Preclosure spectroscopic differences between healed and dehisced traumatic wounds.

BACKGROUND: The complexity and severity of traumatic wounds in military and civilian trauma demands improved wound assessment, before, during, and after treatment. Here, we explore the potential of 3 charge-coupled device (3CCD) imaging values to distinguish between traumatic wounds that heal following closure and those that fail. Previous studies demonstrate that normalized 3CCD imaging values exhibit a high correlation with oxygen saturation and allow for comparison of values between diverse clinical settings, including utilizing different equipment and lighting. METHODS: We screened 119 patients at Walter Reed National Military Medical Center and at Grady Memorial Hospital with at least one traumatic extremity wound of ≥ 75 cm2. We collected images of each wound during each débridement surgery for a total of 66 patients. An in-house written computer application selected a region of interest in the images, separated the pixel color values, calculated relative values, and normalized them. We followed patients until the enrolled wounds were surgically closed, quantifying the number of wounds that dehisced (defined as wound failure or infection requiring return to the operating room after closure) or healed. RESULTS: Wound failure occurred in 20% (19 of 96) of traumatic wounds. Normalized intensity values for patients with wounds that healed successfully were, on average, significantly different from values for patients with wounds that failed (p ≤ 0.05). Simple thresholding models and partial least squares discriminant analysis models performed poorly. However, a hierarchical cluster analysis model created with 17 variables including 3CCD data, wound surface area, and time from injury predicts wound failure with 76.9% sensitivity, 76.5% specificity, 76.6% accuracy, and a diagnostic odds ratio of 10.8 (95% confidence interval: 2.6-45.9). CONCLUSIONS: Imaging using 3CCD technology may provide a non-invasive and cost-effective method of aiding surgeons in deciding if wounds are ready for closure and could potentially decrease the number of required débridements and hospital days. The process may be automated to provide real-time feedback in the operating room and clinic. The low cost and small size of the cameras makes this technology attractive for austere and shipboard environments where space and weight are at a premium.

Accurate and Rapid Differentiation of Acinetobacter baumannii Strains by Raman Spectroscopy: a Comparative Study.

In recent years, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become the standard for routine bacterial species identification due to its rapidity and low costs for consumables compared to those of traditional DNA-based methods. However, it has been observed that strains of some bacterial species, such as Acinetobacter baumannii strains, cannot be reliably identified using mass spectrometry (MS). Raman spectroscopy is a rapid technique, as fast as MALDI-TOF, and has been shown to accurately identify bacterial strains and species. In this study, we compared hierarchical clustering results for MS, genomic, and antimicrobial susceptibility test data to hierarchical clustering results from Raman spectroscopic data for 31 A. baumannii clinical isolates labeled according to their pulsed-field gel electrophoresis data for strain differentiation. In addition to performing hierarchical cluster analysis (HCA), multiple chemometric methods of analysis, including principal-component analysis (PCA) and partial least-squares discriminant analysis (PLSDA), were performed on the MS and Raman spectral data, along with a variety of spectral preprocessing techniques for best discriminative results. Finally, simple HCA algorithms were performed on all of the data sets to explore the relationships between, and natural groupings of, the strains and to compare results for the four data sets. To obtain numerical comparison values of the clustering results, the external cluster evaluation criteria of the Rand index of the HCA dendrograms were calculated. With a Rand index value of 0.88, Raman spectroscopy outperformed the other techniques, including MS (with a Rand index value of 0.58).

Diagnostic Bacteriology: Raman Spectroscopy.

Current clinical methodology for identification of bacterial infections relies predominantly on culturing microbes from patient material and performing biochemical tests. This can often be an inefficient and lengthy process, which has a significant detrimental effect upon patient care. Techniques used in other aspects of molecular research have the potential to revolutionize the way in which diagnostic tests are used and delivered in the clinical setting. The need for rapid, accurate, and cost-effective molecular techniques in the diagnostic laboratory is imperative to improving patient care, preventing the spread of drug resistance and decreasing the overall burden associated with nosocomial infections. Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS) are powerful vibrational spectroscopy techniques that are being developed for highly sensitive pathogen identification in complex clinical samples. Raman spectroscopy is a molecular technique that is capable of probing samples noninvasively and nondestructively. It has been used with high specificity to assess tissue and bacterial samples at the molecular level with diverse clinical and diagnostic applications. SERS has recently developed out of the advances in the Raman spectroscopy arena. This technique is designed to amplify Raman scattering and allows for better differentiation of bacterial isolates. Although the current parameters for the use of SERS require a pure culture and are relatively monoparametric, current breakthroughs and testing are pushing the technology to new levels and thus changing the face of modern bacterial diagnostics.

Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections.

Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growth in vitro via a strong but incomplete selection event. The cocktail here appears to function in a combinatorial manner, as one constituent phage targets capsulated A. baumannii bacteria and selects for loss of receptor, shifting the population to an uncapsulated state that is then sensitized to the remaining four phages in the cocktail. Additionally, capsule is a known virulence factor for A. baumannii, and we demonstrated that the emergent uncapsulated bacteria are avirulent in a Galleria mellonella model. These results highlight the importance of anticipating population changes during phage therapy and designing intelligent cocktails to control emergent strains, as well as the benefits of using phages that target virulence factors. Because of the efficacy of this cocktail isolated from a limited environmental pool, we have established a pipeline for developing new phage therapeutics against additional clinically relevant multidrug-resistant pathogens by using environmental phages sourced from around the globe.

Noninvasive Multimodal Imaging to Predict Recovery of Locomotion after Extended Limb Ischemia.

Acute limb ischemia is a common cause of morbidity and mortality following trauma both in civilian centers and in combat related injuries. Rapid determination of tissue viability and surgical restoration of blood flow are desirable, but not always possible. We sought to characterize the response to increasing periods of hind limb ischemia in a porcine model such that we could define a period of critical ischemia (the point after which irreversible neuromuscular injury occurs), evaluate non-invasive methods for characterizing that ischemia, and establish a model by which we could predict whether or not the animal's locomotion would return to baselines levels post-operatively. Ischemia was induced by either application of a pneumatic tourniquet or vessel occlusion (performed by clamping the proximal iliac artery and vein at the level of the inguinal ligament). The limb was monitored for the duration of the procedure with both 3-charge coupled device (3CCD) and infrared (IR) imaging for tissue oxygenation and perfusion, respectively. The experimental arms of this model are effective at inducing histologically evident muscle injury with some evidence of expected secondary organ damage, particularly in animals with longer ischemia times. Noninvasive imaging data shows excellent correlation with post-operative functional outcomes, validating its use as a non-invasive means of viability assessment, and directly monitors post-occlusive reactive hyperemia. A classification model, based on partial-least squares discriminant analysis (PLSDA) of imaging variables only, successfully classified animals as "returned to normal locomotion" or "did not return to normal locomotion" with 87.5% sensitivity and 66.7% specificity after cross-validation. PLSDA models generated from non-imaging data were not as accurate (AUC of 0.53) compared the PLSDA model generated from only imaging data (AUC of 0.76). With some modification, this limb ischemia model could also serve as a means on which to test therapies designed to prolong the time before critical ischemia.

Pilot study for detection of early changes in tissue associated with heterotopic ossification: moving toward clinical use of Raman spectroscopy.

Over 60% of combat-wounded patients develop heterotopic ossification (HO). Nearly 33% of them require surgical excision for symptomatic lesions, a procedure that is both fraught with complications and can delay or regress functional rehabilitation. Relative medical contraindications limit widespread use of conventional means of primary prophylaxis, such as nonspecific nonsteroidal anti-inflammatory medications and radiotherapy. Better methods for risk stratification are needed to both mitigate the risk of current means of primary prophylaxis as well as to evaluate novel preventive strategies currently in development. We asked whether Raman spectral changes, measured ex vivo, could be associated with histologic evidence of the earliest signs of HO formation and substance P (SP) expression in tissue biopsies from the wounds of combat casualties. In this pilot study, we compared normal muscle tissue, injured muscle tissue, very early HO lesions (< 16 d post-injury), early HO lesions (> 16 d post-injury) and mature HO lesions. The Raman spectra of these tissues demonstrate clear differences in the Amide I and III spectral regions of HO lesions compared to normal tissue, denoted by changes in the Amide I band center (p < 0.01) and the 1340/1270 cm(-1) (p < 0.05) band area and band height ratios. SP expression in the HO lesions appears to peak between 16 and 30 d post-injury, as determined by SP immunohistochemistry of corresponding tissue sections, potentially indicating optimal timing for administration of therapeutics. Raman spectroscopy may therefore prove a useful, non-invasive and early diagnostic modality to detect HO formation before it becomes evident either clinically or radiographically.

