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Keywords:

  • multiphoton flow cytometry;
  • noninvasive;
  • intravital cytometry;
  • dendrimer;
  • circulating tumor cell (CTC);
  • two-photon excitation

Abstract

  1. Top of page
  2. Abstract
  3. Advantages of Multiphoton Excitation
  4. Initial Multiphoton Cytometry Experiments
  5. Two-Photon Flow Cytometry Instrumentation
  6. Fluorescent Labeling
  7. Short and Long Time Scale Monitoring In Vivo
  8. Circulating Tumor Cells
  9. Invasive In Vivo Flow Cytometry With Fiber Optic Probes
  10. Conclusions and Future Directions
  11. Acknowledgements
  12. Literature Cited

A handful of research teams around the world have recently begun to utilize multiphoton techniques in cytometry, especially for in vivo applications. These approaches offer similar enhancements to flow cytometry as the multiphoton phenomenon brought to the field of microscopy at the turn of the 20th century, with at least six advantages over single-photon excitation. Here, we review the published literature on multiphoton cytometry in vivo or in vitro from the initial experiments in 1999 to present. Multiphoton cytometry instrumentation set-ups vary from adapted multiphoton microscopy to a dedicated system, with laser pulse power and repetition rate serving as important variables. Two-beam geometry enables quantitation of cell size. Labeling strategies include conjugated fluorophore targeting, with folate and/or dendrimer platforms. With two-color measurement, ratiometric labeling is also possible, where one dye serves as a trigger to indicate the amount of excitation a cell receives, and another informs of cellular function. With two-color labeling, geometric fluorophore distribution proves important in theory and experiment for detection sensitivity curves and detected event intensity correlation. The main biological achievements to date using this young technology are reviewed, with emphasis on real-time monitoring of minute-by-minute and long-term cell dynamics as well as the clinically significant surveillance of circulating tumor cells. For this goal, minimally invasive two-photon flow cytometry with a fiber probe may overcome the primary issue of sample volume. The technique of multicolor, multiphoton flow cytometry greatly enhances the capabilities of flow cytometry to investigate the dynamics of circulating cells in cancer and other important diseases, and may in the future benefit from advances in microscopy such as super-resolution imaging, coherent control, and bioluminescence. © 2011 International Society for Advancement of Cytometry

The first experiments in the nascent field of in vivo flow cytometry demonstrated promising confocal detection of circulating cells in the vasculature of living mice via a single-color fluorescent biomarker (1–3). In essence, the regulated stream of cells in a conventional flow cytometer was replaced by the blood flow of a native microvessel to carry labeled cells of interest through the laser-interrogated region. In parallel, photothermal (4–6) and photoacoustic (7, 8) methods were developed, which do not rely on fluorescence. In all cases, however, it is necessary to account for variations in an animal's physiology, such as vasoconstriction or fluctuations in heart rate, which can affect circulating cell counts. Due to this limit on the accuracy of single-color detection, fluorescence-based in vivo cytometry has increasingly benefited from multicolor detection paradigms (9–15). Multicolor detection enables simultaneous enumeration of different populations of circulating cells in an animal, and thereby removes the effects of varying physiological conditions by a direct comparison of multiple cell populations in the same vessel. The optical engineering of multicolor detection systems is simplest when a single laser line can be used to excite all detected wavelengths. While recent advances in fluorescent proteins and other labeling compounds (16–18) might enable single-photon fluorescence approaches, multiphoton fluorescence is currently the most practical method to excite multiple well-separated wavelengths of fluorescence with a single laser line. Multiphoton flow cytometry is a useful technique both in vitro as well as for real-time studies of multiple circulating cell populations in vivo, which will advance the ability to probe the biology of circulating cells during disease progression and response to therapy. The discussion below is intended to provide an overview of applications and merits of both in vitro and in vivo multiphoton cytometry.

Advantages of Multiphoton Excitation

  1. Top of page
  2. Abstract
  3. Advantages of Multiphoton Excitation
  4. Initial Multiphoton Cytometry Experiments
  5. Two-Photon Flow Cytometry Instrumentation
  6. Fluorescent Labeling
  7. Short and Long Time Scale Monitoring In Vivo
  8. Circulating Tumor Cells
  9. Invasive In Vivo Flow Cytometry With Fiber Optic Probes
  10. Conclusions and Future Directions
  11. Acknowledgements
  12. Literature Cited

Multiphoton cytometry and other offspring of nonlinear microscopy predominantly rely upon two-photon fluorescence or second- or third-harmonic generation, which are the physical manifestation of second- (χ 2) or third-order (χ 3) nonlinear optical susceptibility. These phenomena become significant in the laboratory only under high-peak intensity light, typically achieved when short laser pulses on the order of tens to hundreds of femtoseconds are focused by a high numerical aperture microscope objective (19). Other nonlinear methods such as sum-frequency generation (20), coherent anti-Stokes Raman spectroscopy (21), and stimulated Raman scattering (22) are rapidly being integrated into the microscopist's toolbox, and will undoubtedly continue to touch cytometric applications as well (23).

Multiphoton excitation brings to cytometry at least six advantages of multiphoton microscopy (19, 24–28):

