Posts Tagged ‘laser photonics’

Laser Therapy Opens Blood-Brain Barrier

Curator: Larry H. Bernstein, MD, FCAP


Laser Surgery Opens Blood-Brain Barrier to Chemotherapy

ST. LOUIS, March 11, 2016 — A laser probe has been used to open the brain’s protective cover, enabling delivery of chemotherapy drugs to patients with glioblastoma — the most common and aggressive form of brain cancer.

In a pilot study conducted by the Washington University School of Medicine in St. Louis, Mo., 14 patients with glioblastoma underwent minimally invasive laser surgery to treat a recurrence of their tumors. Heat from the laser was already known to kill brain tumor cells but, unexpectedly, the researchers found that the technology penetrated the blood-brain barrier.

“The laser treatment kept the blood-brain barrier open for four to six weeks, providing us with a therapeutic window of opportunity to deliver chemotherapy drugs to the patients,” said neurosurgery professor Eric Leuthardt, MD, who also treats patients at Barnes-Jewish Hospital. “This is crucial because most chemotherapy drugs can’t get past the protective barrier, greatly limiting treatment options for patients with brain tumors.

The team is still closely following the patients, though early results indicate they are doing better on average, in terms of survival and clinical outcomes, than what the researchers would expect with other treatment methods.

Glioblastomas are one of the most difficult cancers to treat. Most patients diagnosed with this type of brain tumor survive just 15 months, according to the American Cancer Society.

The research is part of a larger phase II clinical trial that will involve 40 patients. Twenty patients were enrolled in the pilot study, 14 of whom were found to be suitable candidates for the minimally invasive laser surgery, a technology that Leuthardt helped pioneer.

The laser technology was approved by the FDA in 2009 as a surgical tool to treat brain tumors. The Washington team’s research marks the first time the laser has been shown to disrupt the blood-brain barrier, which shields the brain from harmful toxins but inadvertently blocks potentially helpful drugs, such as chemotherapy.

As part of the trial, doxorubicin, a widely used chemotherapy, was delivered intravenously to 13 patients in the weeks following the laser surgery. Preliminary data indicate that 12 patients showed no evidence of tumor progression during the short, 10-week time frame of the study. One patient experienced tumor growth before chemotherapy was delivered; the tumor in another patient progressed after chemotherapy was administered, the researcher reported.

The laser surgery was well-tolerated by the patients in the trial; most went home one to two days afterward, and none experienced severe complications. The surgery was performed while a patient lies in an MRI scanner, providing the neurosurgical team with a real-time look at the tumor. Using an incision of only 3 mm, a neurosurgeon robotically inserted the laser to heat up and kill brain tumor cells at a temperature of about 150 °F.

“The laser kills tumor cells, which we anticipated,” said Leuthardt. “But, surprisingly, while reviewing MRI scans of our patients, we noticed changes near the former tumor site that looked consistent with the breakdown of the blood-brain barrier.”

Leuthardt confirmed and further studied these imaging findings with study co-author Dr. Joshua Shimony, a professor of radiology at Washington University.

The researchers, including co-corresponding author Dr. David Tran, a neuro-oncologist now at the University of Florida, performed follow-up testing, which showed that the degree of permeability through the blood-brain barrier peaked one to two weeks after surgery but that the barrier remained open for up to six weeks.

Other successful attempts to breach the barrier have left it open for only a short time — about 24 hours — not long enough for chemotherapy to be consistently delivered, or have resulted in only modest benefits, the researchers said. The laser technology leaves the barrier open for weeks — long enough for patients to receive multiple treatments with chemotherapy. Further, the laser only opens the barrier near the tumor, leaving the protective cover in place in other areas of the brain. This has the potential to limit the harmful effects of chemotherapy drugs in other areas of the brain, the researchers said.

The findings also suggest that other approaches, such as cancer immunotherapy — which harnesses cells of the immune system to seek out and destroy cancer — could also be useful for patients with glioblastomas.

The researchers are planning another clinical trial that combines the laser technology with chemotherapy and immunotherapy, as well as trials to test targeted cancer drugs that normally can’t breach the blood-brain barrier.

The research was published in Plos One (doi: 10.1371/journal.pone.0148613).