Raman spectroscopic analysis of combat-related heterotopic ossification development.

Over 60% of our severely combat-injured patient population develops radiographically apparent heterotopic ossification. Nearly a third of these require surgical excision of symptomatic lesions, a procedure that is fraught with complications, and delays or regresses functional rehabilitation in many cases. Unfortunately, for the combat injured, medical contraindications and logistical limitations limit widespread use of conventional means of primary prophylaxis. Better means of risk stratification are needed to both mitigate the risk of current means of primary prophylaxis as well as to evaluate novel preventive strategies currently in development. We asked whether Raman spectral changes, measured ex vivo, correlated with histologic evidence of the earliest signs of HO formation using tissue biopsies from the wounds of combat casualties. In doing so, we compared normal muscle tissue to injured muscle tissue, unmineralized HO tissue, and mineralized HO tissue. The Raman spectra of these tissues demonstrate clear differences in the amide I and amide III spectral regions of HO tissue compared to normal tissue, denoted by changes in the 1640/1445cm(-1)(p<0.01), and 1340/1270cm(-1) (p<0.01) band area ratios (BARs). Additionally, analysis of the bone mineral in HO by Raman spectroscopy appears capable of determining bone maturity by measuring both the 945/960cm(-1) and the 1070/1445cm(-1) BARs. Raman may therefore prove a useful, non-invasive, and early diagnostic modality to detect HO formation prior to it becoming evident clinically or radiographically. This technique could ostensibly be utilized as a non-invasive means to risk stratify individual wounds at a time thought to be amenable to various means of primary prophylaxis.

Evidence of a heterogeneous tissue oxygenation: renal ischemia/reperfusion injury in a large animal model.

Renal ischemia that occurs intraoperatively during procedures requiring clamping of the renal artery (such as renal procurement for transplantation and partial nephrectomy for renal cancer) is known to have a significant impact on the viability of that kidney. To better understand the dynamics of intraoperative renal ischemia and recovery of renal oxygenation during reperfusion, a visible reflectance imaging system (VRIS) was developed to measure renal oxygenation during renal artery clamping in both cooled and warm porcine kidneys. For all kidneys, normothermic and hypothermic, visible reflectance imaging demonstrated a spatially distinct decrease in the relative oxy-hemoglobin concentration (%HbO2) of the superior pole of the kidney compared to the middle or inferior pole. Mean relative oxy-hemoglobin concentrations decrease more significantly during ischemia for normothermic kidneys compared to hypothermic kidneys. VRIS may be broadly applicable to provide an indicator of organ ischemia during open and laparoscopic procedures.

Vibrational spectroscopy: a tool being developed for the noninvasive monitoring of wound healing.

Wound care and management accounted for over 1.8 million hospital discharges in 2009. The complex nature of wound physiology involves hundreds of overlapping processes that we have only begun to understand over the past three decades. The management of wounds remains a significant challenge for inexperienced clinicians. The ensuing inflammatory response ultimately dictates the pace of wound healing and tissue regeneration. Consequently, the eventual timing of wound closure or definitive coverage is often subjective. Some wounds fail to close, or dehisce, despite the use and application of novel wound-specific treatment modalities. An understanding of the molecular environment of acute and chronic wounds throughout the wound-healing process can provide valuable insight into the mechanisms associated with the patient's outcome. Pathologic alterations of wounds are accompanied by fundamental changes in the molecular environment that can be analyzed by vibrational spectroscopy. Vibrational spectroscopy, specifically Raman and Fourier transform infrared spectroscopy, offers the capability to accurately detect and identify the various molecules that compose the extracellular matrix during wound healing in their native state. The identified changes might provide the objective markers of wound healing, which can then be integrated with clinical characteristics to guide the management of wounds.

Evaluation of intestinal viability using 3-charge coupled device image enhancement technology in a pediatric laparoscopic appendectomy model.