  • 1
    A single femtosecond laser beam can excite multiple fluorescent dyes, which enables multicolor detection to simultaneously enumerate multiple cell populations, provide a reference channel, or investigate multiple independent properties of the same cell. Thus, multiphoton excitation enhances cytometry by enabling simplified optical setup of multicolor techniques.
  • 2
    Under pulsed excitation, tissue damage should fall off nonlinearly further away from the focus, and therefore collateral damage due to photodestruction is presumably reduced outside the interrogated region, enabling imaging of living specimens with minimal photodamage. Indeed, near-infrared (NIR) ultrashort laser pulse damage behavior to cells has empirically been observed to obey a P2/τ dependence, where P is average power, and τ is pulse duration, pointing to a two-photon damage mechanism (29). It has been experimentally demonstrated that two-photon imaging is less damaging to hamster embryos than confocal microscopy, despite in excess of four orders of magnitude higher radiation dose used for two-photon fluorescence (30). This advantage would be particularly useful upon integration of multiphoton excitation into cell sorting applications of cytometry. For example, damage minimization is of the utmost importance in retrieving stem cells. If nonlinear coherent phenomena such as higher harmonic generation are used instead of multiphoton excitation, detection should in theory be achievable without any energy deposition into the sample whatsoever. Harmonic microscopy has been successfully used to explore transparent biological media (31) and also embryogenesis (32) but remains to be applied to cytometry.
  • 3
    Relative to single-photon events, the wavelength of two-photon excitation is dramatically red-shifted into the near-infrared window (Fig. 1a), where there is reduced intrinsic tissue absorption (33), autofluorescent background (Fig. 1b), and Rayleigh-scattering (34). As in vivo flow cytometry generally requires propagation through such optically imperfect media, this application draws great benefit from the red shift. As a caveat, we note that in turbid media where Mie scattering is dominant, the penetration depth under two-photon excitation is not necessarily larger than that under single-photon excitation due to the anisotropy of scattered photons (35). Regardless, near-infrared wavelengths are generally considered less likely to adversely affect cell viability than shorter wavelengths (26).
  • 4
    Highly sensitive detection of multiphoton fluorescence is practical due to the large separation between the excitation and fluorescence emission wavelengths. Relative to the much smaller Stokes shift typical of single-photon excitation, this enables convenient filtering of all scattered excitation light while collecting the entire fluorescence spectrum with high efficiency.
  • 5
    Selective excitation of a subcellular (femtoliter) volume is achieved with multiphoton excitation, enabling intrinsic optical sectioning, reducing photobleaching, and obviating the need for a confocal pinhole or other method to reject out-of-focus fluorescence. Coupled to the longer wavelength of excitation, submicron resolution is achievable in turbid tissue, hundreds of microns deep with a simple optical setup (25). This selective excitation can even be used to perform photochemistry or nanosurgery within a subfemtoliter volume of a cell or tissue, which might be achieved some day in the most advanced forms of coupled cytometry and cell sorting.
  • 6
    As nonlinear microscopy is based on fundamentally different physical processes than single-photon fluorescence, it offers unique opportunities for biological detection rooted in nonlinear properties of tissue. Harmonic microscopy has been used preclinically as a label-free contrast mechanism to distinguish different types of collagen (36) and to detect infection via intrinsic parasite byproducts (37). While fluorescence has hitherto been the major platform of advances in cytometry, nonfluorescent mechanisms of contrast might provide a novel avenue of enhancement in the future.
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Figure 1. (a) The near infrared window for in vivo imaging lies between the hemoglobin (<650 nm) and water (>900 nm) absorption peaks. (b) Blood autofluorescence spectrum with peak emission wavelengths of selected fluorophores applied to multiphoton cytometry (QD quantum dots). [Reproduced with permission from Weissleder R, Nat Biotechnol 2001, 19, 316-317 (33) and Tkaczyk et al., SPIE 2007, 6631, 66310T (10)]. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Disadvantages of multiphoton excitation of fluorescence include: (1) the present small selection of commercially available extrinsic labeling compounds with a large absorption cross-section in the NIR (38); (2) requirement for high peak photon density in the probed region, which in different setups can necessitate varying levels of technical attentiveness to details unimportant in one-photon imaging, such as dispersion (26, 28, 39); and (3) the present high cost of a femtosecond laser system and optics such as microscope objectives with acceptable distortion of temporal pulse shape and transmissivity in the near-infrared region. Technological development could palliate these issues in the future (40–42).

Initial Multiphoton Cytometry Experiments

  1. Top of page
  2. Abstract
  3. Advantages of Multiphoton Excitation
  4. Initial Multiphoton Cytometry Experiments
  5. Two-Photon Flow Cytometry Instrumentation
  6. Fluorescent Labeling
  7. Short and Long Time Scale Monitoring In Vivo
  8. Circulating Tumor Cells
  9. Invasive In Vivo Flow Cytometry With Fiber Optic Probes
  10. Conclusions and Future Directions
  11. Acknowledgements
  12. Literature Cited

The earliest applications of multiphoton excitation to cytometry (43, 44) predated in vivo photonic flow cytometry by several years, narrowly falling in the same decade as the invention of multiphoton microscopy (45). Inspired by single-molecule techniques, Hänninen et al. used two-photon fluorescence correlation spectroscopy (FCS) (43, 46) to enhance rare event detection in in vitro flow cytometry, which would not otherwise have been possible without dilution or other treatment of the whole blood medium they employed. Indeed, rare cell populations have engendered intense interest in the medical community (47). For example, astounding predictive value has been assigned to as few as five circulating tumor cells per 7.5 mL blood sample in breast cancer patients (48). Though currently no reports of in vivo application of FCS to cytometry exist, the advantages of multiphoton FCS (49) may enhance investigations of rare circulating cell lines in the future.

Ex Vivo Imaging Cytometry and Microfluidics

More recently, Peter So's team at the Massachusetts Institute of Technology has pioneered the extension of rapid multiphoton microscopy techniques for ex vivo imaging cytometry, for enhanced histology and molecular imaging (50–54). A prototype has been built for coupled single-photon in vivo flow cytometry with imaging cytometry (55), but an in vivo multiphoton imaging cytometer remains to be developed. Nonlinear approaches have also been extended to microfluidic flow systems with results relevant to in vitro cytometry (19, 23, 56–58). As discussed by Zhong et al. (59), ratiometric measurements in a two-beam geometry in particular could aid development of miniaturized and versatile microfluidic-based multiphoton cytometers.

In Vivo Cytometry

Development of the multiphoton approach to in vivo cytometry (9, 10, 12, 59–66) began concurrently with the initial single-photon fluorescent, photoacoustic, and photothermal methods. At the time of writing, six teams (12, 43, 44, 58, 65) have or are developing two-photon flow cytometry systems, with the cytometers at the University of Michigan (12), Purdue University (65), and Harvard University (personal communication) geared towards in vivo use.

Two-Photon Flow Cytometry Instrumentation

  1. Top of page
  2. Abstract
  3. Advantages of Multiphoton Excitation
  4. Initial Multiphoton Cytometry Experiments
  5. Two-Photon Flow Cytometry Instrumentation
  6. Fluorescent Labeling
  7. Short and Long Time Scale Monitoring In Vivo
  8. Circulating Tumor Cells
  9. Invasive In Vivo Flow Cytometry With Fiber Optic Probes
  10. Conclusions and Future Directions
  11. Acknowledgements
  12. Literature Cited

Multiphoton in vivo flow cytometry has generally followed an analogous experimental procedure to its confocal counterpart (1, 2). Cell lines of interest are injected into anesthetized immunocompromised mice via tail vein injections. Typically these cells are fluorescently labeled prior to injection, but in vivo tagging is also possible (62, 65). A small vessel, typically a 40 to 100 micron artery in the mouse ear, is imaged via a microscope objective through which a femtosecond laser is aligned. The excited two-photon fluorescence is used to count the frequency with which cells pass through the focus. Published approaches to multiphoton cytometry are summarized in Table 1.

