The tendencies observed for PT imaging and PT thermolens signals at different laser energies appeared also in the formation of PA signals. The PA signal from QDs had a initial classic bipolar shape 9
transformed into a pulse train due to reflection and resonance effects (). An increase in laser energy led to increased PA signals, which were enhanced by bubble formation effects leading to non-regular signal shapes (). Specifically, the PA signal amplitude gradually increased with an increase of laser fluence in the linear range of 0.1–1 J/cm2
(). Then, a nonlinear increase started at 1.5 J/cm2
with a more profound PA amplitude enhancement (10–20 times) at relatively high fluences in the range of 3–7 J/cm2
, which was associated with the afore-mentioned microbubble formation phenomena. Saturation of the PA signal was observed at energy fluences above 15 J/cm2
, likely associated with laser-induced destruction of QDs. This was indeed confirmed by the behavior of PA signal amplitudes as a function of laser pulse number at various laser fluences (). There was no sign of laser-induced alteration of PA signal amplitude and hence absorption QD properties (the PA signal is proportional to coefficient absorption of targets) 3
at relatively low fluence, below 3 J/cm2
. Thus, contrary to fluorescence 1
, the PT signals did not show any blinking behavior. However, at higher fluences, significant decreases in PA signal amplitudes were observed with an increase in pulse number. In particular, at 12.4 J/cm2
, after the first laser pulse the signal decreased ~2 times and almost disappeared after 10 pulses, confirming the possibility of laser-induced disintegration of QDs likely through melting or thermal-based explosion phenomena 12
Figure 4 (a) PA signal amplitudes from QDs as a function of laser fluence after one pulse. (b) PA signal amplitude from QDs as function of laser pulse number at various laser fluences: 12.4 (filled blue diamond), 6.2 J/cm2 (filled yellow square), 4.0 J/cm2 ((filled (more ...)
Fluorescent imaging revealed inhibition of fluorescence with an increase in laser energy, up to complete disappearances of emission at high energy (). It should be noted that this fluorescence inhibition was observed at a relatively low laser pulse energy, around 0.5 J/cm2, at which there was not yet any sign of PA signal degradation (). This indicates that the mechanisms responsible for fluorescent degradation and “PA bleaching” may have different origins, for example, related to photochemical and PT-based phenomena, respectively.
PA spectra of QDs obtained with a pulse laser were in relatively good agreement with the conventional absorption spectra obtained in suspension with a high QD concentration (). Both of them contain an absorption peak near 640 nm and 635 nm, respectively, both blue shifted from the emission line. However, compared to the conventional spectra, the width of the PA spectra peak is more narrow, the PA peak is slightly red shifted, and at wavelengths shorter than 570 nm the PA rate of signal increase is more profound with decreasing wavelength. The observed phenomena leading to the “sharpening” of PA spectra near the absorption spectral maximum should be associated with non-linear PT-based enhancement of PA signals around QDs, since overheating is more profound when the laser wavelength corresponds to the QD absorption wavelength.
Figure 5 Conventional absorption spectra (solid blue curve) and emission spectra of QDs (dash red curve) provided by manufacturers. PA spectra of QDs (thin slide with water suspension) with PA signal amplitude as a function of laser wavelength at one laser pulse, (more ...)
