Browsing by Subject "Microscopy"

Development of a single cell into an adult organism is accomplished through an elaborate and complex cascade of spatiotemporal gene expression. While methods exist for capturing spatiotemporal expression patterns---in situ hybridization, reporter constructs, fluorescent tags---these methods have been highly laborious, and results are frequently assessed by subjective qualitative comparisons. To address these issues, methods must be developed for automating the capture of images, as well as for the normalization and quantification of the resulting data. In this thesis, I design computational approaches for high throughput image analysis which can be grouped into three main areas. First, I develop methods for the capture of high resolution images from high throughput platforms. In addition to the informatics aspect of this problem, I also devise a novel multiscale probabilistic model that allows us to identify and segment objects in an automated fashion. Second, high resolution images must be registered and normalized to a common frame of reference for cross image comparisons. To address these issues, I implement approaches for image registration using statistical shape models and non-rigid registration. Lastly, I validate the spatial expression data obtained from microscopy images to other known spatial expression methods, and develop methods for comparing and calculating the significance between spatial expression patterns. I demonstrate these methods on two model developmental organisms: Arabidopsis and Drosophila.

Electron microscopy is one of the most powerful tools available in observational science. Magnifications of 10,000,000x have been achieved with picometer precision. At this high level of magnification, individual atoms are visible. This is possible because the wavelength of electrons is much smaller than visible light, which also means that the highly focused electron beams used to perform imaging contain significantly more energy than visible light. The beam energy is high enough that it can cause radiation damage to metal specimens. Reducing radiation dose while maintaining image quality has been a central research topic in electron microscopy for several decades. Without the ability to reduce the dose, most organic and biological specimens cannot be imaged at atomic resolution. Fundamental processes in materials science and biology arise at the atomic level, thus understanding these processes can only occur if the observational tools can capture information with atomic resolution.

The primary objective of this research is to develop new techniques for low dose and high resolution imaging in (scanning) transmission electron microscopy (S/TEM). This is achieved through the development of new machine learning based compressive sensing algorithms and microscope hardware for acquiring a subset of the pixels in an image. Compressive sensing allows recovery of a signal from significantly fewer measurements than total signal size (under certain conditions). The research objective is attained by demonstrating application of compressive sensing to S/TEM in several simulations and real microscope experiments. The data types considered are images, videos, multispectral images, tomograms, and 4-dimensional ptychographic data. In the simulations, image quality and error metrics are defined to verify that reducing dose is possible with a small impact on image quality. In the microscope experiments, images are acquired with and without compressive sensing so that a qualitative verification can be performed.

Compressive sensing is shown to be an effective approach to reduce dose in S/TEM without sacrificing image quality. Moreover, it offers increased acquisition speed and reduced data size. Research leading to this dissertation has been published in 25 articles or conference papers and 5 patent applications have been submitted. The published papers include contributions to machine learning, physics, chemistry, and materials science. The newly developed pixel sampling hardware is being productized so that other microscopists can use compressive sensing in their experiments. In the future, scientific imaging devices (e.g., scanning transmission x-ray microscopy (STXM) and secondary-ion mass spectrometry (SIMS)) could also benefit from the techniques presented in this dissertation.

Optical imaging is an essential tool for studying life sciences. Existing biomedical optical systems range from microscopes in clinics that use wave optics principles to examine pathological samples at high resolution, to photoplethysmography in everyday smartwatches utilizing diffuse optics technologies for monitoring deep tissue physiology. An optical system, such as a photography solution in a studio, typically consists of three parts: illumination, objects of interest, and recording devices. Over the past decades, thanks to rapid advancements in semiconductor manufacturing, numerous new and exciting optical devices have emerged. These include low-cost, small form-factor LEDs and CMOS camera sensors in budget tablet devices, as well as high-density time-of-flight array detectors in recent generations of iPhones, for example. Moore's Law, on the other hand, has driven significant development in powerful yet inexpensive computational tools. As a result, nowadays, analogous to other medical imaging modalities such as X-ray CT and MRI, multiplexed optical measurements that may not resemble the object of interest can be recorded and post-processed to reconstruct useful images for human perception. In this thesis, several new computational optical imaging techniques at different scales will be discussed. These range from vectorial tomographic microscopies for imaging anisotropic cells and tissue, to high-throughput imaging systems capable of recording eukaryotic colonies at mesoscopic scales, and novel single-photon-sensitive sensing methods for non-invasive imaging of macroscopic transient dynamics deep within turbid volumes.