Table 1. Overview of cytometry variations incorporating multiphoton excitation
TechniqueApplication examplesYear of first publicationReferences
  1. Techniques that have been used in vivo are marked in bold.

Fluorescence correlation spectroscopy (FCS)Rare event detection1999(43)
Point-excitation without beam scanningMonitoring short and long time scale depletion dynamics of injected cells; In vivo enumeration of circulating tumor cells (CTCs); (Most techniques in this table and most published work in multiphoton flow cytometry is a variation of this approach.)2004(9, 12, 61)
Imaging cytometryHistology and molecular imaging2004(50–54)
Two-beam geometryMonitoring cell aggregation, particle sizes2005(9, 59–61)
Labelling in vivoDetecting: native blood cells; injected leukemia cells; lung cancer cells shed by solid tumors2005(62, 65)
Simultaneous multicolor detectionConcurrently monitoring circulation of two breast cancer cell lines in the same mouse; Quantitative two-channel ratiometric measurement of folic-acid-receptor-targeted dendrimer nanoparticle uptake by carcinoma cells2006(10, 12, 59, 63)
MicrofluidicsCharacterization of microfluidic device performance; Monitoring encapsulation of cells; Vibrational spectroscopy2006(19, 23, 56–58, 69)
Extended cavity excitation laser sourceImproved detection of fluorescent proteins in whole blood2007(64, 66)
Modification of commerical scanning microscopeIn vivo enumeration of circulating tumor cells (CTCs); Label-free probing of the interior of multicellular aggregates in stem cell research2007(58, 65)
Fiber-optic probe for access to deep blood vesselsImproved detection efficiency of injected GFP-labelled sarcoma cells2009(67, 68)

Investigators with multiphoton microscopy equipment can use their existing systems for in vivo flow cytometry, with the methods described by He et al. (65). Essentially, one-dimensional multiphoton images are taken across the vessel in the mouse ear every 5 s and analyzed with custom MATLAB software. In direct comparison, this method was found to be moderately more sensitive than a confocal approach.

Alternatively, for the exclusive purpose of multiphoton in vivo flow cytometry, a dedicated system (Fig. 2) has been built at the University of Michigan without scanning of the excitation laser (9, 61). This characteristic is shared with the single-photon confocal systems developed at approximately the same time at Harvard (1) and has the advantage of high potential throughput.

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Figure 2. (a) Multiphoton in vivo flow cytometer with two-channel detection. (b) Two-channel raw data from the two-channel two-photon flow cytometer. Short wavelength channel (S-channel green) and long wavelength channel (L-channel red) traces from an ear blood vessel of a mouse injected with microspheres at time zero (dashed line). The number of photons counted is shown for each 1.31 ms time bin on the x-axis. Inset is zoomed in to a shorter time period. (c) Schematic diagram of two-beam, two-channel, two-photon flow cytometry. [(a) and (b) reproduced with permission from Tkaczyk et al., Opt Commun 2008, 281, 888-894 (12) and Tkaczyk et al., SPIE 2007, 6631, 66310T (10)]. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The designated two-photon flow cytometer is described in detail elsewhere (12). Briefly, 50-femtosecond pulses of 800 nm wavelength from a standard Ti:Sapphire laser (Coherent Mira) are focused with a long-working distance objective (Olympus 40×) onto a custom heated stage, into a blood vessel of a living anesthetized mouse's ear. A CCD camera visualizes the vessel under white light prior to experiments. Less than 20 mW laser power is used at the focus, which has not been observed to cause any photodamage to the ear of the mice. Dichroic mirrors and bandpass filters separate the fluorescent signals from scattered excitation light. Single-photon-counting photomultiplier tubes detect fluorescence in two channels, and a MATLAB program, which is described in detail elsewhere (66), is used to analyze the peaks created by fluorescent particles moving through the laser focus.

Fiber-Optic Multiphoton Flow Cytometry

Invasive in vivo multiphoton flow cytometry has been explored at the University of Michigan. Rather than propagating through free space into a small vessel, laser and fluorescent photons propagate through a fiber optic probe inserted into the animal with a 30-gauge syringe needle. Additionally, pulses are dispersion precompensated with a grating pair to optimize the intensity at output of the fiber. Due to the light weight of the fiber-needle complex, these experiments should be performable in the vessel of a conscious and mobile mouse, by contrast to free space methods (67, 68).

In Vitro Multiphoton Flow Cytometry

Though most efforts in multiphoton flow cytometry since the new millennium have been aimed at developing in vivo approaches, in vitro application could be fruitful as well. An exclusively in vitro instrument has been developed at the University of Wisconsin (58) to probe the interior of multicell aggregates. Eventually, this could be used for rapid characterization of stem cells while preserving their viability. The instrument's flow cell is constructed by photolithography and soft lithography and permits large particles to stream under the drive of two programmable syringe pumps. An existing multiphoton laser scanning microscopy platform is used for detection, along with custom data acquisition and analysis electronics. The system has been used to image intrinsic fluorescence in mouse embryoid bodies, which are aggregates of embryonic stem cells. Numerous additional unique biomedical research and clinical applications are hoped to be enabled by this instrument in the future. Another higher-order nonlinear optical phenomenon, coherent anti-stokes Raman scattering, has also been applied in vitro, demonstrating the possibility of fluorescence-free cytometry (23, 69).

Flow Cytometry in a Two-Beam Geometry

Whereas in conventional flow cytometry, cell size is determined by the forward scattering signals, this method cannot be applied easily in vivo due to the geometry of the detection system. Instead, the two-photon flow cytometry technique has been extended by splitting the excitation beam (Fig. 2c). Cell size is assessed by observing the time duration that the cell flows through an excitation focus and then normalizing this time to the cell velocity. Velocity is obtained by measuring the time delay between the two excitation foci. It should be emphasized that single-cell measurement at each focus is critical for the two-beam measurement, and is ensured by the high spatial selectivity with nonlinear excitation.