BACKGROUND: Intraoperative laparoscopic determination of intestinal viability is currently limited to subjective visible cues. Adjunctive modalities are neither widely available nor practical. Three-charge coupled device (3-CCD) imaging directly correlates the amount of light detected by CCDs to tissue oxygenation. We hypothesize that application of 3-CCD image enhancement detects bowel ischemia in a pediatric laparoscopic appendectomy model. METHODS: We recorded 10 laparoscopic appendectomies for appendicitis. Offline analysis involved selecting regions of interest (ROIs) in the appendix, adjacent colon, and nonappendiceal fat and calculating mean intensity values in selected images before and after division of the mesoappendix. The colon was used as a control, and the intensity values were normalized to fat. RESULTS: As an indicator of decreased perfusion, the mean appendix ROI intensity values decreased over time (R(2) = 0.92) compared with the colon mean ROI intensity values, which remained stable. There was a statistically significant difference between fat-normalized intensity values for ischemic and nonischemic appendix after 1 minute. CONCLUSION: We have demonstrated proof of principle for the determination of bowel ischemia using 3-CCD image enhancement. By quantitatively identifying areas of ischemia, this technique has the potential to significantly change the management of ischemic bowel in the future.

Visual enhancement of laparoscopic partial nephrectomy with 3-charge coupled device camera: assessing intraoperative tissue perfusion and vascular anatomy by visible hemoglobin spectral response.

PURPOSE: We report the novel use of 3-charge coupled device camera technology to infer tissue oxygenation. The technique can aid surgeons to reliably differentiate vascular structures and noninvasively assess laparoscopic intraoperative changes in renal tissue perfusion during and after warm ischemia. MATERIALS AND METHODS: We analyzed select digital video images from 10 laparoscopic partial nephrectomies for their individual 3-charge coupled device response. We enhanced surgical images by subtracting the red charge coupled device response from the blue response and overlaying the calculated image on the original image. Mean intensity values for regions of interest were compared and used to differentiate arterial and venous vasculature, and ischemic and nonischemic renal parenchyma. RESULTS: The 3-charge coupled device enhanced images clearly delineated the vessels in all cases. Arteries were indicated by an intense red color while veins were shown in blue. Differences in mean region of interest intensity values for arteries and veins were statistically significant (p >0.0001). Three-charge coupled device analysis of pre-clamp and post-clamp renal images revealed visible, dramatic color enhancement for ischemic vs nonischemic kidneys. Differences in the mean region of interest intensity values were also significant (p <0.05). CONCLUSIONS: We present a simple use of conventional 3-charge coupled device camera technology in a way that may provide urological surgeons with the ability to reliably distinguish vascular structures during hilar dissection, and detect and monitor changes in renal tissue perfusion during and after warm ischemia.

Monitoring the healing of combat wounds using Raman spectroscopic mapping.

Soldiers wounded in modern warfare present with extensive and complicated acute wounds, confounded by an overwhelming inflammatory response. The pathophysiology of acute wounds is unknown and timing of wound closure remains subjective. Collagen gene expression profiles are presented for 24 patients. Impaired healing wounds showed a twofold decrease in the up-regulation of COL1A1 and COL3A1 genes in the beginning of the wound healing process, compared with normal healing wounds. By the final debridement, however, collagen gene expression profiles for normal and impaired healing wounds were similar for COL1A1 and COL3A1. In addition, Raman spectroscopic maps were collected of biopsy tissue sections, from the first and last debridements of 10 wounds collected from nine patients. Tissue components obtained for the debridement biopsies were compared to elucidate whether or not a wound healed normally. Raman spectroscopy showed a loss of collagen in five patients, indicated by a negative percent difference in the 1,665/1,445 cm(-1) band area ratios. Four healed patients showed an increased or unchanged collagen content. Here, we demonstrate the potential of Raman spectroscopic analysis of wound biopsies for classification of wounds as normal or impaired healing. Raman spectroscopy has the potential to noninvasively monitor collagen deposition in the wound bed, during surgical wound debridements, to help determine the optimal time for wound closure.

Enhanced surgical imaging: laparoscopic vessel identification and assessment of tissue oxygenation.

