Prospective on Imaging Mass Spectrometry in Clinical Diagnostics.

Imaging mass spectrometry (IMS) is a molecular technology utilized for spatially driven research, providing molecular maps from tissue sections. This article reviews matrix-assisted laser desorption ionization (MALDI) IMS and its progress as a primary tool in the clinical laboratory. MALDI mass spectrometry has been used to classify bacteria and perform other bulk analyses for plate-based assays for many years. However, the clinical application of spatial data within a tissue biopsy for diagnoses and prognoses is still an emerging opportunity in molecular diagnostics. This work considers spatially driven mass spectrometry approaches for clinical diagnostics and addresses aspects of new imaging-based assays that include analyte selection, quality control/assurance metrics, data reproducibility, data classification, and data scoring. It is necessary to implement these tasks for the rigorous translation of IMS to the clinical laboratory; however, this requires detailed standardized protocols for introducing IMS into the clinical laboratory to deliver reliable and reproducible results that inform and guide patient care.

A Guide to MALDI Imaging Mass Spectrometry for Tissues.

Imaging mass spectrometry is a well-established technology that can easily and succinctly communicate the spatial localization of molecules within samples. This review communicates the recent advances in the field, with a specific focus on matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) applied on tissues. The general sample preparation strategies for different analyte classes are explored, including special considerations for sample types (fresh frozen or formalin-fixed,) strategies for various analytes (lipids, metabolites, proteins, peptides, and glycans) and how multimodal imaging strategies can leverage the strengths of each approach is mentioned. This work explores appropriate experimental design approaches and standardization of processes needed for successful studies, as well as the various data analysis platforms available to analyze data and their strengths. The review concludes with applications of imaging mass spectrometry in various fields, with a focus on medical research, and some examples from plant biology and microbe metabolism are mentioned, to illustrate the breadth and depth of MALDI IMS.

Selective Skeletal Surveys for Infants With Skull Fractures: Examining the Rates of Return to Medical Care for Concern of Physical Abuse.

OBJECTIVES: This study aimed to describe which infants with a skull fracture (1) receive a child abuse pediatrician (CAP) consultation, (2) receive a skeletal survey, and (3) re-present to medical care before age 3 years with concerns for physical abuse. METHODS: We conducted a retrospective chart review of infants younger than 12 months who presented to the emergency department between January 1, 2005, and December 30, 2015, with a skull fracture. Medical records were reviewed for the skull fracture presentation and for all future medical evaluations at the same institution with concerns for physical abuse until 3 years of age. RESULTS: Of 366 infants with a skull fracture, a CAP was contacted for 272 (74%) and 76 (20.8%) infants who received a skeletal survey. Factors associated with skeletal survey acquisition included younger age (<6 months), no history to explain the skull fracture, other injuries on examination, and social risk factors. Six children (1.6%) re-presented to medical care with concerns of physical abuse before age 3 years. Five of these infants did not have a skeletal survey at the time of their skull fracture, and 1 was likely a case of missed physical abuse at the time of the skull fracture. DISCUSSION: Most skull fractures in infancy occur accidentally, and a skeletal survey may not be necessary for every infant. Obtaining a thorough history including social risk factors, performing a complete physical examination, and consulting with a CAP is an effective first step in the evaluation of physical abuse in infants with skull fractures.

Diagnosis of melanoma by imaging mass spectrometry: Development and validation of a melanoma prediction model.

BACKGROUND: The definitive diagnosis of melanocytic neoplasia using solely histopathologic evaluation can be challenging. Novel techniques that objectively confirm diagnoses are needed. This study details the development and validation of a melanoma prediction model from spatially resolved multivariate protein expression profiles generated by imaging mass spectrometry (IMS). METHODS: Three board-certified dermatopathologists blindly evaluated 333 samples. Samples with triply concordant diagnoses were included in this study, divided into a training set (n = 241) and a test set (n = 92). Both the training and test sets included various representative subclasses of unambiguous nevi and melanomas. A prediction model was developed from the training set using a linear support vector machine classification model. RESULTS: We validated the prediction model on the independent test set of 92 specimens (75 classified correctly, 2 misclassified, and 15 indeterminate). IMS detects melanoma with a sensitivity of 97.6% and a specificity of 96.4% when evaluating each unique spot. IMS predicts melanoma at the sample level with a sensitivity of 97.3% and a specificity of 97.5%. Indeterminate results were excluded from sensitivity and specificity calculations. CONCLUSION: This study provides evidence that IMS-based proteomics results are highly concordant to diagnostic results obtained by careful histopathologic evaluation from a panel of expert dermatopathologists.