Hyperthermic Laser Ablation of Recurrent Glioblastoma Leads to Temporary Disruption of the Peritumoral Blood Brain Barrier

Poor central nervous system penetration of cytotoxic drugs due to the blood brain barrier (BBB) is a major limiting factor in the treatment of brain tumors. Most recurrent glioblastomas (GBM) occur within the peritumoral region. In this study, we describe a hyperthemic method to induce temporary disruption of the peritumoral BBB that can potentially be used to enhance drug delivery.


Twenty patients with probable recurrent GBM were enrolled in this study. Fourteen patients were evaluable. MRI-guided laser interstitial thermal therapy was applied to achieve both tumor cytoreduction and disruption of the peritumoral BBB. To determine the degree and timing of peritumoral BBB disruption, dynamic contrast-enhancement brain MRI was used to calculate the vascular transfer constant (Ktrans) in the peritumoral region as direct measures of BBB permeability before and after laser ablation. Serum levels of brain-specific enolase, also known as neuron-specific enolase, were also measured and used as an independent quantification of BBB disruption.


In all 14 evaluable patients, Ktrans levels peaked immediately post laser ablation, followed by a gradual decline over the following 4 weeks. Serum BSE concentrations increased shortly after laser ablation and peaked in 1–3 weeks before decreasing to baseline by 6 weeks.


The data from our pilot research support that disruption of the peritumoral BBB was induced by hyperthemia with the peak of high permeability occurring within 1–2 weeks after laser ablation and resolving by 4–6 weeks. This provides a therapeutic window of opportunity during which delivery of BBB-impermeant therapeutic agents may be enhanced.

Trial Registration NCT01851733

Citation: Leuthardt EC, Duan C, Kim MJ, Campian JL, Kim AH, Miller-Thomas MM, et al. (2016) Hyperthermic Laser Ablation of Recurrent Glioblastoma Leads to Temporary Disruption of the Peritumoral Blood Brain Barrier. PLoS ONE 11(2): e0148613.

Glioblastoma (GBM) is the most common and lethal malignant brain tumor in adults [1]. Despite advanced treatment, median survival is less than 15 months, and fewer than 5% of patients survive past 5 years [2, 3]. Effective treatment options for recurrent GBM remain very limited and much of research and development efforts in recent years have focused on this area of greatly unmet needs. Up to 90% of recurrent tumors develop within the 2–3 cm margin of the primary site and are thought to arise from microscopic glioma cells that infiltrate the peritumoral brain region prior to resection of the primary tumor [4, 5]. Therefore elimination of infiltrative GBM cells in this region likely will improve long-term disease control.

Inadequate CNS delivery of therapeutic drugs due to the blood brain barrier (BBB) has been a major limiting factor in the treatment of brain tumors. The presence of contrast enhancement on standard brain MRI qualitatively reflects a disrupted state of the BBB. For this reason, drug access to the viable contrast enhanced tumor rim is likely significantly higher than to the peritumoral region, which usually does not have contrast enhancement [6, 7]. Evidence supporting this hypothesis came from studies in which drug levels of cytotoxic agents were sampled in tumors and the surrounding brain tissue at the time of surgery or autopsy. Drug concentrations were at the highest in the enhancing portion of tumors, and then rapidly decreased up to 40 fold lower by 2–3 cm distance from the viable tumor edge [810]. Overall, these observations suggest that the BBB and its integrity negatively correlate with delivery and potentially therapeutic effects of BBB impermeant drugs.

To circumvent the BBB problem in local drug delivery, recent approaches have focused on bypassing it. A previously described method is the use of Gliadel, a polymer wafer impregnated with the chemotherapeutic agent carmustine (BCNU) and placed intra-operatively in the resection cavity to bypass the BBB. This approach resulted in a statistically significant but modest survival advantage in both newly diagnosed and recurrent GBM [1113]. The modest benefit of Gliadel could be due to the short duration of drug delivery as nearly 80% of BCNU is released from the wafer over a period of only 5 days [14]. This observation further supports the notion that the BBB is critical to chemotherapy effect. However, Gliadel is not widely utilized as it requires an open craniotomy and can impair wound healing. Another approach of bypassing the BBB is the convection-enhanced delivery system in which a catheter is surgically inserted into the tumor to deliver chemotherapy [15]. This procedure requires prolonged hospitalization to maintain the external catheter to prevent serious complications and as a result has not been used extensively.