Zhong et al. used this system with a 50 micron separation of foci to examine size distributions of microspheres, KB cells (a human oral epidermoid carcinoma cell line), and Jurkat cells (an acute T cell leukemia cell line) (59). Although the two-beam scanning method did not yield absolute size measurement of single particles or cells, it was able to differentiate 6-μm and 10-μm fluorescent microspheres and also granted sufficient accuracy to detect aggregation of Jurkat cells induced by Concanavalin A, even under nonuniform flow. Platelet, leukocyte, and red blood cell aggregation is an interesting target due to its role in common circulatory system defects such as atherosclerosis, chronic inflammation and thrombosis as well as pathological processes such as radiation damage (70).

This setup is not suitable for very high concentrations of cells as it cannot distinguish two different cells passing through each focus from a single cell passing sequentially through both foci. Cell clipping, where only a portion of the cell passes through the laser focus, and partial distribution of dyes within cells also remain potential sources of artifacts.

Laser Power, Pulse Length, and Repetition Rate

Thermal damage will eventually limit the average laser power that can be delivered to any biological specimen. In this sense, if one is accustomed to confocal systems, the tens of milliwatts of average laser power typically required to achieve peak intensities capable of generating measurable multiphoton excitation in vivo (10, 12, 65) may seem excessive. Indeed, the first detailed description of a photonic in vivo flow cytometer used a confocal approach and only used 0.6 mW of laser power at the blood vessel (1). In direct comparison of confocal and multiphoton cytometry, investigators at Purdue University required at a minimum 30-fold higher average laser power in the two-photon regime (65). However, due to the advantages of multiphoton excitation outlined earlier, for tissue damage this factor does not actually weigh in favor of single-photon approaches. In fact, Squirrell et al. found that in the face of three orders of magnitude higher average laser powers and in excess of 40,000 times higher total irradiation exposure, two-photon microscopy nevertheless had a more favorable effect on embryo viability than a standard confocal approach (30).

Despite higher power tolerances of biological samples to multiphoton excitation, increasing average laser power is not always a feasible method for boosting fluorescent signal. The most obvious method to boost multiphoton processes is to employ shorter pulses. Indeed, whereas a multiphoton cytometer using 200 fs pulses operated at 80 mW average laser power to detect rhodamine tags in vivo (65), shorter pulses (85 fs) can consistently detect circulating cells with various labels in vivo at average powers below 20 mW (10, 12).

Another parameter that can be adjusted is the frequency of laser pulses. At a fixed average laser power, the integrated two-photon fluorescence signal is directly proportional to the repetition period of the laser (39). Thus a convenient way of increasing two-photon processes is application of an extended cavity laser, which should increase two-photon signal in proportion with cavity length. Ted Norris's team at the University of Michigan has applied this principle to multiphoton flow cytometry (66). Relative to a commercial 76-MHz system, a custom-built 20-MHz extended-cavity Ti:Sapphire oscillator (Fig. 3) experimentally confirmed predicted approximate fourfold enhancement of two-photon fluorescent signal in flowing dye as well as fluorescent polystyrene microspheres. At low power, the custom-built 20-MHz laser was able to detect a significantly larger fraction, in either phosphate buffered saline (PBS) or whole blood, of GFP-expressing MCA-207 mouse sarcoma cells cross-labeled with the membrane-binding lipophilic dye DiD (Fig. 3). The sigmoidal (s-shaped) curve of detection sensitivity via GFP versus average power is preserved in both PBS and blood. For maximal detection sensitivity near the detection threshold, this implies a dramatic advantage for modest increases in fluorescent signal at low excitation power. It is noteworthy that square-law scaling of detected fluorescent event brightness, typical in two-photon processes, while present in the case of a dye solution, does not hold for fluorescent spheres or cells. Calculated detection of ideal spheres demonstrates that the power variation in the detection region is the cause of sub-square law scaling in the case of beads. These simulation results, in terms of fluorophore distribution consequences, will be explored in more detail in the following section of this review.

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Figure 3. (a) Custom-built extended cavity laser (xtnd) with a 20 MHz repetition rate. Optical components described in reference (66). (b) At each average laser power, a greater fraction of cells are detected in whole blood by their GFP fluorescence with the two-photon flow cytometer with this source (xtnd), as compared to a commercial NIR femtosecond laser (Mira). The fraction of detected DiD-labeled GFP-expressing MCA-207 cells present as events in the GFP channel are shown for various inident powers. (c) Theoretical prediction of fraction detected for varying laser powers (signal levels), based on parameters determined in experiment (b). These are: a fixed detection threshold of 4 photons for GFP, 10 photons for DiD; a maximum signal produced at the highest incident laser power from the Mira of 100 photons for GFP, 1,000 photons for DiD (circles), and five times higher maximum signal strength for the same incident laser power from the extended cavity (x′s). [Reproduced with permission from Tkaczyk et al., J Biomed Opt 2008, 13, 041319 (66)]. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Fluorescent Labeling

  1. Top of page
  2. Abstract
  3. Advantages of Multiphoton Excitation
  4. Initial Multiphoton Cytometry Experiments
  5. Two-Photon Flow Cytometry Instrumentation
  6. Fluorescent Labeling
  7. Short and Long Time Scale Monitoring In Vivo
  8. Circulating Tumor Cells
  9. Invasive In Vivo Flow Cytometry With Fiber Optic Probes
  10. Conclusions and Future Directions
  11. Acknowledgements
  12. Literature Cited

While label-free detection has been achieved in multiphoton and other nonlinear microscopies, all implementations of fluorescent flow cytometry have hitherto relied on exogenous fluorescent labels or transfection with fluorescent proteins. Longer wavelength fluorescent tags in the NIR window (Fig. 1a) are preferable (71) to avoid masking by two-photon autofluorescence in blood (Fig. 1b). Inevitably with multicolor detection one of the dyes must be of shorter wavelength. Tkaczyk et al. have compared the ability to detect cells with different fluorescent proteins or dyes via two-photon cytometry (10). Unfortunately, despite the possibility of various direct (72), Fourier-transform (73), and laser-pulse-shaping-based (74) measurements of two-photon excitation cross-sections, only a minority of candidate two-photon fluorescence dyes have accurate published spectra (34, 38, 75–79), and even these suffer widely varying reports of absolute cross-section, as reviewed elsewhere (77). Dyes and fluorescent proteins that have been used to date in multiphoton flow cytometry are summarized in Table 2, including those two-photon excitation maxima that could be located in the published literature.