An orthogonal methods assessment of topical drug concentrations in skin and the impact for risk assessment in the viable epidermis.

Systemic toxicity assessments for oral or parenteral drugs often utilize the concentration of drug in plasma to enable safety margin calculations for human risk assessment. For topical drugs, there is no standard method for measuring drug concentrations in the stratum basale of the viable epidermis. This is particularly important since the superficial part of the epidermis, the stratum corneum (SC), is nonviable and where most of a topically applied drug remains, never penetrating deeper into the skin. We investigated the relative concentrations of a prototype kinase inhibitor using punch biopsy, laser capture microdissection, and imaging mass spectrometry methods in the SC, stratum basale, and dermis of minipig skin following topical application as a cream formulation. The results highlight the value of laser capture microdissection and mass spectrometry imaging in quantifying the large difference in drug concentration across the skin and even within the epidermis, and supports use of these methods for threshold-based toxicity risk assessments in specific anatomic locations of the skin, like of the stratum basale.

Substance Use in a Domestic Minor Sex Trafficking Patient Population.

ABSTRACT: Substance use and abuse have been documented as both a risk factor in and consequence of involvement in domestic minor sex trafficking (DMST). Domestic minor sex trafficking is defined as the commercial sexual exploitation of children in exchange for money, food, shelter, or any other valued entity. The current investigation sought to describe substance use in a cohort of DMST patients who present for medical evaluation. Findings revealed that 68 patients referred for DMST involvement reported high rates of alcohol/substance use and parental substance abuse. Further, many patients who had a urine toxicology screen had a positive result, most often identifying the presence of cannabinoids. Our data may inform the importance of comprehensive assessments and specialized interventions for substance abuse in this unique patient population.

When Parents Hit: Providing Education to Physician Trainees About Corporal Punishment Through a Pilot Simulation and Debriefing.

OBJECTIVES: No studies have evaluated how training physicians intervene when corporal punishment (CP) is observed in a simulated hospital setting. The pilot study examined physician trainee performance in a simulation where hitting is observed between caregiver and child during a medical visit and to assess physician self-reported experiences, opinions, and comfort when observing CP in a simulation. METHODS: We ran 7 simulations where one pediatric resident, emergency medicine resident, or pediatric emergency medicine fellow participated in the simulation while a group of similar trainees observed. All participants were given a postsurvey, followed by a semistructured debriefing led by a child abuse pediatrician. RESULTS: Thirty-seven physician trainees participated; 7 engaged in the simulation while 30 observed. The majority (6/7) did not de-escalate the increasingly aggravated parent prior to hitting, 4 of 7 did not recommend that the caregiver refrain from CP, and most (5/7) did not provide education to the parent about more appropriate discipline. The majority (91.4%) believe that a physician should intervene when a parent hits or spanks his/her child in the hospital setting, highlighting the incongruity between this belief and their performance in/knowledge of intervening. All participants stated they would benefit from additional education and training on CP. CONCLUSIONS: The educational experience provided physicians in training with the opportunity to participate in or observe a situation in which CP occurs in the medical setting. The simulation and debriefing were an innovative approach to providing an educational opportunity for physicians to learn from difficult situations and discussions surrounding CP with caregivers.

Pediatric Rib Fractures Identified by Chest Radiograph: A Comparison Between Accidental and Nonaccidental Trauma.

