Compared to the blood test, the lymphatics has been paid much less attention, although recent discoveries in the biology of lymphatics (e.g., new receptors, LYVE-1, VEGF-3, and PROX1) emphasized its the key role in the development of many specific diseases such as metastasis, infections, or immunity disorders (27
). To date, many questions of lymphatic function are still not quite answered. For example, despite dramatically growing evidence of the crucial role of the lymphatic route in the spread of metastatic cells such as melanoma or breast cancer to other organs, researchers knew almost nothing about transport of these cells through lymph vessels before their invasion into lymph nodes (27
). Another very important issue is lymph transportation of immune-related cells. According to a few in vitro
animal studies, lymphatics are mainly composed of immune system-related cells: 85% T lymphocytes, 5% B lymphocytes, and 10% other cell types (dendritic, and macrophages, among others) (31
). Besides differences in composition, the total concentration of cells in lymphatics is lower and varies significantly (103
cells/μl) than in the blood flow (108
cells/μl). Since, the sampling of lymph is not well-established low-accurate procedure, the exact composition of cells in lymphatics (e.g., amount RBCs), as well as cell rate, especially, at pathology, is not still clear. Despite significant progresses in diagnostic techniques (e.g., magnetic resonance imaging, lymphoscintography, or fluorescence lymphography), to date, none has demonstrated the ability to real-time detection of individual cells in the lymph flow.
As we present here, PAFC using a cell focusing phenomenon and PA effect into single cells has potential to address to this and many other related issue, in particular, lymph testing in vivo
. Discovered in 1860 PA effects, as generation of sound by absorption of light, recently has been used for biomedical imaging of biotissue consisted of many cells only (36
). We demonstrated here, that endogenous cellular absorption can serve as intrinsic cell-specific markers having distinguished spectral and spatial signatures and its level is enough to produce sound in single cells readable with conventional ultrasound technique. This is non-invasive procedure because required a laser fluence is in the range of below 34-100 mJ/cm2
, which is the ANSI laser safety standard at the spectral range of 800-1100 nm, respectively (37
). The absorption-sensitivity threshold of the PAFC at the single-cell level was estimated to be 10-2
, that is, at least four orders of magnitude better than that of absorption spectroscopy for the same condition. This high sensitivity can be further enhanced during interaction of pulse laser with absorbing nanoclusters associated with self-assembly of endogenous chromophores (e.g., cytochromes and melanin) or exogenous nanoparticles. Laser-induced optical, thermal, acoustic, and accompanied phenomena from individual closely located chromophore molecules or nanoparticles into clusters may overlap and provide synergistic amplification of PT/PA signals (38
). Because many cellular structures are weakly fluorescent in native state and may create molecular assemblies/clusters, laser energy absorbed by these clustered structures is transformed mostly into heat and then into amplified sound, that makes PA “listening” of individual cells is promising for cell biology as alternative (or supplementary) to fluorescent “seeing” them. As a result, the high absorption-sensitivity of PAFC together with low background absorption in colorless lymphatics (compared to blood vessels) provides the opportunity for label-free identification and quantitation of RBCs, melanoma cells, and different types of WBCs, () including immunity-related cells in normal, apoptotic, and necrotic stages. In particular, PAFC revealed the presence of low concentrations of RBCs in lymphatics, which may have diagnostic value for evaluation some diseases , in particular, venous disfunctions, or diabetes accompinined by increasing blood vessel permeability (33
). The label-free identification of a subpopulation of WBCs as well as other cancer cells (e.g., breast, or prostate) is not realized yet. However, it should be possible, on the basis of spectral fingerprints of the different cytochromes, which concentration and mutual proportion may vary during cell metabolism, apoptosis, or carcinogenesis (40
The most significant achievement is a real-time label-free detection of metastatic melanoma cells in lymph flow. PAFC at a WBC rate of 102
cell/s in vessels with a diameter of 200 μm is able to detect just one melanoma cell during 3 h of observation; this rate is approximately equivalent to one melanoma cell in the background of 106
WBCs. This unprecedented sensitivity threshold in lymphatics in vivo
is unachievable with any other existing technique. In larger lymph vessels, the sensitivity threshold and the observation time should be improved because of the higher WBC rate. To proof of concept, we used superficial lymphatic vessels at a depth of 100-200 μm; however, recent advances with PA imaging of blood vessels 5-6 cm deep (36
) support the potential of the new tool to sense lymph vessels and lymph nodes at depths of a few centimeters with an expected resolution of 0.1-0.3 mm with NIR laser and focused cylindrical ultrasound transducers (25
). We presented a t few data of detection metastasis in sentinal lymph nodes (SLNs ), because this topic is the outside scope of this paper. Nevertheless, based on these data, we can suggest a potential of PAFC for early detection metastasis in vivo
with a sensitivity that is superior to existing conventional assays ex vivo
. In general, for the first time, we obtained in vivo
evidence that (1) lymph vessels transport live metastatic cells and remove destructed melanoma cells; (2) melanoma cells travel in lymph flow preferentially as small aggregates; (3) the lymphatic route involves the earliest stage of metastatic disease, even before detectable metastasis in SNLs are developed; and (4) the lymphatic system can promote dissemination of tumor cells at the late stage of metastatic disease by passing cells to distant organs (e.g. melanoma cells from an ear tumor may be spread to mesenteric lymphatics within the abdominal cavity). We believe this PA visualization of “colorless” lymphatics and, especially, SNLs using conventional blue dyes (e.g., Evans blue, Methylen blue, or Lymphazurin) that absorb in the visible spectral range without interference with cell detection in the NIR range may help resolve complex and relatively unexplored issues, including integration in vivo
non-invasive SLN mapping, single-cell detection, and metastasis assessment. This includes also the diagnostic value of lymphogenously disseminated rare CTCs for metastasis development (1
The specificity of PAFC can be enhanced by the use of multicolor functionalized nanoparticles with different absorption spectra as we demonstrated for selective detection of neutrophils, and necrotic and apoptotic lymphocytes in lymphatics. These nanoparticles, and especially gold-based probes have shown recently excellent potential for optical labeling because they do not photobleach or blink, are photostable, strong absorbers, can easily be conjugated with many proteins and Abs (38
). Furthermore, compared to conventional organic labels, gold is nontoxic, has excellent biocompatibility, and has been used in humans for 50 years (e.g., dental prosthetics, implants, and treatment of arthritis). The gold-based nanoparticle was recently approved for clinical trials, including PT therapy of cancer (41
). We believe that the in vivo
lymph test concept may be further developed to extend its utility beyond diagnosis to include the possibility of in vivo
lymph purging by elimination, at the appropriate laser energy level, of cancer cells labeled with nanoparticles in flow without harmful effects on surrounding normal cells and tissue.