This dissertation describes three computational sensors. The first sensor is a scanning multi-spectral aperture-coded microscope containing a coded aperture spectrometer that is vertically scanned through a microscope intermediate image plane. The spectrometer aperture-code spatially encodes the object spectral data and nonnegative

least squares inversion combined with a series of reconfigured two-dimensional (2D spatial-spectral) scanned measurements enables three-dimensional (3D) (x, y, λ) object estimation. The second sensor is a coded aperture snapshot spectral imager that employs a compressive optical architecture to record a spectrally filtered projection

of a 3D object data cube onto a 2D detector array. Two nonlinear and adapted TV-minimization schemes are presented for 3D (x,y,λ) object estimation from a 2D compressed snapshot. Both sensors are interfaced to laboratory-grade microscopes and

applied to fluorescence microscopy. The third sensor is a millimeter-wave holographic imaging system that is used to study the impact of 2D compressive measurement on 3D (x,y,z) data estimation. Holography is a natural compressive encoder since a 3D

parabolic slice of the object band volume is recorded onto a 2D planar surface. An adapted nonlinear TV-minimization algorithm is used for 3D tomographic estimation from a 2D and a sparse 2D hologram composite. This strategy aims to reduce scan time costs associated with millimeter-wave image acquisition using a single pixel receiver.

The rate of occurrence and number of deaths associated with cancer continues to climb each year despite the continual efforts to battle the disease. When given a cancer diagnosis, it is particularly demoralizing and devastating news to a patient. Generally, cancer is defined as the uncontrolled rapid growth of abnormal cells with metastatic potential. In the cancer types originating from solid tissue or organ sites, a tumor will grow as a result of this rapid proliferation of cells. Surgical resection is a commonly used as part of the treatment regimen prescribed for these types of cancer.

Specifically in breast cancer, which impacts over 200,000 women a year, surgical intervention is used in almost 92% of treated cases. A specific surgical procedure is known as breast conserving surgery (BCS), where the physician removes only the tumor, while retaining as much normal tissue as possible. BCS is used in 59% of cases and is generally more preferable than the more radically mastectomy procedure where the entire breast is removed.

To minimize the chance of local recurrence, it is vital that the tumor is completely removed and residual cancer cells are not still present in the patient. This diagnosis is made by inspecting the edge of the resected tumor mass, typically known as the surgical margin. If tumor cells are still present at the margin, then a positive diagnosis is given and tumor cells likely remain inside the patient. Unfortunately, since margins are typically diagnosed using post-operative pathology a patient with a positive margin must undergo a second re-excision operation to remove additional tissue.

For breast cancer patients undergoing BCS, a staggering 20-70% of patients must undergo additional operations due to incomplete tumor removal during the first procedure.

Currently, there are two intra-operative techniques that are used, frozen section analysis and touch prep cytology. Although both have been proven to be effective in reducing re-excision rates, both techniques require

There remains a clinical unmet need for an intra-operative technology capable of quickly diagnosis tumor margins during the initial surgical operation

Optical technologies provide an attractive method of quickly and non-destructively assessing tissue. These techniques rely the interactions of light with tissue, which include absorption, scattering, and fluorescence. Utilizing proper measurement systems, these interactions can be measured and exploited to yield specific sources of contrast in tissue. In this dissertation, I have focused on developing two specific optical techniques for the purpose of surgical margin assessment.