The role of hyperthermia in inducing BBB disruption has been previously described in animal models of CNS hyperthermia. In a rodent model of glioma, the global heating of the mouse’s head to 42°C for 30 minutes in a warm water bath significantly increased the brain concentration of a thermosensitive liposome encapsulated with adriamycin chemotherapy [16]. To effect more locoregional hyperthermia, retrograde infusion of a saline solution at 43°C into the left external carotid artery in the Wistar rat reversibly increased BBB permeability to Evans-blue albumin in the left cerebral hemisphere [17]. In another approach, neodymium-doped yttrium aluminum garnet (Nd:YAG) laser-induced thermotherapy to the left forebrain of Fischer rats resulted in loco-regional BBB disruption as evidenced by passage of Evans blue dye, serum proteins (e.g. fibrinogen & IgM), and the chemotherapeutic drug paclitaxel for up to several days after thermotherapy [18]. The effect of hyperthermia on the BBB of human brain has not been examined.

Here we describe an approach to induce sustained, local disruption of the peritumoral BBB using MRI-guided laser interstitial thermal therapy, or LITT. The biologic effects and correlation with MRI findings of LITT have been studied in both animal and human models since the development of LITT over twenty years ago. A well-described zonal distribution of histopathological changes with corresponding characteristic MR imaging findings centered on the light-guide track replace the lesion targeted for thermal therapy. The central treatment zone shows development of coagulative necrosis with complete loss of normal neurons or supporting structures immediately following therapy, corresponding to hyperintense T1-weighted signal intensity relative to normal brain [1922]. The peripheral zone of the post-treatment lesion is characterized by avid enhancement with intravenous gadolinium contrast agents, which peaks several days following thermal therapy and persists for many weeks after the procedure. Gadolinium contrast enhancement in the brain following LITT is due to leakage of gadolinium contrast into the extravascular space across a disrupted BBB [2023]. The perilesional zone of hyperintense signal intensity of FLAIR-weighted images develops within 1–3 days of thermal treatment and persists for 15–45 days [22].

We demonstrate that in addition to cytoreductive ablation of the main recurrent tumor, hyperthermic exposure of the peritumoral region resulted in localized, lasting disruption of the BBB as quantified by dynamic contrast-enhanced MRI (DCE-MRI) and serum levels of brain-specific enolase (BSE), thus providing a therapeutic window of opportunity for enhanced delivery of therapeutic agents.

Table 1. Patient Baseline Demographics and Characteristics.
TMZ/RT: Stupp protocol of 60 Gy radiotherapy plus concurrent 75mg/m2 daily temozolomide. Doxorubicin treatment: Timing of 20mg/m2 IV weekly doxobubicin treatment after LITT. Early = Starting within 1 week after LITT; Late = Starting at 6 weeks after LITT.
Quantitative measurement of LITT-induced peritumoral BBB disruption by DCE-MRI

Brain MRI obtained within 48 hours following LITT showed the targeted tumor replaced by a post-treatment lesion corresponding to the volume of treated tissue on intraoperative thermometry maps. The post-treatment lesion lost the original rim of tumor-associated contrast enhancement and instead demonstrated central hyperintense T1-weighted signal compared to the pre-treated tumor and normal brain and a faint, newly developed discontinuous rim of peripheral contrast enhancement extending beyond the original tumor-associated enhancing rim (Fig 2A). These findings are consistent with a loss of viable tumor tissue caused by LITT, thus achieving an effective cytoreduction similar to open surgical resection. Of note, the rim of new peripheral contrast enhancement persisted for at least the next 28 days (Fig 2B–2E). Perilesional edema qualitatively evaluated on FLAIR-weighted images increased from pretreatment imaging at week 2 and persisted at week 4 following LITT (Fig 2F–2I). Perilesional edema decreased on subsequent MRI examinations. These findings qualitatively indicate that peritumoral BBB is disrupted by LITT and that the disruption peaks within approximately 2 weeks after the procedure.


Fig 3 demonstrates the Ktrans time curves for our cohort of patients. In all subjects the Ktrans in the ROIs within the enhancing ring around the ablated tumor is highly elevated in the first few days after the procedure and then progressively decreases at approximately the 4-week time point. The bottom right subplot in Fig 3 is an average of the Ktrans time courses from all the subjects with adjacent curves indicating the plus and minus one standard error of the mean curves. This figure demonstrates the peak Ktrans value immediately after the LITT procedure with persistent elevation out to about 4 weeks. Radiographically, persistent contrast enhancement and FLAIR hyperintensity were observed well past 6 weeks and in many cases more than 10 weeks later. Several patients had recurrent tumor by radiographic criteria (increasing size of the edema and enhancing area around the tumor site) and these patients also demonstrated a corresponding increase in the Ktrans value. These recurrences occurred after the 10-week mark and thus were not included in Fig 3. Importantly no difference in the pattern of Ktrans tracing was consistently observed between the 10 patients receiving late doxorubicin treatment and the 4 patients receiving early doxorubicin treatment. In summary, these results indicate that the peritumoral BBB disruption as measured by Ktrans peaked immediately after LITT and persisted above baseline for an additional 4 weeks.


