Deep tissue fluorescence imaging and in vivo biological applications.

We describe a novel technical approach with enhanced fluorescence detection capabilities in twophoton microscopy that achieves deep tissue imaging, while maintaining micron resolution. Compared to conventional two-photon microscopy, greater imaging depth is achieved by more efficient harvesting of fluorescence photons propagating in multiple-scattering media. The system maintains the conventional two-photon microscopy scheme for excitation. However, for fluorescence collection the detection system harvests fluorescence photons directly from a wide area of the turbid sample. The detection scheme relies on a wide area detector, minimal optical components and an emission path bathed in a refractive-index-matching fluid that minimizes emission photon losses. This detection scheme proved to be very efficient, allowing us to obtain high resolution images at depths up to 3 mm. This technique was applied to in vivo imaging of the murine small intestine (SI) and colon. The challenge is to image normal and diseased tissue in the whole live animal, while maintaining high resolution imaging at millimeter depth. In Lgr5-GFP mice, we have been successful in imaging Lgr5-eGFP positive stem cells, present in SI and colon crypt bases.

Head-up tilt and hyperventilation produce similar changes in cerebral oxygenation and blood volume: an observational comparison study using frequency-domain near-infrared spectroscopy.

PURPOSE: During anesthesia, maneuvers which cause the least disturbance of cerebral oxygenation with the greatest decrease in intracranial pressure would be most beneficial to patients with intracranial hypertension. Both head-up tilt (HUT) and hyperventilation are used to decrease brain bulk, and both may be associated with decreases in cerebral oxygenation. In this observational study, our null hypothesis was that the impact of HUT and hyperventilation on cerebral tissue oxygen saturation (SctO2) and cerebral blood volume (CBV) are comparable. METHODS: Surgical patients without neurological disease were anesthetized with propofol-remifentanil. Before the start of surgery, frequency-domain near-infrared spectroscopy was used to measure SctO2 and CBV at the supine position, at the 30° head-up and head-down positions, as well as during hypoventilation and hyperventilation. RESULTS: Thirty-three patients were studied. Both HUT and hyperventilation induced small decreases in SctO2 [3.5 (2.6)%; P < 0.001 and 3.0 (1.8)%; P < 0.001, respectively] and in CBV [0.05 (0.07) mL x 100 g(-1); P < 0.001 and 0.06 (0.05) mL x 100 g(-1); P < 0.001, respectively]. There were no differences between HUT to 30° and hyperventilation to an end-tidal carbon dioxide (ETCO2) of 25 mmHg (from 45 mmHg) in both SctO2 (P = 0.3) and CBV (P = 0.4). DISCUSSION: The small but statistically significant decreases in both SctO2 and CBV caused by HUT and hyperventilation are comparable. There was no correlation between the decreases in SctO2 and CBV and the decreases in blood pressure and cardiac output during head-up and head-down tilts. However, the decreases in both SctO2 and CBV correlate with the decreases in ETCO2 during ventilation adjustment.

Scanning laser image correlation for measurement of flow.

Scanning laser image correlation (SLIC) is an optical correlation technique for measuring the fluid velocity of particles suspended in a liquid. This technique combines laser scanning of an arbitrary pattern with pair cross-correlation between any two points in the pattern. SLIC overcomes many of the limitations of other optical correlation techniques for flow measurement, such as laser speckle, spatial temporal image correlation spectroscopy, and two-foci methods. One of the main advantages of SLIC is that the concept can be applied to measurements on a range of scales through simple zooming or modifications in the instrumentation. Additionally, SLIC is relatively insensitive to instrument noise through the use of correlation analysis and is insensitive to background. SLIC can provide detailed information about the direction and pattern of flow. SLIC has potential applications ranging from microfluidics to blood flow measurements.

Multimodal CARS microscopy determination of the impact of diet on macrophage infiltration and lipid accumulation on plaque formation in ApoE-deficient mice.

