Erik H. Lindsley

In vivo cytometry: A spectrum of possibilities†

We investigate whether optical imaging can reliably detect abnormalities in tissue, in a range of specimens (live cells in vitro; fixed, fresh ex‐vivo and in vivo tissue), without the use of added contrast agents, and review our promising spectral methods for achieving quantitative, real‐time, high resolution intrasurgical optical diagnostics.

Polarization-Sensitive Hyperspectral Imaging in vivo : A Multimode Dermoscope for Skin Analysis

Attempts to understand the changes in the structure and physiology of human skin abnormalities by non-invasive optical imaging are aided by spectroscopic methods that quantify, at the molecular level, variations in tissue oxygenation and melanin distribution. However, current commercial and research systems to map hemoglobin and melanin do not correlate well with pathology for pigmented lesions or darker skin. We developed a multimode dermoscope that combines polarization and hyperspectral imaging with an efficient analytical model to map the distribution of specific skin bio-molecules. This corrects for the melanin-hemoglobin misestimation common to other systems, without resorting to complex and computationally intensive tissue optical models. For this systems proof of concept, human skin measurements on melanocytic nevus, vitiligo, and venous occlusion conditions were performed in volunteers. The resulting molecular distribution maps matched physiological and anatomical expectations, confirming a technologic approach that can be applied to next generation dermoscopes and having biological plausibility that is likely to appeal to dermatologists.

Real time Megapixel Multispectral Bioimaging

Spectral imaging involves capturing images at multiple wavelengths resulting in a data cube (x, y, λ) that allows materials to be identified by its spectral signature. While hyperspectral imagers can provide high spectral resolution, they also have major drawbacks such as cost, size, and the copious amounts of data in the image cube. Typically, the complete hyperspectral data cube provides little additional information compared to only 3-8 discrete (multiwavelength) imaging bands. We present two new approaches and related technologies where we are able to acquire spectral imaging data stacks quickly and cost-effectively. Our two spectral imaging systems represent different approaches integrated with standard CCD and CMOS imagers: sequential rotating filter wheels (RFWs) and lithographically patterned dichroic filter arrays (DFAs). The RFW approach offers the ability for rapid configuration of a spectral system, and a whole new level of self-contained image acquisition, processing and on-board display. The DFA approach offers the potential for ultra compact imagers with acquisition of images of multiple wavelengths simultaneously, while still allowing for processing and display steps to be built into the camera. Both approaches lend themselves production of multi-wavelength/spectral imaging systems with differing features and advantages.

Automated quantification of DNA demethylation effects in cells via 3D mapping of nuclear signatures and population homogeneity assessment

Todays advanced microscopic imaging applies to the preclinical stages of drug discovery that employ high‐throughput and high‐content three‐dimensional (3D) analysis of cells to more efficiently screen candidate compounds. Drug efficacy can be assessed by measuring response homogeneity to treatment within a cell population. In this study, topologically quantified nuclear patterns of methylated cytosine and global nuclear DNA are utilized as signatures of cellular response to the treatment of cultured cells with the demethylating anti‐cancer agents: 5‐azacytidine (5‐AZA) and octreotide (OCT). Mouse pituitary folliculostellate TtT‐GF cells treated with 5‐AZA and OCT for 48 hours, and untreated populations, were studied by immunofluorescence with a specific antibody against 5‐methylcytosine (MeC), and 4,6‐diamidino‐2‐phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei (n = 163). Cell images were processed utilizing an automated 3D analysis software that we developed by combining seeded watershed segmentation to extract nuclear shells with measurements of Kullback‐Leiblers (K‐L) divergence to analyze cell population homogeneity in the relative nuclear distribution patterns of MeC versus DAPI stained sites. Each cell was assigned to one of the four classes: similar, likely similar, unlikely similar, and dissimilar. Evaluation of the different cell groups revealed a significantly higher number of cells with similar or likely similar MeC/DAPI patterns among untreated cells (approximately 100%), 5‐AZA‐treated cells (90%), and a lower degree of same type of cells (64%) in the OCT‐treated population. The latter group contained (28%) of unlikely similar or dissimilar (7%) cells. Our approach was successful in the assessment of cellular behavior relevant to the biological impact of the applied drugs, i.e., the reorganization of MeC/DAPI distribution by demethylation. In a comparison with other metrics, K‐L divergence has proven to be a more valuable and robust tool for categorization of individual cells within a population, with potential applications in epigenetic drug screening.

Characterization of tumor cells and stem cells by differential nuclear methylation imaging

DNA methylation plays a key role in cellular differentiation. Aberrant global methylation patterns are associated with several cancer types, as a result of changes in long-term activation status of up to 50% of genes, including oncogenes and tumor-suppressor genes, which are regulated by methylation and demethylation of promoter region CpG dinucleotides (CpG islands). Furthermore, DNA methylation also occurs in nonisland CpG sites (> 95% of the genome), present once per 80 dinucleotides on average. Nuclear DNA methylation increases during the course of cellular differentiation while cancer cells usually show a net loss in methylation. Given the large dynamic range in DNA methylation load, the methylation pattern of a cell can provide a valuable distinction as to its status during differentiation versus the disease state. By applying immunofluorescence, confocal microscopy and 3D image analysis we assessed the potential of differential nuclear distribution of methylated DNA to be utilized as a biomarker to characterize cells during development and when diseased. There are two major fields that may immediately benefit from this development: (1) the search for factors that contribute to pluripotency and cell fate in human embryonic stem cell expansion and differentiation, and (2) the characterization of tumor cells with regard to their heterogeneity in molecular composition and behavior. We performed topological analysis of the distribution of methylated CpG-sites (MeC) versus heterochromatin. This innovative approach revealed significant differences in colocalization patterns of MeC and heterochromatin-derived signals between undifferentiated and differentiated human embryonic stem cells, as well as untreated AtT20 mouse pituitary tumor cells compared to a subpopulation of these cells treated with 5-azacytidine for 48 hours.

