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There is a rapidly growing interest in the advanced analysis of histological data and the development of appropriate detection technologies in particular for mapping of nanoparticle distributions in tissue in nanomedicine applications. We evaluated photothermal (PT) scanning cytometry for color-coded imaging, spectral identification, and quantitative detection of individual nanoparticles and abnormal cells in histological samples with and without staining. Using this tool, individual carbon nanotubes, gold nanorods, and melanoma cells with intrinsic melanin markers were identified in unstained (e.g. sentinel lymph nodes) and conventionally-stained tissues. In addition, we introduced a spectral burning technique for histology through selective laser bleaching areas with nondesired absorption background and nanobubble-based PT signal amplification. The obtained data demonstrated the promise of PT cytometry in the analysis of low-absorption samples and mapping of various individual nanoparticles' distribution that would be impossible with existing assays. Comparison of PT cytometry and photoacoustic (PA) cytometry previously developed by us, revealed that these methods supplement each other with a sensitivity advantage (up to 10-fold) of contactless PT technique in assessment of thin (≤100 μm) histological samples, while PA imaging provides characterization of thicker samples which, however, requires an acoustic contact with transducers. A potential of high-speed integrated PT–PA cytometry for express histology and immunohistochemistry of both intact and stained heterogeneous tissues with high sensitivity at the zepromolar concentration level is further highlighted. © 2010 International Society for Advancement of Cytometry.
Histopathology is the well-established tool for diagnosis of diseases and evaluation of therapeutic interventions through the microscopic examination of tissue samples taken from anatomic areas of interest. Typically, sample preparation includes surgical extraction, fixation, embedding to paraffin or freezing, sectioning, and staining of tissues with conventional dyes or immunohistochemical labeling with specific antibodies (1). Bright-field optical microscopy is still the mainstay for examination of histological samples (2). A trained pathologist may readily reach a diagnosis based on tissue morphology or with the use of specific staining to identify tissue components. However, this remains a time-consuming and labor-intensive procedure requiring extensive training. Processing and staining, required for bright-field microscopy, may also introduce artifactual changes not present in the native tissue. Various optical and nonoptical imaging techniques have been developed to enhance histological analysis including conventional fluorescence microscopy (3); multispectral transmission microscopy (4); stimulated fluorescence microscopy (5); Raman spectroscopy (6); MRI (7); X-ray technique (8); TEM (9); and mass spectrometry (10, 11).
Recently, the rapidly growing application of nanotechnology in biology and medicine has placed new demands on histological analysis (12). Various nanoparticles with different sizes, shapes, and composition have been developed for gene/drug delivery, diagnosis, and therapy (13–16). Before in vivo clinical application of nanoparticles is feasible, it is imperative to determine critical parameters such as the clearance rate, biodistribution, and acute and chronic toxicity of nanoparticles in animal models (15, 16). The TEM (9), X-ray (17) and MRI (18, 19) have been used for imaging of gold, magnetic, and other nanoparticles in various tissues. However, most nonoptical methods are complex techniques requiring expensive equipment and increased costs of time and labor. Some methods may be limited to assessment of a single nanoparticle type. For example, only magnetic nanoparticles may be evaluated using MRI. Optical techniques such as diffusion optical spectroscopy (20) and optical coherence tomography (21) may visualize relatively large nanoparticles in tissue; however, dramatic decrease of scattering efficiency for small nanoparticles limits their image contrast in the presence of strong scattering background from tissue (22). For small nanoparticles, methods based on direct measurement of absorption become more sensitive (22).
We believe that many of these limitations may be overcome by using photoacoustic (PA), and especially photothermal (PT) imaging techniques which recently were shown to demonstrate greater sensitivity and spatial resolution compared with other optical modalities (22–32). To this end, we developed PA scanning cytometry/microscopy for imaging of unlabeled melanoma cells in a thin layer of whole blood (33), label-free mapping of real melanoma metastasis at the single cell level both in vivo and in vitro in sentinel lymph nodes (SLN) at different stages of cancer progression (25, 34, 35), as well as detection of tumor cells molecularly targeted by conjugated nanoparticles in lymph node samples (25, 34–36). The advantages of the PA method include the combination of high sensitivity with increased depth of penetration and the flexibility in sample preparation. PA imaging does not require fixation, sectioning, and staining as in conventional histology (33–, 38).