Replacement of water with ethanol led to an increase in both PT and PA signals from QDs of about 5–7 fold at the same level of laser energy. This is in line with our previous data 10
, where we observed that ethanol provides more profound heating and more effective bubble formations in laser pulse mode because of its better thermodynamic parameters compared to water (see below), including higher (~3.5 times) coefficient of thermal expansion. We further found that the PA signals from the coated QDs exceeded PA signals from uncoated QDs by several times, at the same laser parameters (639 nm, 0.5 J/cm 2
). According to our previous findings 6
, this signal enhancement could be a consequence of various features of the external low-absorbing layers: 1) the layer functions as a thermal insulator, thus providing more effective heat accumulation in the internal core and thus elongating the thermal relaxation time τT
of QDs (for the core alone the τT
lies in sub-nanosecond range), for better thermal confinement with an 8-ns laser pulse; 2) it improves acoustic and thermal confinements due to larger nanoparticle size; and 3) due to fast thermal expansion it provides more effective generation of PA waves, especially when overheating of the QD core is accompanied by non-linear phenomena such as nanoparticle melting, explosion, and fragmentation 12
. Because bubble formation is the main mechanism of cell damage in laser pulse mode 8
, the demonstrated capability of QDs as a PT sensitizer and “bubble” contrast agent allows an extension of QD applications to PT therapy of cancer cells and infections. When QDs are in close proximity of each other, the overlapping of micro-or nano-bubbles from individual QDs () may enhance therapeutic efficiency 13
. Hence, in addition to thermal, acoustic and other confinements as discussed above, we can introduce bubble confinement,
when the minimal size of bubbles matches to distances between QDs. Laser-induced removal of the protective coating around QDs and/or some disintegration of QD crystals (e.g., through core melting or explosion 12
) at increased laser energy may be considered as another potential killing mechanism, since uncoated QDs are associated with increased toxicity 1
. However, this issue requires further study, including damage localization, and clearance of toxic products from organism.
The obtained results clearly demonstrate the potential of QDs to serve as PT and PA contrast agents using an advanced multimodal PT-PA-fluorescent microscope. The highest absorption sensitivity at the single QD level with high spatial resolution (350 nm) was achieved in the pump-probe time-resolved PTI mode (), with image contrast comparable to that of fluorescent imaging (). Of note, the PTI mode provided images of some QDs that did not appear in fluorescent images. These data are in line with results from other groups 4
that indicate that low-intensity fluorescent QDs hold absorption properties that are detectable with the PT technique. The additional advantage of the PTI-mode is its fast image acquisition algorithm (10−1
s, depending on CCD camera speed and laser pulse rate), which requires just one laser pulse with a broad beam diameter (10–30 μm) to create a cell image with a resolution approaching the optical diffraction limit 8
. In comparison, conventional image algorithms use time-consuming scanning of a focused laser beam across cells and requires seconds if not minutes to obtain a single cell image 1
The minimal number of QDs producing readable signals in PT thermolens or PA modes () was estimated to be 140 and 290 QDs in the detected (i.e. irradiated) volume, respectively. This was verified by measuring signals of a serial dilution of an initial suspension with a known QD concentration, as provided by the manufacturer. In addition, the number of QDs in the laser beam volume at low QD concentration was estimated by simultaneous fluorescent imaging. According to literature, in most applications the typical number of QDs inside labeled cells is around several hundred and higher, up to 104
, and references therein). Thus, PT thermolens or PA methods should be able to provide detection of single cells containing ≥300–500 QDs in vitro
and potentially in vivo
Interestingly, the PA detection limit for QDs alone was worse compared to our recently demonstrated PA detection of a just few gold nanorods or their small clusters 6
, which absorb much stronger in NIR range than QDs. Therefore, based on our previous 6
and current findings, we further identified several ways to improve the QD detection limit by increasing conversion of laser energy into PT, PA, and bubble formation phenomena. In particular, the PA signal is proportional to the absorbed laser energy transformed through non-radiative relaxation into heat, and then through thermal expansion phenomena into PA waves. More specifically, according to theoretical models 8
, the PA signal is proportional to laser energy, the absorption cross-section, the coefficient of thermal expansion and inversely proportional to density and heat capacity. In addition, bubble formation, as a PA signal enhancer and cell killer, is more effective at a low boiling point and vaporization heat. Thus, the proposed technology has a great potential for further the improvement by optimization of all these parameters.
First, an increase in sensitivity could be achievable by decreasing the laser pulse duration to the picosecond territory, which would match better to the thermal and, especially, acoustic confinements, since the thermal τT
and acoustic τA
relaxation times lie in the sub-nanosecond range (see above). Alternatively, the QD relaxation times could be increased by using larger QDs (τT
~ R), or by providing conditions for self-assembly of QDs into clusters, in which overlapping of laser-induced optical, thermal, acoustic and bubble formation phenomena will lead to synergistic enhancement of PT and PA cooperative effects 10
. The formation of QD clusters that have increased average sizes compared to single QDs would allow the use of nanosecond lasers, broadly used in medicine because they are simpler, less expensive, and less harmful for healthy biotissue than picosecond and femtosecond lasers.