BACKGROUND: Inherent to minimally invasive procedures are loss of tactile feedback and loss of three-dimensional assessment. Tasks such as vessel identification and dissection are not trivial for the inexperienced laparoscopic surgeon. Advanced surgical imaging, such as 3-charge-coupled device (3-CCD) image enhancement, can be used to assist with these more challenging tasks and, in addition, offers a method to noninvasively monitor tissue oxygenation during operations. STUDY DESIGN: In this study, 3-CCD image enhancement is used for identification of vessels in 25 laparoscopic donor and partial nephrectomy patients. The algorithm is then applied to two laparoscopic nephrectomy patients involving multiple renal arteries. We also use the 3-CCD camera to qualitatively monitor renal parenchymal oxygenation during 10 laparoscopic donor nephrectomies (LDNs). RESULTS: The mean region of interest (ROI) intensity values obtained for the renal artery and vein (68.40 +/- 8.44 and 45.96 +/- 8.65, respectively) are used to calculate a threshold intensity value (59.00) that allows for objective vessel differentiation. In addition, we examined the renal parenchyma during LDNs. Mean ROI intensity values were calculated for the renal parenchyma at two distinct time points: before vessel stapling (nonischemic) and just before extraction from the abdomen (ischemic). The nonischemic mean ROI intensity values are statistically different from the ischemic mean ROI intensity values (p < 0.05), even with short ischemia times. CONCLUSIONS: We have developed a technique, 3-CCD image enhancement, for identification of vasculature and monitoring of parenchymal oxygenation. This technique requires no additional laparoscopic operating room equipment and has real-time video capability.

Non-invasive monitoring of tissue oxygenation during laparoscopic donor nephrectomy.

BACKGROUND: Standard methods for assessment of organ viability during surgery are typically limited to visual cues and tactile feedback in open surgery. However, during laparoscopic surgery, these processes are impaired. This is of particular relevance during laparoscopic renal donation, where the condition of the kidney must be optimized despite considerable manipulation. However, there is no in vivo methodology to monitor renal parenchymal oxygenation during laparoscopic surgery. METHODS: We have developed a method for the real time, in vivo, whole organ assessment of tissue oxygenation during laparoscopic nephrectomy to convey meaningful biological data to the surgeon during laparoscopic surgery. We apply the 3-CCD (charge coupled device) camera to monitor qualitatively renal parenchymal oxygenation with potential real-time video capability. RESULTS: We have validated this methodology in a porcine model across a range of hypoxic conditions, and have then applied the method during clinical laparoscopic donor nephrectomies during clinically relevant pneumoperitoneum. 3-CCD image enhancement produces mean region of interest (ROI) intensity values that can be directly correlated with blood oxygen saturation measurements (R2 > 0.96). The calculated mean ROI intensity values obtained at the beginning of the laparoscopic nephrectomy do not differ significantly from mean ROI intensity values calculated immediately before kidney removal (p > 0.05). CONCLUSION: Here, using the 3-CCD camera, we qualitatively monitor tissue oxygenation. This means of assessing intraoperative tissue oxygenation may be a useful method to avoid unintended ischemic injury during laparoscopic surgery. Preliminary results indicate that no significant changes in renal oxygenation occur as a result of pneumoperitoneum.

Contrast enhancement for in vivo visible reflectance imaging of tissue oxygenation.

Results are presented illustrating a straightforward algorithm to be used for real-time monitoring of oxygenation levels in blood cells and tissue based on the visible spectrum of hemoglobin. Absorbance images obtained from the visible reflection of white light through separate red and blue bandpass filters recorded by monochrome charge-coupled devices (CCDs) are combined to create enhanced images that suggest a quantitative correlation between the degree of oxygenated and deoxygenated hemoglobin in red blood cells. The filter bandpass regions are chosen specifically to mimic the color response of commercial 3-CCD cameras, representative of detectors with which the operating room laparoscopic tower systems are equipped. Adaptation of this filter approach is demonstrated for laparoscopic donor nephrectomies in which images are analyzed in terms of real-time in vivo monitoring of tissue oxygenation.

Infrared spectroscopic imaging for noninvasive detection of latent fingerprints.

The capability of Fourier transform infrared (FTIR) spectroscopic imaging to provide detailed images of unprocessed latent fingerprints while also preserving important trace evidence is demonstrated. Unprocessed fingerprints were developed on various porous and nonporous substrates. Data-processing methods used to extract the latent fingerprint ridge pattern from the background material included basic infrared spectroscopic band intensities, addition and subtraction of band intensity measurements, principal components analysis (PCA) and calculation of second derivative band intensities, as well as combinations of these various techniques. Additionally, trace evidence within the fingerprints was recovered and identified.