We characterized several cellular and structural features of early stage Type II/III atherosclerotic plaques in an established model of atherosclerosis-the ApoE-deficient mouse-by using a multimodal, coregistered imaging system that integrates three nonlinear optical microscopy (NLOM) contrast mechanisms: coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excitation fluorescence (TPEF). Specifically, the infiltration of lipid-rich macrophages and the structural organization of collagen and elastin fibers were visualized by CARS, SHG, and TPEF, respectively, in thick tissue specimens without the use of exogenous labels or dyes. Label-free CARS imaging of macrophage accumulation was confirmed by histopathology using CD68 staining. A high-fat, high-cholesterol Western diet resulted in an approximate 2-fold increase in intimal plaque area, defined by CARS signals of lipid-rich macrophages. Additionally, analysis of collagen distribution within lipid-rich plaque regions revealed nearly a 4-fold decrease in the Western diet-fed mice, suggesting NLOM sensitivity to increased matrix metalloproteinase (MMP) activity and decreased smooth muscle cell (SMC) accumulation. These imaging results provide significant insight into the structure and composition of early stage Type II/III plaque during formation and allow for quantitative measurements of the impact of diet and other factors on critical plaque and arterial wall features.

Coherent movement of cell layers during wound healing by image correlation spectroscopy.

We have determined the complex sequence of events from the point of injury until reepithelialization in axolotl skin explant model and shown that cell layers move coherently driven by cell swelling after injury. We quantified three-dimensional cell migration using correlation spectroscopy and resolved complex dynamics such as the formation of dislocation points and concerted cell motion. We quantified relative behavior such as velocities and swelling of cells as a function of cell layer during healing. We propose that increased cell volume ( approximately 37% at the basal layer) is the driving impetus for the start of cell migration after injury where the enlarged cells produce a point of dislocation that foreshadows and dictates the initial direction of the migrating cells. Globally, the cells follow a concerted vortex motion that is maintained after wound closure. Our results suggest that cell volume changes the migration of the cells after injury.

Spatiotemporal image correlation spectroscopy measurements of flow demonstrated in microfluidic channels.

Accurate blood flow measurements during surgery can improve an operation's chance of success. We developed near-infrared spatio-temporal image spectroscopy (NIR-STICS), which has the potential to make blood flow measurements that are difficult to accomplish with existing methods. Specifically, we propose the technique and we show feasibility on phantom measurements. NIR-STICS has the potential of measuring the fluid velocity in small blood vessels (less than 1 mm in diameter) and of creating a map of blood flow rates over an area of approximately 1 cm(2). NIR-STICS employs near-infrared spectroscopy to probe inside blood vessel walls and spatiotemporal image correlation spectroscopy to directly-without the use of a model-extract fluid velocity from the fluctuations within an image. We present computer simulations and experiments on a phantom system that demonstrate the effectiveness of NIR-STICS.

Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy.

Paxillin is an adaptor molecule involved in the assembly of focal adhesions. Using different fluorescence fluctuation approaches, we established that paxillin-EGFP is dynamic on many timescales within the cell, ranging from milliseconds to seconds. In the cytoplasmic regions, far from adhesions, paxillin is uniformly distributed and freely diffusing as a monomer, as determined by single-point fluctuation correlation spectroscopy and photon-counting histogram analysis. Near adhesions, paxillin dynamics are reduced drastically, presumably due to binding to protein partners within the adhesions. The photon-counting histogram analysis of the fluctuation amplitudes reveals that this binding equilibrium in new or assembling adhesions is due to paxillin monomers binding to quasi-immobile structures, whereas in disassembling adhesions or regions of adhesions, the equilibrium is due to exchange of large aggregates. Scanning fluctuation correlation spectroscopy and raster-scan image correlation spectroscopy analysis of laser confocal images show that the environments within adhesions are heterogeneous. Relatively large adhesions appear to slide transversally due to a treadmilling mechanism through the addition of monomeric paxillin at one side and removal of relatively large aggregates of proteins from the retracting edge. Total internal reflection microscopy performed with a fast acquisition EM-CCD camera completes the overall dynamic picture and adds details of the heterogeneous dynamics across single adhesions and simultaneous bursts of activity at many adhesions across the cell.

Detection of cerebral ischemia in neurovascular surgery using quantitative frequency-domain near-infrared spectroscopy.