To optimize the ELISA assay for BSE, we collected sera from 3 patients with a newly diagnosed low-grade (WHO grade 2) glioma before and after their planned craniotomy and surgical resection, and determined serum concentrations of BSE. WHO grade 2 gliomas were chosen for the optimization because as they are generally non-contrast enhanced tumors on brain MRI, tumor-associated BBB is relatively intact and consequently, serum concentrations of brain-specific factors are predicted to be low pre-operatively and to then rise post-operatively due to the BBB compromise from the surgery. Serum BSE concentrations were low prior to surgery and then, as predicted, consistently increased after open craniotomy and tumor resection, thus indicating that this method had adequate sensitivity in detecting changes in serum levels of BSE due to disruption of the BBB (Fig 4).

Fig 4. Optimization of the BSE ELISA assay for measuring BBB disruption.

Serum concentrations of BSE before and after open craniotomy for surgical debulking in 3 subjects (A, B, and C) with a low-grade glioma, WHO grade II. *p<0.05.


Fig 5. BBB disruption induced by LITT as measured by serum biomarkers
Serum concentrations of BSE for each of the 14 evaluable subjects in the study (A-N) and as the mean + SEM (O) as a function of time in days from the LITT procedure. In 7/14 subjects, serum BSE levels slightly decreased immediately after LITT, then in 13/14 subjects, serum BSE levels rose shortly after LITT, peaked between 1–3 weeks after LITT, and then decreased by the 6-week time point. In Patient #12, serum BSE concentration increased at week 10 coincident with an increased Ktrans at the same time point, consistent with a recurrent tumor as demonstrated on diagnostic MR imaging. Patient #15’s serum BSE concentration began to rise by week 4, consistent with early multifocal recurrent disease as demonstrated on diagnostic MR imaging.

LITT is a minimally invasive neurosurgical technique that achieves effective tumor cytoreduction of brain tumors using a laser to deliver hyperthermic ablation. Here we have demonstrated that an unexpected, potentially useful effect of LITT is its ability to also disrupt the BBB in the peritumoral region that extends outwards 1–2 cm from the viable tumor rim. Importantly, the disruption persists in all 14 evaluable, treated patients for up to 4 weeks after LITT as measured quantitatively by DCE-MRI and up to 6 weeks as measured by serum levels of the brain-specific factor BSE. These observations indicate that after LITT there is a window during which enhanced local delivery of therapeutic agents into the desired location (i.e. peritumoral region) can potentially be achieved.

In all of the patients in this series, the peaks of serum concentrations of BSE showed wider variations and were delayed from several days to 1–2 weeks following the peak of BBB disruption as measured by Ktrans. The wider variations and delay of BSE concentrations lead to relatively low correlation coefficients between the two parameters and could be explained by: 1) the higher data point resolution for the serum values versus DCE-MRI values (weekly versus biweekly, respectively); 2) interval physiologic breakdown of thermally ablated tissue coupled with subsequent diffusion and equilibration between the intracranial and peripheral compartments; and 3) high inter-tumor heterogeneity among patients resulting in a wide variation in the rates at which ablated tissues of different compositions are broken down and released into the circulation. Whether these differences may be in part due to tumor-related factors such as IDH1/2 mutations and MGMT promoter methylation is unclear due to the small number of subjects. More importantly, both methods showed that the peritumoral BBB disruption induced by LITT was temporary, decreasing soon after peaking and being resolved by 4–6 weeks in most patients. In addition, although no significant difference in all the BBB measurement parameters was observed between the early and late doxorubicin treatment arms, the number of evaluable subjects was too small to allow generalization at this time.


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Spectroscopy Advances

Larry H. Bernstein, MD, FCAP, Curator



Learn the basics of fluorescence compensation’s role in flow cytometry




Compensation is a critical topic for flow cytometry, yet it is poorly understood. Within a flow cytometer, the appropriate ranges of excitation and emission wavelengths are selected by bandpass filters. But when emission spectra overlap, fluorescence from more than one fluorochrome may be detected. To correct for this spectral overlap, the fluorescence compensation process ensures that the fluorescence detected in a particular detector derives from the fluorochrome that is being measured.