Identifying chemical changes in subchondral bone taken from murine knee joints using Raman spectroscopy.

Application of Raman spectroscopy to analysis of subchondral bone is described. The effect of cartilage health on subchondral bone has been widely studied using radiological and histological methods; however, there is no method to directly assay mineral components. We present Raman spectra of femur condyles and observe mineral bands that arise from the subchondral bone. In two separate experiments, transgenic mouse models of early-onset osteoarthritis (OA) and lipoatrophy were compared to tissue from wild-type mice. Raman spectroscopy was used to identify chemical changes in the mineral of subchondral bone that may accompany or precede morphological changes that can be observed by histology. The transgenic mice were compared to age-matched wild-type mice. Subtle alterations in the mineral or collagen matrix were observed by Raman spectroscopy using established Raman markers such as the carbonate-to-phosphate ratio, mineral-to-matrix ratio (MTMR), and amide I ratio. The Raman microscope configuration enabled rapid collection of Raman spectra from the mineralized layer that lies under an intact layer of non-mineralized articular cartilage. The effect of the cartilage layer on collection of spectra is discussed. The technique proposed is capable of providing insight into the chemical changes that occur in subchondral bone on a molecular level.

Raman spectroscopic evidence for octacalcium phosphate and other transient mineral species deposited during intramembranous mineralization.

UNLABELLED: To understand early mineralization events, we studied living murine calvarial tissue by Raman spectroscopy using fibroblast growth factor 2 (FGF2)-soaked porous beads. We detected increased levels of a transient phase resembling octacalcium phosphate in sutures undergoing premature suture closure. INTRODUCTION: Several calcium phosphates have been postulated as the earliest inorganic precursors to bone mineral. They are unstable and have not been previously detected in tissue specimens. Whether the same intermediates are formed in sutures undergoing premature closure is also unknown. METHODS: Six coronal suture tissue specimens from fetal day 18.5 B6CBA F1/J wild-type mice were studied. Three sutures specimens were treated with FGF2-soaked heparin acrylic beads to induce accelerated mineralization and premature suture closure. Three control specimens were treated with empty heparin acrylic beads. All sutures were maintained as organ cultures to permit repeated spectral analyses at 12-24 h intervals over a 72-h period. RESULTS: During the first 24 h, the spectra contained bands of octacalcium phosphate (OCP) or an OCP-like mineral. The main phosphorus-oxygen stretch was at 955 cm(-1), instead of the 957-959 cm(-1) seen in bone mineral, and there was an additional band at 1010-1014 cm(-1), as expected for OCP. A broad band was found at 945 cm(-1), characteristic of a highly disordered or amorphous calcium phosphate. An increased amount of mineral was observed in FGF2-treated sutures, but no qualitative differences in Raman spectra were observed between experimental and control specimens. CONCLUSIONS: Inorganic mineral deposition proceeds through transient intermediates, including an OCP-like phase. Although this transient phase has been observed in purely inorganic model systems, this study is the first to report OCP or an OCP-like intermediate in living tissue. Raman microspectroscopy allows observation of this transient mineral and may allow observation of other precursors as well.

Raman imaging demonstrates FGF2-induced craniosynostosis in mouse calvaria.

Craniosynostosis is a severe craniofacial disease where one or more sutures, the fibrous tissue that lies between the cranial bones, fuses prematurely. Some craniosynostosis syndromes are known to be caused by mutations in fibroblast growth factor (FGF) receptors. Mutated FGF receptors are thought to cause constitutive signaling. In this study, heparin acrylic beads released fibroblast growth factor 2 (FGF2) to mimic constitutive signaling by mutated receptors, delivering FGF2 in addition to already existing normal tissue amounts. Fetal day 18.5 mouse sutures were treated with FGF2-soaked beads and cultured in serum free media for 48 h. We have shown previously that this treatment leads to fusion and increased Msx2 expression, but here we use near-infrared Raman imaging to simultaneously examine the mineral components and matrix components of cranial tissue while providing light microscopic spatial information. FGF2-treated mouse sutures show increased v1 phosphate and v1 carbonate bandwidths, indicating a slightly chemically modified mineral being rapidly deposited. In addition, FGF2-treated mouse sutures show a marked increase in mineral-to-matrix ratios compared to control mouse sutures, typical of increased mineralization.