OBJECT: There is great value in monitoring for signs of ischemia during neurovascular procedures. Current intraoperative monitoring techniques provide real-time feedback with limited accuracy. Quantitative frequency-domain near-infrared spectroscopy (Q-NIRS) allows measurement of tissue oxyhemoglobin (HbO2), deoxyhemoglobin (HHb), and total hemoglobin (tHb) concentrations and brain tissue oxygen saturation (SO2), which could be useful when monitoring for evidence of intraoperative ischemia. METHODS: Using Q-NIRS, the authors monitored 25 neurovascular procedures including aneurysm clip placement, arteriovenous malformation resection, carotid endarterectomy, superficial temporal artery-middle cerebral artery (MCA) bypass surgery, external carotid artery-MCA bypass surgery, encephaloduromyosynangiosis, and balloon occlusion testing. The Q-NIRS technology provides measurable cerebral oxygenation values independent from those of the scalp tissue. Thus, alterations in the variables measured with Q-NIRS quantitatively reflect cerebral tissue perfusion. Bilateral monitoring was performed in all cases. Five of the patients exhibited evidence of clinical ischemic events during the procedures. One patient suffered blood loss with systemic hypotension and developed diffuse brain edema intraoperatively, one patient suffered an ischemic event intraoperatively and developed an occipital stroke postoperatively, and one patient showed slowing on electroencephalography intraoperatively during carotid clamping; in two patients balloon occlusion testing failed. In all cases of ischemic events occurring during the procedure, Q-NIRS monitoring showed a decrease in HbO2, tHb, and SO2, and an increase in HHb. CONCLUSIONS: . Quantitative frequency-domain near-infrared spectroscopy provides quantifiable and continuous real-time information about brain oxygenation and hemodynamics in a noninvasive manner. This continuous intraoperative oxygenation monitoring is a promising method for detecting ischemic events during neurovascular procedures.

Effects of vasodilation on intrinsic optical signals in the mammalian brain: a phantom study.

Using a broadband spectral technique, we recently showed [J. Biomed. Opt. 10, 064009 (2005)] that during visual stimulation of the cat brain there were not only changes in oxy- and deoxyhemoglobin levels, reminiscent of the optical blood oxygenation level dependence (BOLD) effect reported in humans, but also the apparent water content of the tissue and the optical scattering contribution decreased during stimulation. These relatively fast changes (in seconds) in water tissue content are difficult to explain in physiological terms. We developed a simple model to explain how local vasodilation, which occurs as a result of the stimulation, could cause this apparent change in water content. We show that in a phantom model we can obtain spectral effects similar to those observed in the cat brain such as the apparent decrease of the water spectral component without changing the water content of the bath in which the phantom measurements were performed. Furthermore, using the phantom model, we show that the relative apparent changes in the spectral components due to vasodilation during stimulation are roughly comparable in magnitude to the changes in tissue chromophores due to the optical equivalent of the BOLD effect reported in the literature.

Spectrally resolved neurophotonics: a case report of hemodynamics and vascular components in the mammalian brain.

We developed a spectral technique that is independent of the light transport modality (diffusive or nondiffusive) to separate optical changes in scattering and absorption in the cat's brain due to the hemodynamic signal following visual stimulation. We observe changes in oxyhemoglobin and deoxyhemoglobin concentration signals during visual stimulation reminiscent of the functional magnetic resonance imaging (fMRI) blood oxygenation level dependence (BOLD) effect. Repeated measurements at different locations show that the observed changes are local rather than global. We also determine that there is an apparent large decrease in the water concentration and scattering coefficient during stimulation. We model the apparent change in water concentration on the separation of the optical signal from two tissue compartments. One opaque compartment is featureless (black), due to relatively large blood vessels. The other compartment is the rest of the tissue. When blood flow increases due to stimulation, the opaque compartment increases in volume, resulting in an overall decrease of tissue transmission. This increase in baseline absorption changes the apparent relative proportion of all tissue components. However, due to physiological effects, the deoxyhemoglobin is exchanged with oxyhemoglobin resulting in an overall increase in the oxyhemoglobin signal, which is the only component that shows an apparent increase during stimulation.

Mitigating thermal mechanical damage potential during two-photon dermal imaging.

Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 microm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with one-photon absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized.

Partitioning of NaPi cotransporter in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched membrane domains modulates NaPi protein diffusion, clustering, and activity.