This article discusses the principles of compensation to help those who use flow cytometry to master it for research experiments.


Instrumentation advances add flexibility and quantitation to flow cytometry

Giacomo Vacca and Jessica P. Houston

Despite its usefulness for bioimaging, fluorescence tagging has critical limitations. But a variant called fluorescence lifetime inherently solves longstanding problems and offers additional benefits that allow the use of more parameters. With new instrumentation developments, these capabilities are now enabling quantitative flow cytometry, which promises to advance cell biology in important ways.

Fluorescence labels have advanced cell biology and cytometry by enabling the study of cellular function and structure. The ability to attach a wide range of fluorophores to different antibodies has made it possible to classify and map the diverse populations of white blood cells that make up the human immune system, for instance. And the stunning images produced by confocal fluorescence microscopy have not only elucidated the relationships of subcellular structures previously only guessed at, but have also captured the popular imagination (witness the 2014 Nobel Prize in chemistry for super-resolved fluorescence microscopy). Fluorescence-based tools are here to stay.

For all its successes, however, fluorescence tagging seems to have hit a ceiling—even as applications demand ever-greater performance.

One challenge is that fluorophores, the molecules responsible for fluorescent behavior, are not always as well-behaved as we would like them to be. Overwhelmingly organic in nature, their synthesis can be complex, their stability is not guaranteed, and unlike the on-off states of digital switches, their fluorescence performance is governed by absorption and emission spectra-mechanisms firmly rooted in the analog world and subject to nonlinearities. The net effect is considerable uncertainty in the amount of light one can expect a given fluorophore to emit, even with a fixed excitation. When fluorophores are combined—for example, in applications of fluorescence resonance energy transfer (FRET)—the uncertainties add, preventing the results from being reliable quantitatively.

Another problem is that most fluorophores have relatively broad emission spectra—on the order of 30–50 nm. The availability of light sources and detectors (combined with the absorption curve of water) constrains the usable portion of the spectrum to some 400 nm, spanning the visible range and little more. To make the most of this limited range, microscopists and cytometrists have pushed for the development of large libraries of fluorophores with almost any peak emission wavelength in the visible spectrum. But while hundreds of choices exist, only a handful can be used simultaneously, with their spectra spread sufficiently to allow for differential detection. This forces experiments to be run sequentially instead of in parallel—a nuisance in microscopy and a severe limitation in flow cytometry, where sequential interrogation of a single cell is incompatible with the nature of the technique. In other words, the multiplexing capability of fluorescence-based approaches is capped at a low level.

Why fluorescence lifetime?

Detection of fluorescence lifetime offers a solution to both problems. When a fluorophore absorbs an incoming excitation photon, it spends a certain amount of time in the excited state before emitting a longer-wavelength photon and returning to its ground state. A collection of identical fluorophores will generate a distribution of time intervals between absorption and emission; this statistical distribution takes the shape of an exponential decay curve (see Fig. 1a), and can be characterized by its 1/e point τ—the lifetime of this fluorescent transition. Critically, the lifetime value τ is completely independent of intensity (see Fig. 1b). This characteristic makes lifetime measurements insensitive to fluctuations in the intensity of the light source, as well as to variations in the amount of light ultimately absorbed by the fluorophores.

FIGURE 1. A collection of fluorophores, excited by a short optical pulse (thick line), produces collective fluorescence emission that follows an exponential decay curve with 1/e lifetime τ (a). Regardless of the intensity of the excitation pulse, the decays from identical fluorophores all have the same characteristic lifetime τ (b).

By virtue of its insensitivity to intensity fluctuations, fluorescence lifetime lends itself particularly well to quantitative studies. In the case of FRET, the difference between intensity-based and lifetime-based measurements is even more critical, as FRET assays rely on the interaction between two fluorophores. Not only do intensity fluctuations cause uncertainty in the absorption process, but the energy transfer process itself, between the two fluorophores in the FRET pair, is affected by intensity variations—further confounding the results. Measuring fluorescence lifetime makes it possible to eliminate these spurious effects and carry out quantitative FRET applications; for instance, measurements on protein folding and conformation, and on protein-protein interactions.