PT technique has higher absorption sensitivity compared with PA technique for thin samples (39, 40) as in the case of conventional histological sections with thickness of 5–50 μm. The applications of PT method have been recently reported toward transparent biological samples such as individual cells (28, 41, 42) and some thin tissues (40); however, the use of PT method for analysis of conventional histological samples has not yet been addressed. Herein, we demonstrate the excellent capability of contactless high-sensitivity PT scanning cytometry/microscopy for analysis of both conventionally fixed and stained histological samples as well as fresh tissue sections without fixation or processing.
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- LITERATURE CITED
To our best knowledge, this is the first demonstration of the use of pump-probe PT thermal lens method for examination of real histological samples. PT technique was validated for imaging and quantification of stains in a tissue sample, mapping of melanin content in melanoma metastasis, assessment of model samples spiked with CNTs and GNRs, and eventual detection of CNTs in mouse histological tissue sections after intravenous injection of CNTs. Our data indicate that sensitivity of the PT method is sufficient to detect individual melanoma cells even with very low melanin content as well as to detect individual submicrometer sized nanoparticle clusters. We were also able to demonstrate PT spectral identification of individual nanoparticles that were not visible with conventional assays in tissue sections. Integration of PT scanning image cytometry, PT spectroscopy, and conventional optical bright-field TDM microscopy provided opportunity for detailed observation of morphological tissue features and ultra-high sensitivity detection, quantification and spatial localization of nanoparticles. No additional sample preparation is required for PT analysis of histological samples; moreover, high PT contrast allows nondestructive mapping of a wide range of nanoparticles in the sample without the need for enhancement of nanoparticle optical properties [e.g., with silver staining kit (49) used to image gold nanoparticles].
Given the extremely fast growth in a number of nanotechnology applications in biology and medicine, we expect wide range of PT cytometry applications including detection of various types of light-absorbing nanoparticles such as gold nanoparticles or quantum dots (29). Besides biodistribution of nanomaterials in different tissues, possible applications could include study of cell-nanoparticle interaction, and verification of the relationship between morphological tissues changes and presence of nanoparticles with a focus on acute and long-term toxicity. Routine detection of nanoparticles in histological sections via conventional methods is challenging (see Introduction Section), and in samples with a low density of nanomaterials, many sections must be examined at high magnification and/or with the use of enhancing kits. PT technique may reduce analysis duration providing rapid examination with higher sensitivity compared with existing assays. Our data demonstrates that in current configuration detection limits for CNTs were at the level of individual 150–200 nm nanoparticles in the detection volume.
We also consider PT cytometry as a tool for verification and refinement of conventional histopathology data. First, PT contrast superior to that of conventional optical microscopy could be used for detailed analysis of weakly stained histological structures. We expect significant increase in the amount of information acquired from low contrast or weak stains in immunohistochemistry with the use of PTI and functionalized nanoparticles (to be addressed in our future study). We recently demonstrated the unprecendented high sensitivity of PT cytology in detection of small amounts of antibody-nanoparticle conjugates bound with specific antigens of breast cancer cells (36). The same protocol can be applied for PT cytometry of histological tissue sections. Second, PT analysis is capable of localizing and quantifying label-free subcellular molecular chromophores. As we demonstrated previously (28), sensitivity of pulsed PT method is sufficient for mapping low amount (up to 15 zeptomol) and distribution of intrinsic cell chromophores such as cytochrome c in unstained cells. This methodology could be easily extended to histological samples. Third, the observed effects of dye PT bleaching under intensive laser irradiation may become a powerful tool for identification of weakly absorbing components against strongly absorbing nondesired background by selective laser-based photobleaching of the stains in the sample. In this case, the PT technique simultaneously erases nondesired dye and controls amount of the remaining dye through acquiring PT signals. This approach is similar both to spectral hole burning of chromophores (50), and photobleaching of fluorescent labels in tissue samples (51) and in cells (52); nevertheless, to the best of our knowledge, controllable laser-based erasing/burning of nondesired background stains in histological samples is proposed here for the first time.