Table 2. Two- (Ex 2P) and one-photon excitation (Ex) and emission (Em) peaks (nm) and physical intracellular distributions of fluorophores investigated for two-photon flow cytometry
FluorophoreEx 2PExEmDistributionCommentReferences
  1. Single-photon data is from manufacturers, and two-photon data is from Ref. (34, 38, 75–77). Variants of red fluorescent protein are marked in boldface. [Expanded with permission from Tkaczyk et al., SPIE 2007, 6631, 66310T (10)].

GFP927488510CytoplasmFluorescent protein(10, 66, 68)
CFSE 494521Cytoplasm (59)
AlexaFluor488 (folate conjugate) 495519Folate receptors (65)
FITC640, 790494520Folate receptorsConjugated to folate (65) and/or dendrimer (59)(59, 65)
DiI<690549565Membrane (10, 65)
tdTomato684554581CytoplasmFluorescent protein(10)
DSRed RFP700558583CytoplasmFluorescent protein(10)
T4 555586CytoplasmFluorescent protein(10)
Rhodamine-X (folate conjugate) 572590Folate receptors (65)
CellTracker Red CMTPX (CT) 577602Cytoplasm (59)
mPlum724590649CytoplasmFluorescent protein(10)
Mitotracker DeepRed 640662MitochondriaAlso used with folate-dendrimer conjugate(59)
DiD 644665Membrane (10, 12, 66)

Targeted Fluorescent Conjugates

The need to label already circulating cells was realized early in the development of in vivo flow cytometry and demonstrated amongst the earliest experiments of confocal (1) and two-photon cytometry (62). The simplest method is to simply inject free dye into the blood stream. In one experiment, DeepRed, a cell-permeating probe that binds to the outer membrane of mitochondria in living cells, was injected into mice during two-photon cytometry, after which fluorescent peaks began to appear (59). Potential clinical use requires targeting cells of interest. One platform that has been investigated is polyamidoamine dendrimers (80), which have shown great promise as fluorescently labeled nontoxic targeting molecules. Conjugated with trastuzumab, a humanized monoclonal antibody to Her2, dendrimer labeling of live cancer cells increased fivefold relative to fluorescent dendrimer alone, as measured by two-photon flow cytometry in vitro (10). In vitro investigations of multiply colored folic acid dendrimer conjugates were also performed (59). Folic acid conjugates are a promising choice for in vivo labeling of tumor cells as folate receptors tend to be overexpressed amongst human cancers. He et al. showed the feasibility of labeling CTCs in vivo using folate-conjugated dyes (65) for two-photon cytometry. Folic acid conjugates avoid two potential pitfalls with fluorescent antibody-conjugates: (1) labeled antibodies circulate for several days before removal, potentially elevating fluorescent background; (2) tumor-specific antibodies could lead to underestimates of CTC burden as they have been known to promote phagocytosis. It has been found experimentally that folate-dye conjugates improve sensitivity relative to fluorescently labeled antibodies, and folate-FITC has already been tested in humans (65).

Ratiometric Labeling

One of the challenges of in vivo flow cytometry is nonuniform excitation of cells as they flow through the laser focus at random positions and velocities, in marked distinction from the precise alignment in conventional flow cytometry. This results in vivo in a broad fluorescence intensity distribution for uniformly stained cells, and the intensity of the fluorescence signal level alone is not a useful parameter to differentiate single cells. A dual-dye staining method has been demonstrated as a method to overcome this obstacle. Cells are labeled with two different dyes, one of which is always fluorescent (the “trigger”) upon excitation by a laser beam, whereas the other fluoresces only when the cell function changes (the “reporter”). As a cell flows through the excitation region, the fluorescence signal from the “trigger” dye reflects the amount of excitation the cell receives, while the fluorescence signal from the “reporter” dye informs of changes in cell function. Two spectral channels can detect fluorescence signals from two dyes that emit at distinct wavelengths, and the ratio of the “reporter” over “trigger” can be used to quantitatively measure cellular parameters, expressed as an angular measurement. To make quantitative ratiometric measurements, it is essential that the fluorescence amplitudes of signal and reporter be correlated (i.e., have a narrow angular distribution). This is not possible if one attempts to compare fluorophores of differing spatial distributions in a dual-labeled cell (59, 66). Figure 4 demonstrates the lack of two-photon fluorescence intensity correlations between dyes of different cellular distribution, both experimentally in vitro as well as in computer simulation.

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Figure 4. Decorrelation in experiment and computer simulation of two-photon fluorescence intensity between dyes of differing cellular distribution. CFSE is distributed throughout the cell and is detected in the short wavelength (S) channel, whereas the mitochondrial dye DeepRed is localized to the outer cytosol and fluoresces in the long wavelength (L) channel. (a) Two-channel dot plot of two-photon excited fluorescent signals from peripheral mononuclear blood cells stained with CFSE and DeepRed. (b) Simulated dot plot of dual-stained cells. [Reproduced with permission from Zhong et al., J Biomed Opt 2008, 13, 034008-1-034008-19 (59)]. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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One solution to establish the required correlation between the trigger and reporter dyes is to use fluorochromes targeted to the same receptor. In this manner, quantitative two-channel ratiometric measurement of uptake of fluorescently labeled dendrimers targeted to folic acid receptors was achieved under nonuniform flow conditions of KB cells, an epidermoid carcinoma cell line (59). In future investigations, if the fluorescence signal from one of the dye-conjugated nanoparticles is associated with cellular function, such as apoptosis, the ratiometric method could thus be used to quantitatively measure cellular function.

Effects of Fluorophore Distribution on Two-Photon Flow Cytometry Data

The geometrical distribution of fluorophores can significantly affect two-photon flow cytometry data. As mentioned earlier, decorrelation of signal strength in the two channels precludes successful implementation of the ratiometric labeling method for quantitative cytometry. Tkaczyk et al. have constructed a deterministic mathematical model (Fig. 5), which explains several unique features of the two-photon cytometry signals of fluorophores distributed in the cytosol or on the cell membrane. These include; the uncorrelated signal strengths in two detection channels; a sigmoidal sensitivity curve for detection under varying powers for cell detection thresholds as low as a single photon; and sub-square law scaling of unsaturated two-photon fluorescence signal (66).