The first is diffuse reflectance spectroscopy (DRS) which is a specific method to extract quantitative biological composition of tissues has been used to discern tissue types in both pre-clinical and clinical cancer studies. Typically, diffuse reflectance spectroscopy systems are designed for single-point measurements. Clinically, an imaging system would provide valuable spatial information on tissue composition. While it is feasible to build a multiplexed fiber-optic probe based spectral imaging system, these systems suffer from drawbacks with respect to cost and size. To address these I developed a compact and low cost system using a broadband light source with an 8-slot filter wheel for illumination and silicon photodiodes for detection. The spectral imaging system was tested on a set of tissue mimicking liquid phantoms which yielded an optical property extraction accuracy of 6.40 ± 7.78% for the absorption coefficient (µa) and 11.37 ± 19.62% for the wavelength-averaged reduced scattering coefficient (µs').

While DRS provided one potential approach to margin diagnosis, the technique was inherently limited in terms of lateral resolution. The second optical technique I chose to focus on was fluorescence microscopy, which had the ability to achieve lateral resolution on the order of microns. Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. I developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1×1.6 mm (3.4 mm2) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm−1 used in conjunction with a 4x 0.1 NA objective. This yielded an optical section thickness of 128 µm and an 8.9x contrast enhancement over uniform illumination. I successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, I demonstrated that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy.

However the development of the SIM system was only the first step in showing potential application to surgical margin assessment. The nest study presented in this dissertation was to demonstrate clinical viability on a sample size of 23 animals. The biological samples used in this study were a genetically engineered mouse model of sarcoma, where a spontaneous solid tumor was grown in the hind leg. After the tumor was surgically removed from the animal and the relevant margin was stained with acridine orange (AO), a simple and widely available contrast agent that brightly stains cell nuclei and fibrous tissues. The margin was imaged with the SIM system with the primary goal of visualizing specific morphological changes in cell nuclei. To automatically segment AO-stained regions, an algorithm known as maximally stable extremal regions (MSER) was optimized and applied to the images.

As an intermediate step prior to diagnosing whole margins, a tissue-type classification model was developed to differentiate localized regions (75x75 µm) of tumor from skeletal muscle and adipose tissue based on the MSER nuclei segmentation output. A logistic regression model was used which yielded a final output in terms of probability (0-100%) the tumor within the localized region. The model performance was tested using an ROC curve analysis that revealed a 77% sensitivity and 81% specificity. For margin classification, the whole margin image was divided into localized regions and this tissue-type classification model was applied. In a subset of 6 margins (3 negative, 3 positive), it was shown that at a tumor probability threshold of 50% only 8% of all regions from a negative margins exceeded this threshold, while over 25% of all regions exceeded the threshold in the positive margins.

Optical interrogation techniques are particularly well-suited for imaging tissue morphology, biological dynamics, and disease pathogenesis by providing noninvasive access to subcellular-resolution diagnostic information. State-of-the-art spectral domain optical coherence tomography (SDOCT) systems provide real-time optical biopsies of in vivo tissue, and have demonstrated clinical potential, particularly for applications in ophthalmology.

Recent advances in microscopy and endoscopy have led to improved resolution and compact optical designs, beyond those of conventional imaging systems. Application of encoded and multiplexed illumination and detection schemes may allow for the development of optical tools that surpass classical imaging limitations. Furthermore, complementary technologies can be combined to create multimodal optical imaging tools with advantages over current-generation systems.

In this dissertation, the development of coherence-gated and resolution-multiplexed technologies, aimed towards applications in human vitreoretinal imaging is described. Technology development in coherence-gated systems included increasing the imaging range of SDOCT by removing the complex conjugate artifact, improving acquisition speed using a scanning spectrometer design and a two-dimensional detector array, and hardware and algorithmic implementations that facilitated imaging of Doppler flow.

Structured illumination microscopy techniques were applied for resolution enhancement, and a spectrally encoded ophthalmic imaging system was developed for en face confocal fundus imaging through a single-mode fiber. These devices were resolution-multiplexed extensions of existing ophthalmic imaging devices, such as scanning laser ophthalmoscopes (SLO), which demonstrated improved resolution and more compact optical designs as compared to their conventional counterparts.