Fluorescence lifetime has some additional benefits. The lifetime of a fluorescent transition can be influenced by the local molecular environment of the fluorophore. For example, certain fluorophores exhibit a dependence of their lifetime on the pH of the medium surrounding them;1 or they can be sensitive to the concentration of certain ions, such as Ca++.2 This sensitivity can be exploited to use lifetime as a local, molecular-scale probe of environmental conditions.

Measurements of fluorescence lifetime have been implemented successfully on imaging platforms. In particular, a technique called time-correlated single-photon counting (TCSPC) has been widely used to generate fluorescence lifetime analysis on advanced microscopes. Developers of TCSPC detection systems, such as Becker & Hickl GmbH and PicoQuant GmbH (both of Berlin, Germany), offer a rich portfolio of products aimed at imaging applications of fluorescence lifetime. The plethora of results3 made possible by their technologies is a testament to the power of fluorescence lifetime imaging microscopy (FLIM) techniques. FLIM-FRET in particular has enjoyed growing popularity.4

However, the slow data acquisition rates of TCSPC, added to the inherent low throughput of microscopy, severely limit the efficiency of analysis. Processing a field of view with perhaps 50 cells can take minutes; while FLIM provides spatially resolved information on each cell, it comes at a steep price in terms of throughput trade-offs. FLIM is a great technique for detailed analysis of handfuls of cells, but it is poorly suited to the rapid analysis or screening of thousands to millions of cells.

For those applications where localization information is not critical, it is highly desirable to trade spatial resolution for analysis throughput. Flow cytometry does just that: By interrogating single cells in a narrow stream flowing at high speeds past a laser interrogation point, it can routinely deliver analysis rates on the order of 10,000 cells per second-three to four orders of magnitude faster than is possible with microscopy-based tools. And while one of the trade-offs is imaging information, flow cytometry offers multiplexed analysis to compensate, in part, for that.

Fluorescence lifetime flow cytometry

It is therefore natural to want to combine the benefits of fluorescence lifetime with the high throughput of flow cytometry. Early work in this direction was spearheaded by John Steinkamp and collaborators at Los Alamos National Laboratory (LANL) in the 1990s,5-8 and briefly explored commercially by BD Biosciences (San Jose, CA) through the work of Robert Hoffman and collaborators9 around the same time. Those undertakings were largely based on analog-electronics implementations of a fluorescence lifetime technique referred to as “phase lifetime” or “frequency-domain lifetime.” More recently, researchers at New Mexico State University (NMSU) have revitalized the field with the introduction of new digital fluorescence lifetime technologies for cell sorting and analysis.10 These efforts also involve frequency modulation approaches, and have led to phasor analysis and non-modulated approaches to lifetime.11,12

The early pioneering experiments were crucial for establishing the viability of performing fluorescence lifetime measurements in flow, but traditional instrumentation and signal processing tools used to perform the measurements were complex, expensive, and did not lend themselves to commercialization. For this reason, the new digital approaches are appealing. The frequency-domain approaches, however, can reliably report only a single lifetime component (an area NMSU researchers are now exploring), whereas for the technique to have practical value, flow cytometrists would need it to resolve multiple lifetime components within any single cell.

An academic/commercial collaboration (between NMSU and Kinetic River Corp.) supported by the NSF CAREER DBI 1150202 project has produced a new approach that, like its TCSPC FLIM counterpart, is based on the time domain: Fluorescence lifetime values are extracted directly from the fluorescence decay curves emitted in response to pulsed excitation. This has allowed resolution of multiple components of fluorescence lifetime within individual decay curves13—to our knowledge, the first such feat performed at the speeds of particle flow typical of flow cytometry (see Fig. 2).

FIGURE 2. Time-domain fluorescence lifetime flow cytometry results from ethidium bromide (EB) in Chinese Hamster Ovary cells (CHO-K1). The multiexponential decay curve in this case comprises two distinct lifetimes (τ1 and τ2), each contributing to the overall curve in proportion to the corresponding population of fluorophores (unbound and DNA-bound EB, respectively).

The first instrument to emerge from this collaboration, the Danube I, proved that our approach is feasible. It has been in use at NMSU since late 2012. That prototype is capable of multi-exponential decay measurements; it has a lifetime resolution of around 2 ns—sufficient for proving the concept and even for certain applications, but not enough to measure the shorter lifetimes of many fluorophores of interest. A second-generation instrument, the Danube II (completed in the summer of 2014), delivers shorter pulses and higher sensitivity, and the NMSU lab plans to introduce a range of laser modulation and digital signal processing approaches onto this instrument for more novel ways to measure fluorescence lifetimes. We expect this system to yield sub-nanosecond lifetime resolution.