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Figure 5. Determinisic model to calculate expected two-photon fluorescence flow signals for different dye distributions. The right-handed x-y-z coordinate system is defined with z the axial direction of the propagating laser beam and x the direction of fluid flow in the capillary. (x0, y0, z0) is a variable for the location of the center of the fluorophore-containing object. (a) Magnitude of the beam squared-intensity function B in the x = 0 plane for a beam waist radius w of 2 μm (1/e2 point). (b) Various level sets of B in 3 spatial dimensions. (c) Instantaneous signal (arbitrary units) for a 10-μm infinitesimally thick spherical shell centered at (x0, y0, z0) = (5, 5, 0) μm in a Gaussian beam (waist w = 2 μm). (d) Signal (arbitrary units) for a shell centered at (x0, y0, z0) = (x0, 5, 0) μm as a function of x0 origin, which increases as the object flows in the x-direction. Net signal is the integral of the curve. [Reproduced with permission from Tkaczyk et al., J Biomed Opt 2008, 13, 041319 (66)]. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The model employs simplified geometrical spatial distributions of fluorophores in an idealized spherical cell (radius 10 μm). A solid sphere is used to approximate cytosolic fluorophores (such as GFP), whereas a spherical shell is used for membrane-bound dyes (such as DiI or DiD). Gaussian beam propagation is assumed, and for each possible origin in the capillary through which the idealized cell flows, the total anticipated two-photon fluorescence signal level in the GFP channel (Fig. 6a) or DiD channel (Fig. 6b) is calculated. The experimentally observed histograms (probability distribution functions) of detected event signal strengths for GFP and DiD is in good agreement with the model result. The heterogeneity of intensity distributions manifests itself in both theory and experiment as a lack of correlation between the intensities of both channels on a scatter plot. Similar decorrelation was already demonstrated via Monte Carlo simulation (Fig. 4) of dyes distributed uniformly throughout the cell (such as CFSE) vs. outer cytosolic dyes (such as the mitochondrial dye DeepRed).

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Figure 6. Model-predicted net signal (arbitrary units) from 10 μm radius (a) sphere or (b) shell (t = 0.5 μm) with different origins (y0, z0) in the capillary cross-section, flowed down the x direction. Beam propagates in z direction with a 2 μm spot size. [Reproduced with permission from Tkaczyk et al., J Biomed Opt 2008, 13, 041319 (66)]. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Fluorophore distribution also explains the sigmoidal curve for fraction of all cells detectable via the GFP signal, which in turn is the reason a reduced-repetition-rate laser offers such a sensitivity advantage. The sigmoidal curve is realized theoretically for different laser sources by assuming a fixed minimum threshold for detection of a cell by its two-photon fluorescence signal, which in turn varies with squared-laser-power (Fig. 3). Even with balanced signal strength in both channels and detection thresholds as low as a single photon, the calculation shows preservation of the sigmoidal curve for differing fluorophore geometries. Effectively, the detectable fraction of events is the area of the y0-z0 plane in Figure 6 above a certain finite signal level S, which with increasing incident power falls at a lower level relative to the maximum S. Nonlinearities are inherited from this signal landscape. This is also the source of the observed sub-quadratic scaling of the two-photon signal of flowing cells or spheres with increasing excitation power. Intuitively, for increasing excitation intensity, the detectable two-photon excitation region becomes larger, subsuming more dim events (in the cooler-colored regions of Fig. 5b). This reduces the signal level below the quadratic dependence that would occur without this thresholding.

Short and Long Time Scale Monitoring In Vivo

  1. Top of page
  2. Abstract
  3. Advantages of Multiphoton Excitation
  4. Initial Multiphoton Cytometry Experiments
  5. Two-Photon Flow Cytometry Instrumentation
  6. Fluorescent Labeling
  7. Short and Long Time Scale Monitoring In Vivo
  8. Circulating Tumor Cells
  9. Invasive In Vivo Flow Cytometry With Fiber Optic Probes
  10. Conclusions and Future Directions
  11. Acknowledgements
  12. Literature Cited

Even with single-color detection, monitoring cells over short or long time scales is possible. Minute-by-minute depletion dynamics after injection of yellow-green fluorescent microspheres are shown in Figure 7a. Fluorescent peaks are detectable within 30 s of injection and fell off within 10 min. Though such temporal resolution is not achievable by conventional ex vivo flow cytometry, validation with selected blood samples from the mouse was made. In an analogous experiment, splenocyte dynamics were monitored for 20 min (12). The same system was used to monitor DiD-stained red blood cells (RBCs) for periods of greater than 2 weeks. It was observed that the operator could significantly increase the number of detected events by manually adjusting the laser focus during the course of data acquisition, presumably to the maximum flow position in the artery. This suggests the possibility of using, in a multicolor approach, one fluorescent detection channel as an internal standard to quantify sampled volume via calculations from a well-mixed, large stably circulating population of cells such as RBCs.

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Figure 7. (a) Depletion dynamics of circulating fluorescent microspheres, enumerated in vivo with two-photon flow cytometry. The number of detected events (log scale) are plotted over 10 minutes after the injection at time 0. (b) Simultaneous monitoring of two populations of circulating cancer cells. Detected events are shown after the injection of 5 × 105 Qdot585 stained (low metastatic potential) MCF-7 cells simultaneously with 1 × 106 Qdot665 stained (high metastatic potential) MDA-MB-435 cells into the same mouse at time 0. Twice as many MDA-MB-435 cells were used because initial studies suggested that fewer could be detected immediately after injection. The frequency was calculated as the number of events counted in the short wavelength S-channel (open markers) or long wavelength L-channel (closed markers) within 430 s. The experiment was repeated in three different mice, and the average results are shown, with error bars indicating the variation over the three animals. Both axes are log scale. Another set of mice were injected with tumor cells labeled with the reverse pattern of quantum dots (MCF-7 labeled with Qdot585 and MDA-MB-435 labeled with Qdot655) to confirm that the particular fluorophore assignment does not influence the detectability of these cell lines in vivo, with similar results. [Reproduced with permission from Tkaczyk et al., Opt Commun 2008, 281, 888-894 (12) and Tkaczyk et al., SPIE 2007, 6631, 66310T (10)].