Finally, several multimodal ophthalmic diagnostic tools were developed that combined the advantages of OCT with existing imaging devices. These included a combined SLO-OCT system and a vitreoretinal surgical microscope combined with OCT. These devices allowed for concurrent ophthalmic imaging using complementary modalities for improved visualization and clinical utility.

Mercury (Hg) is a toxic pollutant whose speciation will highly impact its fate and transformation in the environment. One understudied form is that of liquid elemental mercury (Hg0L), a dense non-aqueous phase liquid which, upon release to the environment, can persist in soil for decades. Over time, this residual Hg0L is gradually released to groundwater and eventually surface water, serving as a long-term source of contamination. Hg that reaches natural waters can eventually be transformed into methylmercury (MeHg), a powerful neurotoxin that can biomagnify in aquatic food chains and endanger human health. The presence of Hg0L in the environment brings up a number of interrelated topics relevant in Hg science, namely mobilization, reactivity, and exposure. Hg0L contamination exists worldwide due to the historic and current use of Hg0L in various industrial and mining practices. Many former Hg processing sites, such as chlor alkali plants, contain Hg-contaminated soils. Today, however, artisanal and small-scale gold mining (ASGM) is the primary utilizer of Hg0L. This largely unregulated, rudimentary mining process uses large quantities of Hg0L to separate and amalgamate gold from excavated soils. This gold-Hg amalgam is later heated to evaporate the Hg0L, resulting in large losses of Hg to the atmosphere. As a result of the ongoing and past use of Hg0L, threats of Hg exposure due to contaminated soil environments will remain prevalent. This dissertation addresses three questions in order to better understand Hg0L in the environment and reliable ways to assess human exposure to the metal: (1) How does Hg0L interact with sulfide, and what impact do these interactions have on Hg0L dissolution and mobility? (2) What is the Hg speciation in streambank soils downstream of a site with historical Hg0L soil contamination, and what is the potential for future mobility and transformation of Hg in these soils? (3) How does exposure to Hg0L impact the efficacy of hair total Hg (THg) as a proxy for MeHg exposure in ASGM communities? These questions are explored through the lens of two different sites: Madre de Dios, Peru, a region contaminated from rapidly growing ASGM activity, and East Fork Poplar Creek (EFPC), a superfund site with historical Hg0L contamination in Oak Ridge, TN. The spillage of Hg0L, with a density that is 13 times the density of liquid water, results in deep penetration of the metal into soils and entrapment of Hg0L droplets in soil pore spaces. Over time, this residual Hg0L can undergo various chemical and biological transformations that will ultimately impact its fate in the subsurface. Of particular interest is the corrosion of Hg0L via reaction with reduced inorganic sulfur species to form mercury sulfide (HgS), a process that enables long term sequestration of mercury in soils and generally reduces its mobility and bioavailability. The second chapter of this dissertation examines the natural corrosion of Hg0L in the presence of sulfide by quantifying rates of Hg release under different sulfide doses and aging environments. For droplets aged in ambient air, no differences in Hg release were observed among all sulfide doses. However, for droplets aged in the presence of a strong oxidant (H2O2) and reacted under low sulfide doses, we observed an increase in Hg release relative to droplets aged in air. However, the release of Hg from droplets aged in H2O2 was suppressed upon addition of adequate sulfide. These results suggest two critical factors dictate the corrosion of Hg0L in the presence of sulfide: surface oxidation of the droplet and sufficient sulfide dose. Surface oxidation controls the release of Hg into solution; without adequate oxidation, no Hg will be available to be sequestered through precipitation of HgS. However, in the case of an oxidized Hg0L surface, sufficient sulfide is needed to prevent large amounts of Hg from being released into solution. These results suggest that the mobilization of Hg0L will largely depend on aging conditions in the subsurface. Hg mobilized from the dissolution of Hg0L will likely be transported to other environmental compartments downstream. For sites located near alluvial systems, bank soils are one likely receptor of this mobilized Hg. The solid-phase speciation of Hg in these receiving streambanks are critical because erosion and flooding events may mobilize Hg into the adjacent waterbody and eventually be methylated. Therefore in Chapter 3 of this dissertation, we characterized mercury speciation in Hg-enriched soils (100-1100 mg/kg Hg) collected from the incised bank of the East Fork Poplar Creek (EFPC) in Oak Ridge, TN (USA). A combination of characterization techniques were used including scanning electron microscopy, X-ray absorption fine structure spectroscopy, and transmission electron spectroscopy. Altogether the data demonstrated the predominance of nanoparticulate HgS with crystal lattice defects in the streambank soils of this industrially impacted stream. The results of this work support further investigation of the impact of these nanocrystalline lattice defects on particle surface reactivity, including Hg dissolution rates and bioavailability at key microbial interfaces. Increased availability of Hg to methylators may give rise to hotspots of MeHg bioaccumulation. In order to identify and monitor human populations with elevated MeHg exposure, total mercury content (THg) in hair is used as a proxy for chronic MeHg exposure. However, while this biomarker may be useful for populations who are primarily exposed to Hg as dietary MeHg, recent studies have indicated that for those in mixed exposure communities such as ASGM areas, THg in hair may comprise a substantial proportion of inorganic mercury (iHg). With the enactment of the Minamata Convention on Mercury prompting renewed efforts for biomonitoring of vulnerable populations, the efficacy of hair THg as a proxy for MeHg exposures to populations needs to be evaluated for ASGM settings. Chapter four investigates the efficacy of hair THg as an indicator of MeHg exposure by quantifying both THg and MeHg contents in hair from a representative subset of participants in a large, population-level mercury exposure assessment near ASGM areas in Madre de Dios (MDD), Peru. We observed that 100% of hair samples contained MeHg, and 86% had levels reflective of dietary exposure, or non-occupational exposure. For a small subset of individuals living in mining towns (N = 15; ~20% of within mining sample), we observed the opposite result: hair THg mostly comprised of inorganic mercury. However, this subset did not influence population level trends; hair MeHg-THg correlations were high (r >0.7) for all communities, regardless of location or nativity. Our results support the use of hair THg for monitoring of MeHg exposure of populations in ASGM settings in addition to urine, the current recommended biomarker for ASGM communities. Altogether, this research seeks to improve our risk characterization of environments and populations impacted by soil Hg0L contamination by addressing gaps in our knowledge on Hg0L mobilization, downstream reactivity, and exposure evaluation. This work will contribute to our understanding of the formation and identification of biological Hg hotspots resulting from Hg0L contaminated sites. Additionally, our enhanced understanding of hair biomarkers will help countries with active ASGM sites develop monitoring programs to identify vulnerable populations and assess the effectiveness of programs designed to decrease environmental Hg release.