The short distance between, and proper alignment of, donor and acceptor ensure that, upon excitation by light in the donor absorption spectrum, efficient FRET takes place, transferring excitation energy to the acceptor molecule. As the donor produces fluorescence emission, the lifetime of the transition is reduced by the presence of the acceptor. Figure 4b shows how, when a protease severs the linker, the donor and acceptor molecules no longer undergo FRET, and the lifetime of the donor emission returns to its longer native value.

FIGURE 4. Fluorescence lifetime can study apoptosis mediated by proteases. See text for details.12

More broadly, the subject of fluorescence lifetime flow cytometry is enjoying a veritable renaissance. At the last Annual Congress of Cytometry (CYTO, May 2014, Ft. Lauderdale, FL), a new workshop on fluorescence lifetime,15 co-chaired by Silas Leavesley, of South Alabama University (Mobile, AL), featured a distinguished panel (see Fig. 3) that included Prof. Suzuki, who gave the latest results on her work on FRET bioprobes; Patrick Jenkins of NMSU and the Fred Hutchinson Cancer Research Center (Seattle, WA), who talked about using fluorescence lifetime to study cell signaling in yeast cells using FRET; János Szöllosi of Debrecen University (Hungary), an expert in applying FRET-FLIM to cell signaling pathways in cancer research; and Ralph Jimenez of JILA/NIST and the University of Colorado (Boulder), who presented his work on the relationship of fluorescence lifetime and photostability in fluorescent proteins. Jenkins also demonstrated sorting of cells based on differences in fluorescence lifetime alone.16 And at the most recent Cytometry Development Workshop (October 30–November 2, 2014; La Jolla, CA), fluorescence lifetime flow cytometry was the topic area with the largest number of presented talks, according to the workshop summary by Morgan Richert of the Scintillon Institute.

The early interest in multi-exponential fluorescence lifetime flow cytometry has focused on quantitative FRET—a broad set of applications where the ability to accurately resolve multiple fluorescence decay components can make the most immediate difference (see Fig. 4). As researchers become more familiar with fluorescence lifetime, other kinds of applications will follow suit. Researchers at NMSU are pursuing measurements of autofluorescence to differentiate normal from cancerous cells. By measuring the different lifetimes of endogenously fluorescent compound NADH in its free and bound states, one can distinguish cells with normal metabolism and ones with abnormally high metabolic rates—a hallmark of cancer. Being able to do so on a flow cytometry platform would allow rapid discrimination of cancer cells out of large samples of normal background cells. NMSU researchers are also now sorting cells using the fluorescence lifetime as a single parameter.12 Therefore, sorting out cells with altered metabolism based on fluorescence decay times is near.

FIGURE 5. (a) Traditional multiplexing schemes for fluorescence in cytometry and microscopy have relied on tight packing of fluorescence emission bands (colored curves) into the limited spectral range of visible light (wavelength axis λ). This approach is constrained by the breadth of the emission spectra and their overlaps (shaded areas), which cause unwanted spillover and require burdensome compensation. (b) By using fluorescence lifetime, one is able to open up an entire new dimension in multiplexing (vertical axis τ), and for each fluorescence spectral band (FL1,FL2, etc.) exploit several lifetime bands (e.g., τ1, τ2, τ3). Each intersection point (like the thick black square indicated for FL2 and τ2) represents a unique combination (a “channel”) of wavelength and lifetime. By spreading out the fluorescence bands, the spectral spillover problem is reduced or eliminated, while still increasing the number of effective channels available.

An even broader goal is to use fluorescence lifetime as a separate dimension to massively increase the multiplexing capabilities of flow cytometers (see Fig. 5b). By using fluorescence lifetime as a parameter, spectrally overlapping emissions can be distinguished. Even just doubling the number of channels available for detection—a rather conservative estimate of the potential benefit of multiplexing with fluorescence lifetime—would be a huge boon to immunologists and other cell biology researchers currently forced to work with a limited number of tags on the single cramped dimension of the visible spectrum.