OBJECTIVE: The objective of this study was to determine the prevalence of rib fractures (RFs) identified by chest x-ray (CXR) among children younger than 2 years who sustained accidental versus nonaccidental injuries. It is hypothesized that RFs are uncommon among all accidental pediatric trauma mechanisms (eg, falls, motor vehicle crashes) as compared with the prevalence of RFs in the setting of nonaccidental trauma (NAT). METHODS: A retrospective chart review of sequential CXRs of children younger than 2 years evaluated at a pediatric level 1 trauma center for accidental trauma and possible NAT was conducted from January 1, 2011, to October 31, 2016. Data collected included demographics, CXR indication and findings, history of cardiopulmonary resuscitation, trauma mechanism, associated injuries, final diagnoses, and outcomes. RESULTS: Two (<1%) of 226 CXRs obtained to evaluate accidental trauma demonstrated acute RFs. Ten (19.6%) of 51 CXRs obtained in the setting of concern for NAT revealed RFs (9/10 identified only healing RFs and 1/10 identified acute RFs). Among patients with a final diagnosis of NAT (ie, not neglect, accidental trauma, etc; n = 38), the overall prevalence increased to 26.3%. CONCLUSIONS: The presence of RFs in pediatric accidental trauma is uncommon even in the setting of high-force mechanisms, and when identified, these RFs are acute. Comparatively, the overall prevalence of RFs identified on CXR among cases with a final diagnosis of NAT was much higher and almost exclusively healing RFs. These data provide support that identification of RFs is highly concerning for NAT even if an accidental mechanism is provided. When RFs are identified, a full NAT work-up should be considered.

A Comprehensive Pediatric Acute Sexual Assault Protocol: From Emergency Department to Outpatient Follow-up.

OBJECTIVE: Close medical follow-up after pediatric acute sexual assault is recommended and may mitigate adverse consequences and decrease long-term comorbidities. The objectives are to (1) examine adherence to a comprehensive outpatient medical follow-up protocol after evaluation in the emergency department in a pediatric population and (2) identify characteristics associated with patient adherence to inform the utilization of a medical follow-up protocol after pediatric acute sexual assault. METHODS: A retrospective medical record review was conducted of patients younger than 18 years presenting to the emergency department from January 1, 2010, to December 31, 2013, with a discharge diagnosis suggestive of sexual assault/abuse. We examined differences in demographics, assault characteristics, and medical/legal needs of patients who were evaluated in follow-up versus patients who were not. RESULTS: Of 182 patients, 60.4% completed follow-up appointments with the child protection center. Younger patients had follow-up rates higher than older patients (70.2% vs 50%; odds ratio [OR], 0.42). For patients where child protective services or law enforcement were called, follow-up rates were 74.2% and 64.7%, respectively (OR, 2.5; OR, 3.1). All patients with anogenital injuries on initial examination were seen in follow-up. The majority of patients who followed-up were accompanied by a caregiver/relative (95%). CONCLUSIONS: (1) Caregivers should be integrated into the evaluation to facilitate compliance with follow-up; (2) child abuse specialists may be consulted to facilitate specific interventions and recommendations; (3) professionals should work as a multidisciplinary team; and (4) the patient's psychological status should be evaluated, and mental health interventions recommended.

Heme sensing and detoxification by HatRT contributes to pathogenesis during Clostridium difficile infection.

Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium that infects the colon, causing symptoms ranging from infectious diarrhea to fulminant colitis. In the last decade, the number of C. difficile infections has dramatically risen, making it the leading cause of reported hospital acquired infection in the United States. Bacterial toxins produced during C. difficile infection (CDI) damage host epithelial cells, releasing erythrocytes and heme into the gastrointestinal lumen. The reactive nature of heme can lead to toxicity through membrane disruption, membrane protein and lipid oxidation, and DNA damage. Here we demonstrate that C. difficile detoxifies excess heme to achieve full virulence within the gastrointestinal lumen during infection, and that this detoxification occurs through the heme-responsive expression of the heme activated transporter system (HatRT). Heme-dependent transcriptional activation of hatRT was discovered through an RNA-sequencing analysis of C. difficile grown in the presence of a sub-toxic concentration of heme. HatRT is comprised of a TetR family transcriptional regulator (hatR) and a major facilitator superfamily transporter (hatT). Strains inactivated for hatR or hatT are more sensitive to heme toxicity than wild-type. HatR binds heme, which relieves the repression of the hatRT operon, whereas HatT functions as a heme efflux pump. In a murine model of CDI, a strain inactivated for hatT displayed lower pathogenicity in a toxin-independent manner. Taken together, these data suggest that HatR senses intracellular heme concentrations leading to increased expression of the hatRT operon and subsequent heme efflux by HatT during infection. These results describe a mechanism employed by C. difficile to relieve heme toxicity within the host, and set the stage for the development of therapeutic interventions to target this bacterial-specific system.

The Prevalence of Rib Fractures Incidentally Identified by Chest Radiograph among Infants and Toddlers.