In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaPi) despite an increase in its apical membrane abundance. This novel posttranslational regulation of NaPi activity is mediated by the increased glycosphingolipid content of the potassium-deficient apical membrane. However, the mechanisms by which these lipids modulate NaPi activity have not been determined. We determined if in potassium deficiency NaPi is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaPi in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaPi partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaPi was localized in the lipid raft fractions. By combining fluorescence correlation spectroscopy and photon counting histogram methods for control and potassium-deficient apical membranes reconstituted into giant unilamellar vesicles, we showed a 2-fold decrease in lateral diffusion of NaPi protein and a greater than 2-fold increase in size of protein aggregates/clusters in potassium deficiency. Our results indicate that NaPi protein is localized in membrane microdomains, that in potassium deficiency a larger proportion of NaPi protein is present in these microdomains, and that NaPi lateral diffusion is slowed down and NaPi aggregation/clustering is increased in potassium deficiency, both of which could be associated with the decreased Na/Pi cotransport activity in potassium deficiency.

Age-correlated changes in cerebral hemodynamics assessed by near-infrared spectroscopy.

Cerebral hemodynamic responses due to normal aging may interfere with hormonal changes, drug therapy, diseases, life style, and other factors. Age-correlated alterations in cerebral vasculature and autoregulatory mechanisms are the subject of interest in many studies. Near-infrared spectroscopy (NIRS) is widely used for monitoring cerebral hemodynamics and oxygenation changes at the level of small vessels. We believe that the compensatory ability of cerebral arterioles under hypoxic conditions and the dilatatory ability of cerebral vessels due to vasomotion may decline with normal aging. To test this hypothesis we used frequency-domain NIRS to measure changes in cerebral tissue oxygenation and oxy- and deoxy-hemoglobin concentrations caused by hypoxia during breath holding. We also assessed cerebral vasomotion during profound relaxation. Thirty seven healthy volunteers, 12 females and 25 males, ranging from 22 to 56 years of age (mean age 35 +/- 11 years) participated in the study. We observed age-correlated changes in the cerebral hemodynamics of normal subjects: diminished cerebral hemodynamic response to hypoxia due to breath holding in middle-aged subjects (38-56 years) and reduced amplitude of cerebral hemodynamic changes due to vasomotion during rest. Snoring related changes in cerebral hemodynamics did not allow us to observe the effect of age in a group of snorers. The prolonged supine position influenced measured changes due to hypoxia. In this investigation NIRS methodology allowed detection of age-correlated changes in cerebral oxygenation and hemodynamics. Other variables, such as snoring or posture impacted the observations in our group of healthy volunteers.

Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS).

Giant unilamellar vesicles (GUVs) have been widely used as a model membrane system to study membrane organization, dynamics, and protein-membrane interactions. Most recent studies have relied on imaging methods, which require good contrast for image resolution. Multiple sequential image processing only detects slow components of membrane dynamics. We have developed a new fluorescence correlation spectroscopy (FCS) technique, termed scanning FCS (i.e., SFCS), which performs multiple FCS measurements simultaneously by rapidly directing the excitation laser beam in a uniform (circular) scan across the bilayer of the GUVs in a repetitive fashion. The scan rate is fast compared to the diffusion of the membrane proteins and even small molecules in the GUVs. Scanning FCS outputs a "carpet" of timed fluorescence intensity fluctuations at specific points along the scan. In this study, GUVs were assembled from rat kidney brush border membranes, which included the integral membrane proteins. Scanning FCS measurements on GUVs allowed for a straightforward detection of spatial-temporal interactions between the protein and the membrane based on the diffusion rate of the protein. To test for protein incorporation into the bilayers of the GUVs, antibodies against one specific membrane protein (NaPi II cotransporter) were labeled with ALEXA-488. Fluorescence images of the GUVs in the presence of the labeled antibody showed marginal fluorescence enhancement on the GUV membrane bilayers (poor image contrast and resolution). With the application of scanning FCS, the binding of the antibody to the GUVs was detected directly from the analysis of diffusion rates of the fluorescent antibody. The diffusion coefficient of the antibody bound to NaPi II in the GUVs was approximately 200-fold smaller than that in solution. Scanning FCS provided a simple, quantitative, yet highly sensitive method to study protein-membrane interactions.