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In vivo spectroscopy technique could advance medical diagnostics and research

A team of researchers at Purdue University (West Lafayette, IN) has demonstrated that an in vivo spectroscopy technique can reveal the chemical composition of living tissue for medical diagnostics and cellular studies.
The development is potentially important because knowing the chemical content of tissue is needed for early detection of disease, and the system also can be used to study molecular dynamics in living cells as they are occurring, according toJi-Xin Cheng, a professor in Purdue’s Weldon School of Biomedical Engineering and Department of Chemistry and Scientific Director of the Label-free Imaging lab at Purdue’s Discovery Park. Conventional imaging technologies such as magnetic resonance imaging (MRI) and computed tomography (CT) do not reveal the chemical composition of tissues, he says.

Although optical spectroscopy has been routinely used to study molecules in a sample cell, it is currently not practical to perform in vivo spectroscopy, or the analysis of how light interacts with molecules in living tissue. This is because photons strongly scatter when light shines through tissues, making detection of the signal through a spectrometer inefficient, Cheng says.

The new technique works by “coding” individual photons from a pulsing laser with a megahertz radio frequency and then collecting those photons with a detector after they have interacted with tissue. The system was demonstrated in human breast cancer detection. Ordinarily, the cancer tissue samples would have to be processed for histological examination, which could take up to a week. The new technology yields results in about 2 s.

Ji-Xin Cheng leads a Purdue team demonstrating an in vivo spectroscopy technique developed with a $1 million W. M. Keck Foundation grant. The technology could bring advanced medical diagnostics. (Vincent Walter/Purdue University image)
The technique also was used to map vitamin E in the skin of laboratory mice. “People use vitamin E on their skin as a topical treatment, and, like any drug, we would like to know where it goes after it is applied to study drug delivery mechanisms,” Cheng says. “We converted Raman spectroscopy, which is generally used to study molecules in solutions or fixed tissues, to an in vivo imaging platform that is able to monitor how a living cell executes its functions in real time. It’s a proof of concept. The approach allows us to get a spectrum of individual molecules, revealing the chemical composition of the tissues.”

The innovation offers “label-free” detection, or imaging that does not require the use of fluorescent dyes or other preparations to detect structures and features. Because processing kills the tissue and is time-consuming, Cheng says, the label-free technology allows the study of unaltered living tissues and cells, making for more rapid and accurate studies.

Flow Cytometry: Small, inexpensive flow cytometer automates fast tumor cell counting
Cancerous growths release circulating tumor cells (CTCs) into the bloodstream, and their number indicates the effectiveness of therapy: A decrease during treatment means success. Traditional flow cytometers can quantify tumor cells from a blood sample, but they are as large as a washer/dryer combo, can cost more than $300,000, and produce results only after several hours. By contrast, the inexpensive, shoebox-size PoCyton, by the Fraunhofer Institute for Chemical Technology (ICT-IMM; Mainz, Germany), works about 20 times faster and its automated measurements need no calibration.
The measuring channel (visible at right) forms the key component of the PoCyton flow cytometer. (Image courtesy of Fraunhofer ICT-IMM)

Flow cytometry involves injecting fluorescent dye into the blood; the dye molecules bind to tumor cells, leaving all other cells unmarked. While adding dye to a blood sample is typically a manual process, the PoCyton automates it. Within the system, the tagged blood is funneled through a narrow focal area, causing suspended cells to pass in front of a laser spot detector one by one. The light causes the cancer cells to fluoresce, enabling the device to detect and count them. This narrow passage is the key to the PoCyton process: its geometry ensures that every object flowing past the detector is registered, and no cell is hidden behind another. This is critical because even in a severely sick patient, only five in approximately one billion suspended objects in a 10 mL sample of blood is a CTC. The researchers report that the device provides adequate sensitivity, and they are now working to create a fully functional prototype.

Besides counting of CTCs, the PoCyton could enable detection of legionella bacteria in drinking water, which can cause Legionnaire’s disease.


Spectroscopy method to help develop motion-sensitive human-computer technologies
The Fraunhofer Institute of Industrial Engineering (IAO) opened its NeuroLab test environment, where researchers are applying neuroscience knowledge — including a spectroscopy method — to ergonomic workplace design issues, with focus on assistance systems in vehicles, in human-robot collaboration, and in knowledge work. View more >>


Spectroscopy helps show relationship between male brain activity and fitness
An optical neuroimaging technique called functional near-infrared spectroscopy (fNIRS) helped to show the direct relationship between brain activity, brain function, and physical fitness in a group of older Japanese men. View more


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