OBJECTIVES: To determine the prevalence of incidental rib fractures identified by chest radiograph (CXR) obtained for indications unrelated to accidental trauma or nonaccidental trauma (NAT), and describe the histories associated with cases of incidental rib fractures and their proposed etiologies. It is hypothesized that incidental rib fractures are rare and alternative explanations for rib fractures occasionally used in a medico-legal context such as minor accidental trauma, undiagnosed medical conditions, and transient metabolic bone disturbances are unlikely to be the etiology of incidental rib fractures. STUDY DESIGN: A retrospective chart review of sequential CXRs of children ages 0 to <2 years was conducted from January 1, 2011 to October 31, 2016. CXRs were obtained in the emergency department, general pediatric or intensive care units, or outpatient pediatric clinics. Data collected included demographics, CXR indication and findings, history of cardiopulmonary resuscitation, laboratory and additional imaging results, and incidental rib fracture descriptions and proposed etiologies. RESULTS: A total of 7530 patients underwent 9720 CXRs associated with unique clinical encounters. Five CXRs had incidental rib fractures identified, making the prevalence of CXRs with incidental rib fractures in this cohort <0.1%. Of 5 identified incidental cases, mean age was 3.6 months, 3 were concerning for NAT, 1 was confirmed NAT, and 1 had radiographic findings consistent with osteopenia of prematurity. CONCLUSIONS: Identification of incidental rib fracture on CXR is rare. When detected in the absence of corresponding trauma history and/or objective laboratory or radiographic metabolic abnormalities, work-up for NAT should be pursued.

Yeast and Filaments Have Specialized, Independent Activities in a Zebrafish Model of Candida albicans Infection.

Candida albicans dimorphism is a crucial virulence factor during invasive candidiasis infections, which claim the lives of nearly one-half of those afflicted. It has long been believed that filaments drive tissue invasion and yeast mediates bloodstream dissemination, but observation of these activities during infection has been prevented by technical limitations. We used a transparent zebrafish infection model to analyze more comprehensively how C. albicans utilizes shape to disseminate and invade. This model facilitated the use of diverse, complementary strategies to manipulate shape, allowing us to monitor dissemination, invasion, and pathogenesis via intravital imaging of individual fungal cells throughout the host. To control fungal cell shape, we employed three different strategies: gene deletion (efg1Δ/Δ cph1Δ/Δ, eed1Δ/Δ), overexpression of master regulators (NRG1 or UME6), and modulation of the infection temperature (21°C, 28°C, or 33°C). The effects of these orthogonal manipulations were consistent, support the proposed specialized roles of yeast in dissemination and filaments in tissue invasion and pathogenesis, and indicate conserved mechanisms in zebrafish. To test if either morphotype changes the effectiveness of the other, we infected fish with a known mixture of shape-locked strains. Surprisingly, mixed-strain infections were associated with additive, but not synergistic, filament invasion and yeast dissemination. These findings provide the most complete view of morphotype-function relationships for C. albicans to date, revealing independent roles of yeast and filaments during disseminated candidiasis.

Enhanced Spatially Resolved Proteomics Using On-Tissue Hydrogel-Mediated Protein Digestion.

The identification of proteins from tissue specimens is a challenging area of biological research. Many current techniques for identification forfeit some level of spatial information during the sample preparation process. Recently, hydrogel technologies have been developed that perform spatially localized protein extraction and digestion prior to downstream proteomic analysis. Regiospecific protein identifications acquired using this approach have thus far been limited to 1-2 mm diameter areas. The need to target smaller populations of cells with this technology necessitates the production of smaller diameter hydrogels. Herein, we demonstrate hydrogel fabrication processes that allow hydrogel applications down to a diameter of ∼260 µm, approximately 1/15 of the area of previous approaches. Parameters such as the percent polyacrylamide used in hydrogel construction as well as the concentration of trypsin with which the hydrogel is loaded are investigated to maximize the number of protein identifications from subsequent liquid chromatography tandem MS (LC-MS/MS) analysis of hydrogel extracts. An 18% polyacrylamide concentration is shown to provide for a more rigid polymer network than the conventional 7.5% polyacrylamide concentration and supports the fabrication of individual hydrogels using the small punch biopsies. Over 600 protein identifications are still achieved at the smallest hydrogel diameters of 260 µm. The utility of these small hydrogels is demonstrated through the analysis of sub regions of a rat cerebellum tissue section. While over 900 protein identifications are made from each hydrogel, approximately 20% of the proteins identified are unique to each of the two regions, highlighting the importance of targeting tissue subtypes to accurately characterize tissue biology. These newly improved methods to the hydrogel process will allow researchers to target smaller biological features for robust spatially localized proteomic analyses.