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Circulating Tumor Cells

  1. Top of page
  2. Abstract
  3. Advantages of Multiphoton Excitation
  4. Initial Multiphoton Cytometry Experiments
  5. Two-Photon Flow Cytometry Instrumentation
  6. Fluorescent Labeling
  7. Short and Long Time Scale Monitoring In Vivo
  8. Circulating Tumor Cells
  9. Invasive In Vivo Flow Cytometry With Fiber Optic Probes
  10. Conclusions and Future Directions
  11. Acknowledgements
  12. Literature Cited

Recent studies in cancer biology emphasize the importance of detecting and quantifying various types of cells, including rare populations of tumor and endothelial cells, in determining prognosis and response to therapy (81). Great technological progress has been made in isolating CTCs, as reviewed by Smerage and Hayes (82). Modern available assays include combinations of conventional flow cytometry, immunohistochemistry, immunofluorescent microscopy, immunomagnetic separation (83), PCR, RT-PCR (84), high-throughput optical-imaging systems (85) including fiber-optic scanning (86), and most recently, microchip technology (87). The last is the only technology capable of isolating viable cells from whole blood without any additional processing or preparation. As a whole, there remain notable deficiencies with the existing assays that enumerate total malignant cells in a single blood sample (88, 89). The first limitation is specificity. While elevated numbers of CTCs generally are associated with worse prognosis, not all patients with detectable breast cancer cells in blood progress rapidly (48). This result suggests that there are subsets of CTCs that are more biologically relevant for prognosis and response to therapy. The second limitation is sensitivity. Only approximately 50% of patients with metastatic breast cancer have detectable circulating tumor cells at any given time, even though all eventually have progressive disease. These observations suggest that entry of breast cancer cells into the circulation may be intermittent, so analyses based on a single blood sample may give incorrect information. Deficiencies in existing assays for CTCs potentially have an even greater impact for patients with early stage primary tumors or after adjuvant therapy due to decreased tumor burden. Collectively, these limitations emphasize the need to develop new methods including in vivo photonic flow cytometry with the sensitivity and specificity to detect subsets of CTCs that are linked more directly to prognosis or response to therapy.

Developers of multiphoton flow cytometry have been motivated by the pressing clinical need for better CTC assays. He et al. were able to track leukemia cells after tail vein injection (of 106 cells) via folate-FITC or folate-rhodamine (65). With this in vivo labeling technique, they were also able to monitor CTCs arising spontaneously from murine lung cancer xenografts by 2 weeks after implantation, with exponential rise over the following 2 weeks. Metastatic disease, however, was not detectable in necropsied tissues until a full 5 weeks after implantation. This demonstrates the ability of multiphoton in vivo flow cytometry to quantify CTCs weeks before microscopic metastasis. With a folate-AlexaFluor 488 conjugate, they also detected CTCs (confirmed to be malignant by a monoclonal antibody) in whole blood from ovarian cancer patients, but not in healthy controls. More recently, with the invasive fiber optic multiphoton in vivo flow cytometry technique, Chang et al. monitored the minute-by-minute depletion dynamics of MCA-207 mouse sarcoma cells in a live mouse (68).

Monitoring of Two Circulating Cell Lines Concurrently

With multicolor, two-photon in vivo flow cytometry, different populations of cells can be compared simultaneously in a single animal with a single laser source, overcoming many variables intrinsic to in vivo experiments, such as animal-to-animal differences in perfusion. This technique was used to measure dynamics in the circulation of living mice of two different human breast cancer cell lines, MDA-MB-435 and MCF-7, labeled with different quantum dots (12). The first are known to spontaneously form metastases in mice (90), while MCF-7 cells have very low metastatic potential (91). As shown in Figure 7b, the dynamics of each cell line in the circulation were markedly different. Ten minutes after injection, numbers of MCF-7 cells were more than 10-fold greater than MDA-MB-435 cells, despite the small difference in total numbers of cells injected. However, by 1,000 min after injection, MCF-7 cells had cleared almost completely from the circulation, while MDA-MB-435 cells remained detectable. These data are similar to those reported by Georgakoudi et al. who showed using single channel in vivo flow cytometry that exit of prostate cancer cells from the circulation correlates with relative differences in metastatic potential (2). To our knowledge, at present only the two-photon in vivo flow cytometry approach has been used to compare simultaneously during one experimental run different circulating cancer cell lines in a single animal.

Invasive In Vivo Flow Cytometry With Fiber Optic Probes

  1. Top of page
  2. Abstract
  3. Advantages of Multiphoton Excitation
  4. Initial Multiphoton Cytometry Experiments
  5. Two-Photon Flow Cytometry Instrumentation
  6. Fluorescent Labeling
  7. Short and Long Time Scale Monitoring In Vivo
  8. Circulating Tumor Cells
  9. Invasive In Vivo Flow Cytometry With Fiber Optic Probes
  10. Conclusions and Future Directions
  11. Acknowledgements
  12. Literature Cited

For clinical implementation, the major limitation of in vivo flow cytometry is sample volume. The blood volume sampled by fluorescent, photothermal, and photoacoustic detection techniques generally has had an upper limit of 0.5 μL/min (consider a blood vessel of ∼ 50 μm in diameter with an average flow velocity of 5 mm/s) (92), compared to CTC concentrations under 1 per mL in early and even intermediate stages of cancer. Thus, to observe a single CTC, a free space laser system would need to be engineered to maintain continuously for days precise focus within a cancer patient's blood vessel. However, free space propagation is not the only option. As cancer patients often have indwelling lines through which they receive chemotherapy, it is in fact practical to use fiber optics in these lines to access larger blood vessels with higher flow rates for extended periods of time, thereby increasing the number of detected cells.