Histatin-5 (Hist-5) is a polycationic, histidine-rich antimicrobial peptide with potent antifungal activity against the opportunistic fungal pathogen Candida albicans. Hist-5 can bind metals in vitro, and metals have been shown to alter the fungicidal activity of the peptide. The goal of this work is to gain insight into the role of metals in the biological activity Hist-5. Toward this goal, we developed and characterized a novel fluorescently labeled Hist-5 peptide (Hist-5*) to visualize changes in internalization and localization of the peptide in fungal cells as a function of metal availability in the growth medium. Here, we provide evidence for Zn-modulated antifungal activity of Hist-5 in which the availability of Zn2+ in the surrounding environment inhibits Hist-5 cellular uptake and cidality. Cellular growth assays revealed a concentration-dependent inhibitory effect of Zn2+ on Hist-5 antifungal activity. Imaging by confocal microscopy showed that equimolar concentrations of Zn2+ kept the peptide localized along the cell periphery rather than internalizing, thus preventing cytotoxicity and membrane disruption. We found that modulation of extracellular Zn2+ concentration by metal chelating molecules or proteins reversed Zn-induced surface adhesion of Hist-5, leading us to propose a dynamic role for Zn2+ as an inhibitory switch to regulate Hist-5 fungicidal activity. We next present data to support the hypothesis that Hist-5 interacts with intracellular Cu to increase the fungicidal activity Hist-5. Combined fluorescence spectroscopy and microscopy experiments showed reversible Cu-dependent quenching of Hist-5* fluorescence, indicating a direct interaction between Hist-5 and intracellular Cu. X-ray fluorescence microscopy images revealed peptide-induced changes to cellular Cu distribution and cell-associated Cu content. Finally, we present progress towards expanding the scope in which we understand and assess Hist-5 biological activity by investigating the activity of the peptide under biologically relevant conditions and testing its fungicidal activity against other fungal species.