Adhesive fiber stratification in uropathogenic Escherichia coli biofilms unveils oxygen-mediated control of type 1 pili.

Bacterial biofilms account for a significant number of hospital-acquired infections and complicate treatment options, because bacteria within biofilms are generally more tolerant to antibiotic treatment. This resilience is attributed to transient bacterial subpopulations that arise in response to variations in the microenvironment surrounding the biofilm. Here, we probed the spatial proteome of surface-associated single-species biofilms formed by uropathogenic Escherichia coli (UPEC), the major causative agent of community-acquired and catheter-associated urinary tract infections. We used matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) to analyze the spatial proteome of intact biofilms in situ. MALDI-TOF IMS revealed protein species exhibiting distinct localizations within surface-associated UPEC biofilms, including two adhesive fibers critical for UPEC biofilm formation and virulence: type 1 pili (Fim) localized exclusively to the air-exposed region, while curli amyloid fibers localized to the air-liquid interface. Comparison of cells grown aerobically, fermentatively, or utilizing an alternative terminal electron acceptor showed that the phase-variable fim promoter switched to the "OFF" orientation under oxygen-deplete conditions, leading to marked reduction of type 1 pili on the bacterial cell surface. Conversely, S pili whose expression is inversely related to fim expression were up-regulated under anoxic conditions. Tethering the fim promoter in the "ON" orientation in anaerobically grown cells only restored type 1 pili production in the presence of an alternative terminal electron acceptor beyond oxygen. Together these data support the presence of at least two regulatory mechanisms controlling fim expression in response to oxygen availability and may contribute to the stratification of extracellular matrix components within the biofilm. MALDI IMS facilitated the discovery of these mechanisms, and we have demonstrated that this technology can be used to interrogate subpopulations within bacterial biofilms.

Next-generation technologies for spatial proteomics: Integrating ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR imaging mass spectrometry for protein analysis.

MALDI imaging mass spectrometry is a powerful analytical tool enabling the visualization of biomolecules in tissue. However, there are unique challenges associated with protein imaging experiments including the need for higher spatial resolution capabilities, improved image acquisition rates, and better molecular specificity. Here we demonstrate the capabilities of ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR IMS platforms as they relate to these challenges. High spatial resolution MALDI-TOF protein images of rat brain tissue and cystic fibrosis lung tissue were acquired at image acquisition rates >25 pixels/s. Structures as small as 50 µm were spatially resolved and proteins associated with host immune response were observed in cystic fibrosis lung tissue. Ultra-high speed MALDI-TOF enables unique applications including megapixel molecular imaging as demonstrated for lipid analysis of cystic fibrosis lung tissue. Additionally, imaging experiments using MALDI FTICR IMS were shown to produce data with high mass accuracy (<5 ppm) and resolving power (∼75 000 at m/z 5000) for proteins up to ∼20 kDa. Analysis of clear cell renal cell carcinoma using MALDI FTICR IMS identified specific proteins localized to healthy tissue regions, within the tumor, and also in areas of increased vascularization around the tumor.

Activation of heme biosynthesis by a small molecule that is toxic to fermenting Staphylococcus aureus.

Staphylococcus aureus is a significant infectious threat to global public health. Acquisition or synthesis of heme is required for S. aureus to capture energy through respiration, but an excess of this critical cofactor is toxic to bacteria. S. aureus employs the heme sensor system (HssRS) to overcome heme toxicity; however, the mechanism of heme sensing is not defined. Here, we describe the identification of a small molecule activator of HssRS that induces endogenous heme biosynthesis by perturbing central metabolism. This molecule is toxic to fermenting S. aureus, including clinically relevant small colony variants. The utility of targeting fermenting bacteria is exemplified by the fact that this compound prevents the emergence of antibiotic resistance, enhances phagocyte killing, and reduces S. aureus pathogenesis. Not only is this small molecule a powerful tool for studying bacterial heme biosynthesis and central metabolism; it also establishes targeting of fermentation as a viable antibacterial strategy.

