instance,
heme proteins are known to have extremely fast internal conversion rate and short
excited state lifetime (<50 fs) of Soret band such that almost all absorbed photon
energy is converted to heat [99, 100]. Therefore, PT effect provides ideal contrast for
the detection of these molecules at fairly low concentrations (100 µM hemoglobin
solution) [98]. Besides, gold nanoparticles and nanorods are a group of nano-objects
that often being studied using visible excited PTM since the plasmonic resonances
at 550–800 nm increases local electromagnetic fields and enhances the PT signals
allowing nanoparticles as small as 1.4 nm to be detected [80].
Another advantage is that optics are well-designed and readily operated for visible
beams compared to mid-IR sources that require special materials for coatings and
substrates. There are no specific sample preparation requirements for visible excited
PTM compared to fluorescence microscopy or IR microscopy. Liquid sample cells
or sandwiched glass slides are usually used for transparent samples at forward detection mode. Although some experiments were conducted in glycerol or other organic
solvents in order to enhance the SNR, the practical steps are nothing more complicated than preparing samples suspended in water. For opaque samples, backward
detection mode allows visible excited PT imaging of only the surface of the samples
due to dramatic power attenuation as both pump and probe beams propagate deep
into the samples. But the operation is relatively easy in this case. Such advantage significantly simplifies the customized development, broader applications, and routine
maintenance of the PT microscopes.
Third, laser engineering has come a long way in developing visible laser sources
with higher power, lower noise, and broader tunable range, which all benefits the
overall performances of PTM with visible pump beams. At first, the pump and probe
beams power in samples are limited to 2 and 0.1 mW, respectively [96]. The application of high-power lasers increases the power dissipated to samples to 10 times
for each beam, which leads to 100 times PT signal and 10 times SNR, given the
quadratic relation of laser power and signal level in PT process [74]. Before tunable
lasers were implemented, PTM is excited by monochromatic visible pump beams
including He–Ne laser, Ar–ion laser, and 532 nm diode laser, through which only
one type of molecules can be determined at a time. Then, dual pump beams appa-