Localized irregularities in hemoglobin flow and oxygenation in calf muscle in patients with peripheral vascular disease detected with near-infrared spectrophotometry.

PURPOSE: Near-infrared spectrophotometry is used to measure flow, concentration, and oxygenation of hemoglobin in arterioles, capillaries, and venules several centimeters deep in tissue. The purpose of this study was to investigate the distribution of flow, concentration, and oxygenation of hemoglobin in calf muscle in patients with documented peripheral arterial occlusive disease (PVD), patients with risk factors for PVD,and healthy younger subjects at rest. METHOD: With a frequency-domain near-infrared spectrophotometer and a specially designed probe, we generated maps at 22 locations simultaneously of hemoglobin flow, concentration, and oxygenation, with the venous occlusion method. Eight legs of 7 patients with diagnosed PVD (PVD group), 10 legs of 8 patients with normal ankle-brachial index but with risk factors for PVD (RF group), and 16 legs of 8 healthy subjects (H group) were studied. RESULTS: Global mean values were significantly (P <.05) different between the three groups for oxygen consumption (PVD group, 0.027 +/- 0.009 mL/100 g/min; RF group, 0.038 +/- 0.017 mL/100 g/min; H group, 0.022 +/- 0.020 mL/100 g/min), venous oxygen saturation (PVD, 59.7% +/- 15.4%; RF, 69.6% +/- 10.5%; H, 80.8% +/- 4.5%), and, at 60 s of venous occlusion, concentration changes in oxyhemoglobin (PVD, 4.48 +/- 3.25 micromol/L; RF, 8.44 +/- 2.33 micromol/L; H, 6.85 +/- 4.57 micromol/L), deoxyhemoglobin (PVD, 3.60 +/- 0.73 micromol/L; RF, 4.39 +/- 1.30 micromol/L; H, 2.36 +/- 1.79 micromol/L), and total hemoglobin (PVD, 8.07 +/- 3.83 micromol/L; RF, 12.83 +/- 2.75 micromol/L; H, 9.21 +/- 6.34 micromol/L). No significant difference was found between the three groups for hemoglobin flow (PVD, 0.92 +/- 0.69 micromol/100 mL/min; RF, 1.68 +/- 0.50 micromol/100 mL/min; H, 1.44 +/- 1.17 micromol/100 mL/min) and blood flow (PVD, 0.45 +/- 0.28 mL/100 g/min; RF, 0.77 +/- 0.21 mL/100 g/min; H, 0.62 +/- 0.50 mL/100 g/min). All parameters featured a distribution dependent on location. CONCLUSION: Mean value for venous oxygen saturation was higher in healthy subjects compared to patients with documented PVD. In patients with PVD, areas of lower oxygenation were clearly discernible. At distal locations of calf muscle, significant correlations between reduced hemoglobin flow, venous oxygen saturation, oxyhemoglobin, and total hemoglobin and reduced ankle-brachial index were found. Maps revealed localized irregularities in oxyhemoglobin, total hemoglobin, and venous oxygen saturation in patients with PVD. Near-infrared spectrophotometry is a noninvasive bedside technique that can enable determination of blood flow and oxygenation in tissue and may provide a method for evaluating patients with PVD.

Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.

Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell except for trace amounts in the nuclear membrane and some vesicles. We performed fluorescence correlation spectroscopy measurements and photon-counting histogram analysis in specific domains of live cells. For AK1-EGFP, we observed only one diffusion component in the cytoplasm. For AK1beta-EGFP, we observed two distinct diffusion components on the plasma membrane. One corresponded to the free diffusing protein, whereas the other represented the membrane-bound AK1beta-EGFP. The diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect to that of EGFP, which was 50% more than what we would expect for a free diffusing AK1-EGFP. To rule out the possibility of oligomer formation, we performed photon-counting histogram analysis to direct analyze the brightness difference between AK1-EGFP and EGFP. From our analysis, we concluded that cytoplasmic AK1-EGFP is monomeric. fluorescence correlation spectroscopy proved to be a powerful technique for quantitatively studying the mobility of the target protein in live cells. This technology offers advantages in studying protein interactions and function in the cell.