Multimode optical imaging of small animals : Development and applications

We present an optical system for small animal imaging that can combine various in vivo imaging modalities, including fluorescence (intensity and lifetime), spectral, and trans-illumination imaging. This system consists of light-tight box with ultrafast pulsed or cw laser light excitation, motorized translational and rotational stages, a telecentric lens for detection, and a cooled CCD camera that can be coupled to an ultrafast time-gated intensifier. All components are modular, making possible laser excitation at various wavelengths and pulse lengths, and signal detection in a variety of ways (multimode). Results of drug nanoconjugate carrier delivery studies in mice are presented. Conventional and spectrally-resolved fluorescence images reveal details of in vivo drug nanoconjugate carrier accumulation within the tumor region and several organs in real time. By multi-spectral image analysis of ex vivo specimens from the same mice, we were able to evaluate the extent and topology of drug nanoconjugate carrier distribution into specific organs and the tumor itself.

Spectral imaging for precise surgical intervention in Hirschsprung's Disease

We used advanced spectral imaging for intrasurgical decision making in a preclinical study, on a mouse model of Hirschsprungs Disease. Our imaging device sampled areas from normal and abnormal (aganglionic) colon in these animals. Spectral segmentation and classification of the resulting images showed a clear distinction between the normal and aganglionic regions, as confirmed by pathological analysis and use of mutant mice. We developed a simple algorithm that could distinguish normal from aganglionic colon with high spatial resolution and reproducibility, and the following statistics: sensitivity = 97%, specificity = 94%, positive predictive value = 92%, negative predictive value = 98%. These studies showed translational proof of concept that spectral imaging could be used during operations, in real time, to help surgeons precisely distinguish normal from abnormal tissue without requiring traditional biopsy.

Wide-field two-photon microscopy: features and advantages for biomedical applications

We describe a simple fluorescence microscope based on wide-field two-photon excitation. While still taking advantage of some inherent properties of non-linear (two-photon) microscopy, such as increased penetration depth through tissue and reduced phototoxicity, this approach provides video frame rate imaging, can be easily coupled to fluorescence spectral and lifetime detection modules, and makes efficient use of the high average power currently available from ultrashort pulsed lasers. For a standard histopathology specimen, we were able to identify different structures based on spectral and fluorescence lifetime detection and analysis. We examined the use of 200fs and 2ps pulses from Spectra Physics MaiTai and Tsunami lasers, respectively, with average power ranging from 50mW to 500mW.

Polarization-sensitive digital dermoscopy for image processing-assisted evaluation of atypical nevi: towards step-wise detection of melanoma

We have taken a three-pronged approach to improving the current standard of melanoma detection: (a) we are developing a new hyperspectral imaging-based medical device aimed at noninvasively detecting melanoma (b) we used a commercially available hand-held microscope with polarization control as a dermoscope, to begin establishing an inexpensive, portable imaging capability that could help assess the risk of a particular lesion (pigmented nevus) harboring melanoma (c) we created an updated ABCD algorithm and user interface software that more accurately generates a single risk number (Total Dermoscopy Score), for allowing a trained clinician to better assess the need for seeing the patient whose internet-uploaded nevus images they are evaluating. The hyperspectral instrument (a) is discussed elsewhere, and we focus here on (b) and (c), in the hope of increasing melanoma awareness and early detection.

Contrast enhancement in biomedical optical imaging using ultrabright color LEDs

The recent emergence of bright, inexpensive colored LEDs offers several advantages over traditional light sources, including reduced size and increased portability, low power consumption and heat production, increased durability and longer life, and high temporal resolution. We assembled a modular array of different Phillips LUMILED LUXEON LEDs, white and seven colors with peak wavelengths between 450 and 640 nm and bandwidths of 20-30 nm. LED illumination was fiber-optically coupled to the transmitted light path of an inverted microscope, and digital images of sectioned human tissue stained with absorbing dyes were acquired using combinations of the white and color LEDs. The LED array was also coupled to an endoscope and used to image human and mouse tissue in situ. Image contrast was assessed (1) qualitatively by looking down the microscope and by viewing the digital images, and (2) quantitatively by using entropy analysis in the real and frequency domains to assess the dependence of contrast enhancement on spatial frequency. Contrast in image features of a given color range was enhanced by LEDs conjugate to that color, whereas LED colors spanning a wider range enhanced contrast in the entire image, with white LEDs often maximizing contrast of tissue. This analysis demonstrates the utility of LED illumination in modulating contrast in light microscopy and endoscopy, which may facilitate spectral segmentation and classification of image features.