Imaging mass spectrometry for assessing temporal proteomics: analysis of calprotectin in Acinetobacter baumannii pulmonary infection.

Imaging MS is routinely used to show spatial localization of proteins within a tissue sample and can also be employed to study temporal protein dynamics. The antimicrobial S100 protein calprotectin, a heterodimer of subunits S100A8 and S100A9, is an abundant cytosolic component of neutrophils. Using imaging MS, calprotectin can be detected as a marker of the inflammatory response to bacterial challenge. In a murine model of Acinetobacter baumannii pneumonia, protein images of S100A8 and S100A9 collected at different time points throughout infection aid in visualization of the innate immune response to this pathogen. Calprotectin is detectable within 6 h of infection as immune cells respond to the invading pathogen. As the bacterial burden decreases, signals from the inflammatory proteins decrease. Calprotectin is no longer detectable 96-144 h post infection, correlating to a lack of detectable bacterial burden in lungs. These experiments provide a label-free, multiplexed approach to study host response to a bacterial threat and eventual clearance of the pathogen over time.

Identification of an Acinetobacter baumannii zinc acquisition system that facilitates resistance to calprotectin-mediated zinc sequestration.

Acinetobacter baumannii is an important nosocomial pathogen that accounts for up to 20 percent of infections in intensive care units worldwide. Furthermore, A. baumannii strains have emerged that are resistant to all available antimicrobials. These facts highlight the dire need for new therapeutic strategies to combat this growing public health threat. Given the critical role for transition metals at the pathogen-host interface, interrogating the role for these metals in A. baumannii physiology and pathogenesis could elucidate novel therapeutic strategies. Toward this end, the role for calprotectin- (CP)-mediated chelation of manganese (Mn) and zinc (Zn) in defense against A. baumannii was investigated. These experiments revealed that CP inhibits A. baumannii growth in vitro through chelation of Mn and Zn. Consistent with these in vitro data, Imaging Mass Spectrometry revealed that CP accompanies neutrophil recruitment to the lung and accumulates at foci of infection in a murine model of A. baumannii pneumonia. CP contributes to host survival and control of bacterial replication in the lung and limits dissemination to secondary sites. Using CP as a probe identified an A. baumannii Zn acquisition system that contributes to Zn uptake, enabling this organism to resist CP-mediated metal chelation, which enhances pathogenesis. Moreover, evidence is provided that Zn uptake across the outer membrane is an energy-dependent process in A. baumannii. Finally, it is shown that Zn limitation reverses carbapenem resistance in multidrug resistant A. baumannii underscoring the clinical relevance of these findings. Taken together, these data establish Zn acquisition systems as viable therapeutic targets to combat multidrug resistant A. baumannii infections.

Multimodal MALDI imaging mass spectrometry for improved diagnosis of melanoma.

Imaging mass spectrometry (IMS) provides promising avenues to augment histopathological investigation with rich spatio-molecular information. We have previously developed a classification model to differentiate melanoma from nevi lesions based on IMS protein data, a task that is challenging solely by histopathologic evaluation. Most IMS-focused studies collect microscopy in tandem with IMS data, but this microscopy data is generally omitted in downstream data analysis. Microscopy, nevertheless, forms the basis for traditional histopathology and thus contains invaluable morphological information. In this work, we developed a multimodal classification pipeline that uses deep learning, in the form of a pre-trained artificial neural network, to extract the meaningful morphological features from histopathological images, and combine it with the IMS data. To test whether this deep learning-based classification strategy can improve on our previous results in classification of melanocytic neoplasia, we utilized MALDI IMS data with collected serial H&E stained sections for 331 patients, and compared this multimodal classification pipeline to classifiers using either exclusively microscopy or IMS data. The multimodal pipeline achieved the best performance, with ROC-AUCs of 0.968 vs. 0.938 vs. 0.931 for the multimodal, unimodal microscopy and unimodal IMS pipelines respectively. Due to the use of a pre-trained network to perform the morphological feature extraction, this pipeline does not require any training on large amounts of microscopy data. As such, this framework can be readily applied to improve classification performance in other experimental settings where microscopy data is acquired in tandem with IMS experiments.