Previously reported application of PA scanning cytometry for early detection of metastasis in nonstained biopsy samples of SLNs (34) was herein repeated by PT assessment of the extracted SLNs. Comparison of these approaches demonstrated the complementary nature of PA and PT scanning cytometry technologies: in combination, these methods may cover a wider range of tissue samples and provide more detailed information than if used alone. The advantage of PA cytometry is the ability to image relatively thick samples, while contactless PT cytometry provide higher sensitivity (10–50 folds) for samples less than 120-μm thick. It should be noted that PT and PA methods are based on similar physical processes (optical excitation nonradiative relaxation medium heating detection of thermal-based effects) and both methods target objects with similar properties (absorbing or weekly fluorescent objects). Both may be used for many applications in spectroscopy, microscopy, analytical chemistry, and biomedicine. The difference between the methods is the detection parameter [thermal generation of acoustic waves (PA) or temperature-dependent changes in refraction index (thermal lens, PT)]. The laser-induced temperature profile in the sample could be detected by various methods: thermometry, infrared radiometry, deflection, phase contrast, polarization interference contrast, heterodyne, acoustic, and other schematics (22–25, 32, 34, 39, 42). Detection parameters influence detection schematics, creating advantages and limitations for each method. For example, in PA methods, the ultrasound transducer must be in acoustic contact with samples, while in contactless PT thermal lens method, the distance between photodetector and sample can be tens of centimeters.
The relative ease of use of the PT/PA-based methods and lack of additional histological sample preparation (including staining free analysis) provide key advantages of these methods over other optical modalities. The nanosecond laser-based pump-probe thermal lens schematic used in this work has rather simple optical scheme, is robust, and allows detection sensitivity sufficient for nanoparticles [pump-probe thermal lens method sensitivity is less affected by light scattering than that of conventional spectrophotometry or scattering-based PT methods (22, 39)].
For the current setup with OPO operating at 100-Hz pulse rate, PT mapping of the sample area of 100 × 100 μm2 with 1-μm resolution took at least 3.2 min (total time per pixel was defined by translation stage response time of 20 ms). These parameters can be significantly improved using advanced optical components and lasers. In particular, a short PT/PA signal duration of 2–10 μs in the time-resolved mode could be used for rapid (millisecond scale) examination of histological samples using pulsed nanosecond laser with high pulse rates (up to 100–500 kHz; 53). In this case, high pulse rate allows the use of high-speed mechanical stage operating at linear rate of up to 10 cm/s, while maintaining 1-μm PT/PA image resolution. This scheme could be preferred for large sample areas with fast linear sweeps over the sample surface. On the other hand, for relatively small areas, a 2D PT/PA imaging with the use of an advanced scanning system (54) could acquire PT images containing 10 × 10 (one cell) or 100 × 100 pixels in 1 and 100 ms, respectively (100-kHz laser source, one laser pulse per pixel). The PT schematic could be easily integrated into different microscope systems.
With the current schematic, the PT image is correlated with a 2D depth-integrated absorption distribution in the irradiated volume. High sensitivity of PT/PA technique may provide assessment of weakly absorbing samples that is unachievable with other optical modalities. Further development of the technique could include 3D PT mapping of tissues with confocal PT microscopy (55), PT tomography as analogous to PA tomography (31, 32, 56), far-field PT microscopy beyond diffraction limit (57), and fast scanning imaging with a high pulse rate laser (53). It would be intriguing to combine PT, PA, optical, and fluorescent imaging modalities in a single universal multifunctional setup to increase the range of detectable substances, sensitivity, specificity, and allow for flexible analysis of tissue samples of various thickness.