Chang et al. investigated the potential to perform invasive multiphoton in vivo flow cytometry with fiber-optics (67, 68). Previously, a two-photon optical fiber fluorescence (TPOFF) probe based on a single-mode fiber had been used to quantify fluorescence in solid tumors in live mice on a real-time basis (93, 94). Using a double-clad fiber (DCF) as the probe had enabled more than one order of magnitude increase in the detected two-photon excited fluorescence signals (95–97). This DCF-TPOFF probe was therefore chosen to conduct cell detection with two wavelength detection channels, first in whole blood in vitro, and subsequently in vivo. Fiber-based detection was demonstrated to have several distinct advantages for flow cytometry (68):

  • 1
    Since the detection of fluorescence from the tip of a fiber inserted into whole blood circumvents the usual problems associated with light scattering and absorption, a substantially higher sensitivity and detection efficiency was obtained relative to free-space techniques. In particular, in vitro multiphoton cytometry measurements of green-fluorescent-protein-expressing (GFP) cells performed in free-space suffer a dramatic drop in signal and detection efficiency when whole blood is substituted for a saline medium, due to strong overlap of the fluorescence spectrum with blood absorption and autofluorescence (Fig. 1). By contrast, with the fiber probe, the detected signal strength from fluorescent cells (by GFP or DiD) was comparable in either medium, with similar detection efficiency. Because all the excitation and collection happen only in close proximity to the fiber core, the two-photon-excited fluorescence photons can be collected directly before suffering multiple scattering as in free-space detection schemes. The frequency of detected events in vitro was consistent with predictions of a two-photon interrogation region that is 5 microns in lateral diameter.
  • 2
    The fiber can be inserted into tissue or large blood vessels, enabling long-term monitoring of a faster-moving, larger blood volume. To prove this principle, DiD-labeled untransfected or GFP-transfected cells were injected by various paths into a mouse's blood stream. The invasive fiber probe, inserted into the left liver lobe (chosen due to its high density of blood vessels and abundant blood flow), was successfully used to monitor ensuing circulation dynamics in real time. One technical challenge observed by this method was a high periodic background in the GFP detection channel, likely due to tissue autofluorescence, which was resolvable with the assistance of DiD signals in the second, long-wavelength channel. The long channel signal alone was used in another experiment to monitor minute-by-minute depletion dynamics of MCA-207 mouse sarcoma cells.
  • 3
    The ability to perform measurements in a conscious subject via a light-weight fiber will present a distinct advantage over free-space methods.
  • 4
    Finally, as tumors are very heterogeneous and there are no single markers which are universally expressed in all tumors, we anticipate utility of two-photon excitation to simultaneously detect multiple tumor markers using a single excitation laser beam. Thus, the TPOFF probe provides a promising way of conducting long-term real-time monitoring of circulating cells in animal models or in patients to advance the understanding of cancer biology or post-therapy response surveillance.

Conclusions and Future Directions

  1. Top of page
  2. Abstract
  3. Advantages of Multiphoton Excitation
  4. Initial Multiphoton Cytometry Experiments
  5. Two-Photon Flow Cytometry Instrumentation
  6. Fluorescent Labeling
  7. Short and Long Time Scale Monitoring In Vivo
  8. Circulating Tumor Cells
  9. Invasive In Vivo Flow Cytometry With Fiber Optic Probes
  10. Conclusions and Future Directions
  11. Acknowledgements
  12. Literature Cited

Through various instrumentation and labeling approaches, two-photon excitation of fluorescence has enabled unique cytometry investigations, both in vitro and in vivo. From a technological side, multiphoton cytometry will indubitably benefit from emerging biomedical optics platforms, such as super-resolution microscopy. There is mounting interest in subcellular investigations in cytometry (98), and it is only a matter of time for these techniques (99–101) to gain sufficient image acquisition speed to support high throughput. Presently, methods to achieve image resolution beyond the diffraction limit of light include STED (102) / RESOLFT (103), PALM (104) / STORM (105), structured illumination, and the related spatially nonuniform techniques of SPIN and SPADE (106). As nonlinear implementations exist, a substantial portion of future reviews of multiphoton cytometry may well be devoted to these methods. Indeed, nonlinear microscopy has already been demonstrated in cytometry (23).

With any in vivo cytometry application, the ability to effectively distinguish cells of interest is of the utmost importance. Autofluorescence itself can be used as a contrast agent in multiphoton imaging, for example to image leukocyte trafficking in vivo via endogenous tryptophan fluorescence (107). Laser pulse shaping for coherent control and selective excitation has produced interesting results (108–115) which might be usefully applied to fluorescence in cytometry as well. A coherently controlled flow cytometry system has in fact been built at the University of Michigan. Though the contrast was not sufficient to distinguish DiI- from DiD-labeled cells (unpublished data), the interface of coherent control and cytometry may prove more fertile in future investigations. Barring great advances in molecularly specific control of autofluorescence, targeted labeling will continue to remain a very significant component of most fluorescence-based cytometry strategies. As reviewed here, multiphoton cytometry has already been combined with folate conjugates and dendrimer platforms. Bioluminescence may eventually be integrated as well, which has been shown to enable in vivo detection down to a single genetically modified cell inserted under the skin of a mouse (116). One could also further extend the ratiometric labeling method beyond a dual dye staining scheme. For example, a two-beam, two-photon excitation cytometry system could be developed to detect the spectral shift from a single cell, or even to perform FRET measurements.

Cancer research has been a primary driving force of progress in this young field, particularly due to the clinical need to monitor circulating tumor cells. Clinical tracking of circulating cells in humans remains a major challenge and would be a momentous application of two-photon in vivo cytometry. Preliminary steps for this potentially revolutionary development have been achieved by the ability of multiphoton microscopy to examine T-cell response to antigens (117), host-pathogen interactions (118), and to study adaptive immune response (119), areas of investigation that in turn may also benefit from multiphoton cytometry. The research reviewed here establishes multiphoton cytometry as an effective tool for real-time in vivo studies, advancing the potential to probe the biology of circulating cells during disease progression and response to therapy.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Advantages of Multiphoton Excitation
  4. Initial Multiphoton Cytometry Experiments
  5. Two-Photon Flow Cytometry Instrumentation
  6. Fluorescent Labeling
  7. Short and Long Time Scale Monitoring In Vivo
  8. Circulating Tumor Cells
  9. Invasive In Vivo Flow Cytometry With Fiber Optic Probes
  10. Conclusions and Future Directions
  11. Acknowledgements
  12. Literature Cited

The authors express their gratitude to all the colleagues who have helped to develop the field of multiphoton cytometry. The authors would like to thank Prof. Valery Tuchin for the invitation to write this review as well as the reviewers for their useful suggestions to improve the manuscript.

Literature Cited

  1. Top of page
  2. Abstract
  3. Advantages of Multiphoton Excitation
  4. Initial Multiphoton Cytometry Experiments
  5. Two-Photon Flow Cytometry Instrumentation
  6. Fluorescent Labeling
  7. Short and Long Time Scale Monitoring In Vivo
  8. Circulating Tumor Cells
  9. Invasive In Vivo Flow Cytometry With Fiber Optic Probes
  10. Conclusions and Future Directions
  11. Acknowledgements
  12. Literature Cited