Dermatopathologists need more reliable tools for analyzing biopsies of lesions that are potentially melanomas and determining the best treatment plan for the patient. Previously inaccessible, the chemical and physical properties of melanin provide insight into melanoma biochemistry. Two-color, near-infrared pump-probe microscopy of unstained, human pathology slides reveals differences in the type of melanins and the distribution of melanins between melanomas and benign nevi. Because the pump-probe response of melanin is resilient to aging, even for hundreds of millions of years, this tool could prove useful in retrospective studies to correlate melanin characteristics with patient outcome, thus eliminating the pathologist's uncertainty from the development of this classification method.

Pump-probe spectroscopy of a variety of melanin preparations including melanins with varying amounts of metal ions and toxins, those that have been photo-damaged or chemically oxidized, and melanins with a homogeneous size distribution shows that the pump-probe response is sensitive to these chemical and physical differences, not just melanin type as previously hypothesized. When sampling the response at several pump wavelengths, the specificity of this technique is derived from the absorption spectra of the underlying chromophores. Therefore, hyperspectral pump-probe microscopy of melanin could serve as an indicator of the chemical environment in a variety of biological contexts. For example, the melanin chemistry of macrophages suggests that these cells oxidize, homogenize, and compact melanin granules; whereas melanocytes produce heterogeneous melanins.

Mouse models of cancer are an invaluable tool for studying the mechanism of the disease and the effect of new therapies. Recent years have seen an explosive growth in the development of such models and consequently there is an increased need for better imaging techniques to study them. The goal of this work was to develop a technique that satisfied the requirements for preclinical cancer imaging: high spatial resolution, good soft tissue differentiation, excellent motion immunity, fast and non-invasive imaging to enable high-throughput, longitudinal studies.

T₂-weighted and diffusion-weighted magnetic resonance imaging (MRI) has been shown to be effective for tumor characterization clinically. But translation of these techniques to the mouse is challenging. The higher spatial resolution and faster physiologic motion make conventional approaches very susceptible to phase artifacts. Additionally, at higher magnetic fields required for these studies, T*₂ and T₂ are significantly shorter and T₁ is longer, making in vivo imaging even harder.

A rigorous cancer imaging protocol was developed by optimizing and integrating various components of the system, including MR hardware, animal handling, and pulse sequence design to achieve reliable, repeatable and rapid imaging. The technique presented here relies heavily on the non-Cartesian sampling strategy of PROPELLER (Periodically Rotated Overlapping ParallEL Lines with Enhanced Reconstruction) MRI. The novel data acquisition and reconstruction overcomes the adverse effects of physiological motion, allows for rapid setup and acquisition and provides excellent tissue contrast. The sequence was optimized to enable T₂-weighted and diffusion-weighted imaging in tumor-bearing mice with in-plane resolution of 117μm and slice thickness of 1mm. Multi-slice datasets covering the entire thorax and abdomen were acquired in ∼30 minutes.

The imaging protocol developed here was applied to a high-throughput, longitudinal study in a mouse model of liver metastases. The liver is a common site of distal metastases in colon and rectal cancer, and if detected early has an improved prognosis. Unfortunately, severe respiratory motion make it hard to image. The relative merits of the proposed PROPELLER technique were analyzed with respect to the accepted gold-standard for abdominal cancer imaging, computed tomography (CT).

The non-Cartesian MR microscopy technique proposed here is a valuable tool in the “Cancer analysis toolkit”. It allows for high-throughput, longitudinal experiments in free-breathing mice generating both structural and functional information with minimal artifacts and excellent spatial resolution. This work should find broad applications in various mouse models of cancer for studying the pathology of the disease, its progression as well as its response to treatment.