To verify the concept of the in vivo rapid multispectral lymph test, we applied a two-wavelength mode, although the potential exists to increase the number of spectral channels to include low-cost, compact diode lasers with different wavelengths and high-pulse repetition rates (up to 10-50 kHz). Assuming the appearance of cells in the irradiated volume approximately during 1 ms (for a flow velocity of 5 mm/s and laser beam diameter of 5 μm) and a minimum delay of 10 μs between laser pulses, approximately 10-3 sec/10-5 sec = 100 laser pulses with 100 different wavelengths can be used for spectral cell identification ( in modern FC only 8-16 wavelength).
The integrated PA and fluorescent lymph flow cytometry schematics provided real-time detection of individual lymphocytes in norm and apoptosis by their labeling with conventional fluorescent probes in vivo
(). We did not indicate notable changes in lymphatics function parameters (17
) at used doses that were in line with other data (2
), although a possible toxicity should be further estimated before use in clinics. We are confident that replacement of fluorescent dyes on PA gold nanoparticles could solve this problem. In addition, conventional fluorescent labels can be used as PA/PT labels because the limited quantum yield of most of them, typically in the range of 1-20%, results in the transformation of most absorbed energy into heat (25
). The PA/PT technique is very sensitive to this heat, and this high PA sensitivity makes it possible to use labeling agents at lower concentrations than standard doses, which may reduce the risk of cytotoxicity.
The phasic contractions of lymph vessels acting as a natural concentrator of flowing cells near a vessel's center in combination with lymphatic valves acting as natural nozzles provided cell positioning with minimum radial fluctuation at level 5-10 μm in the lymph vessel with diameter of 150-200 μm. It was sufficient for counting each cells near valve exit using linear laser beam shape with length 40-70 μm and width 5-15 μm.
To exclude counting the same cells during their backward motion, we applied the monitoring cell motion with TDM with further exclusion of data obtained for backward cell motion. Besides, the contractions in some lymphangions and cycles of valve activity (opening-closing) were found to be relatively stable, with an average rate of 12 ± 1/min and 9 ± 1/min, correspondingly (17
). So laser exposure was adjusted to these particular rhythms. In addition, detection of forward moving cells only can be achieved by synchronization of laser exposure with the vessel-wall or valve motion coinciding to forward cell motion, controlled with a pilot laser.
The majority of examined lymphangions had well-developed bicuspid funnel-shaped valves and operated mainly under viscous fluid forces. This anatomic structure was not quite optimal for creation of the natural small nozzles with clear margins. In addition, some external factors that are not well controlled, such as muscle contractions, respiratory movements, and intestinal peristalsis, can influence valve function and phasic constrictions. Nevertheless, most selected active valves provided excellent natural hydrodynamic focusing of cells into single-file flow in analogy with artificial hydrodynamic focusing in conventional FC in vitro. We believe that a similar approach using vein valves for cell focusing can also be applied to FC of blood flow.
Reversible laser-induced short term lymph vessel squeezing () is also very attractive for the purpose of forming single-file cell flow, although its mechanism (e.g., laser activation of pacemakers, or smooth muscle), possible invasive nature, side effects, and control degree require further study. In conclusion, a novel combination of PA microscopy/spectroscopy and FC with many innovations (natural cell focusing in vivo,
PA effects in single cells, endogenous absorption as an intrinsic cell signature, real-time multispectral identification of cells with multicolor nanoparticles, combined navigation/detection schematics using for visualization of “colorless” lymphatics the contrast agents without interference with cell detection; an almost ideal preclinical animal model to test the concept of lymph FC in vivo;
and integration of PA, PT, fluorescent, and TDM techniques, which makes the in vivo
lymph test more universal) may be considered a new, powerful tool in lymphatic research with a broad spectrum of potential applications including real-time detection of various types of cells (e.g., RBCs, lymphocytes, dendritic, metastatic, infected, inflamed, and stem cells) at different states (e.g., norm, apoptotis, or necrosis), infectious agents such as bacteria and viruses, contrast agents, nanoparticles, liposomes, and drugs in a variety of lymphatic vessels (e.g., initial, afferent, and efferent lymphatics, and lymph nodes) under normal and pathological conditions. (e.g., tumors, infections, lymphedema). A quick translation of this technology to use in humans is anticipated with the development of a portable device that can be attached to lymph vessels and nodes to single cell diagnostics, metastasis detection, or assessment of therapy (i.e., radio- and chemosensitivity testing) through the quantification of metastatic and apoptotic cells, or different impacts (e.g., nicotine) (42