To improve PA sensitivity we also propose to: 1) increase absorbed energy through conjugation of relatively low absorbing but highly fluorescent QDs to strongly absorbing nanostructures, both non-fluorescent or fluorescent (e.g., CNTs, gold nanoparticles, or even conventional dyes such as ICG); 2) use insulating layers around QDs or between the QD core and the protective coating that must have low heat diffusion and thus should improve the thermal confinement; and 3) use a layer with a high coefficient of thermal expansion (as is the case for ethanol, 10
) and lower speed of sound (as is the case for rubber-like materials), to improve the acoustic confinement. The combination of QDs with gold nanoparticles with various modifications may significantly enhance PA diagnostics and PT nanotherapy, including the development of integrated QD-gold nanoprobes, analogous to gold-CNT complexes that serve as PT contrast agents 13
. It should be noted that empty nanostructures like CNTs have a lower heat capacity compared to solid nanoparticles (like carbon or graphite), allowing better heating in pulse mode even with the same absorption properties (see above). In the case of combinations with gold, an increase of QD absorption can be achieved by either synthesizing QDs with thin gold layers, or “labeling” QDs with bioconjugated gold nanoparticles of different compositions and shapes. Gold nanoparticles are able to quench QD photoluminescence 14
, and thus increase the efficiency of the transformation of absorbed laser energy into heat, although the quenching effect should be used with precaution if the fluorescent properties of QD are needed. As we have demonstrated previously 11
, the absorbed energy, and hence the PA signals can be enhanced in medium with multiphoton absorption of pulse radiation. This mode can also be realized in QDs, exhibiting two-and multiphoton effects 15
PA spectroscopy revealed () that the optimal spectral range with high PA responses from the used QDs lies in the wavelength range of 620–645 nm, which is close to the NIR window transparency of biological tissues (650–950 nm), where interference with blood absorption is minimal 17
. Progress in the development of NIR fluorescent QDs with absorption in the range of 750 nm and even 850 nm 2
opens up opportunities to use them also with PA/PT techniques.
The advantage of QDs in fluorescent applications is the possibility to use one optical source to excite QDs with different emission wavelengths (colors). This does not have a direct analogy in the utilized PT/PA techniques. Nevertheless, we recently developed time-resolved multispectral PA detection of nanoparticles with different absorption spectra 19
, and this can thus provide a platform to use QDs as multicolor PA/PT contrast agents.
Currently, the time-resolved pump-probe PTI and PT thermolens technique operates more effectively in trans-illumination (forward) mode, which can provide imaging and detection of QDs in relatively transparent structures such as cells in suspension in vitro, and small-animal mesentery/ear models, as well as whole organisms such as Zebrafish and C. elegans in vivo. The PA schematic is more universal and can provide measurements both in forward and backward mode (i.e., laser and transducer are on one side), which is crucial for use in vivo on animal model and potentially on humans.
In summary, we herein demonstrated that QDs, in addition to being excellent fluorescent probes, can also be considered as PA and PT diagnostic probes, as well as PT sensitizers in nanotherapeutic applications. This observation may significantly extend the traditional application of QDs. Furthermore, the combination of QDs with PT/PA techniques that have inherent scattering and autofluorescent backgrounds 8
extends the applications of these techniques themselves. Indeed, detection of both fluorescent light and heat can increase the diagnostic value of integrated PT-PA–fluorescence modalities. For example, in vivo
studies could now be suggested that analyze QD pharmokinetics (e.g., clearance and accumulation rates, tissue biodistribution) in blood, the lymphatic system as well as tissues and organs up to 2–3 cm deep that are accessible with PA techniques 9
; studies using fluorescent/PT/PA imaging for guiding PT therapy, including tracking of individual tumor cells, bacteria or adenoviruses labeled both with QDs and/or gold nanoparticles 10
; mapping of tumor, sentinel lymph nodes and infected sites with integrated imaging modalities, or the use for multimodal molecular targeting and imaging in in vivo
blood and lymph flow cytometry 5