Peter F. Favreau

Excitation-scanning hyperspectral imaging microscope

Abstract. Hyperspectral imaging is a versatile tool that has recently been applied to a variety of biomedical applications, notably live-cell and whole-tissue signaling. Traditional hyperspectral imaging approaches filter the fluorescence emission over a broad wavelength range while exciting at a single band. However, these emission-scanning approaches have shown reduced sensitivity due to light attenuation from spectral filtering. Consequently, emission scanning has limited applicability for time-sensitive studies and photosensitive applications. In this work, we have developed an excitation-scanning hyperspectral imaging microscope that overcomes these limitations by providing high transmission with short acquisition times. This is achieved by filtering the fluorescence excitation rather than the emission. We tested the efficacy of the excitation-scanning microscope in a side-by-side comparison with emission scanning for detection of green fluorescent protein (GFP)-expressing endothelial cells in highly autofluorescent lung tissue. Excitation scanning provided higher signal-to-noise characteristics, as well as shorter acquisition times (300  ms/wavelength band with excitation scanning versus 3  s/wavelength band with emission scanning). Excitation scanning also provided higher delineation of nuclear and cell borders, and increased identification of GFP regions in highly autofluorescent tissue. These results demonstrate excitation scanning has utility in a wide range of time-dependent and photosensitive applications.

Thin-film tunable filters for hyperspectral fluorescence microscopy

Abstract. Hyperspectral imaging is a powerful tool that acquires data from many spectral bands, forming a contiguous spectrum. Hyperspectral imaging was originally developed for remote sensing applications; however, hyperspectral techniques have since been applied to biological fluorescence imaging applications, such as fluorescence microscopy and small animal fluorescence imaging. The spectral filtering method largely determines the sensitivity and specificity of any hyperspectral imaging system. There are several types of spectral filtering hardware available for microscopy systems, most commonly acousto-optic tunable filters (AOTFs) and liquid crystal tunable filters (LCTFs). These filtering technologies have advantages and disadvantages. Here, we present a novel tunable filter for hyperspectral imaging—the thin-film tunable filter (TFTF). The TFTF presents several advantages over AOTFs and LCTFs, most notably, a high percentage transmission and a high out-of-band optical density (OD). We present a comparison of a TFTF-based hyperspectral microscopy system and a commercially available AOTF-based system. We have characterized the light transmission, wavelength calibration, and OD of both systems, and have then evaluated the capability of each system for discriminating between green fluorescent protein and highly autofluorescent lung tissue. Our results suggest that TFTFs are an alternative approach for hyperspectral filtering that offers improved transmission and out-of-band blocking. These characteristics make TFTFs well suited for other biomedical imaging devices, such as ophthalmoscopes or endoscopes.

An approach for characterizing and comparing hyperspectral microscopy systems.

Hyperspectral imaging and analysis approaches offer accurate detection and quantification of fluorescently-labeled proteins and cells in highly autofluorescent tissues. However, selecting optimum acquisition settings for hyperspectral imaging is often a daunting task. In this study, we compared two hyperspectral systems—a widefield system with acoustic optical tunable filter (AOTF) and charge coupled device (CCD) camera, and a confocal system with diffraction gratings and photomultiplier tube (PMT) array. We measured the effects of system parameters on hyperspectral image quality and linear unmixing results. Parameters that were assessed for the confocal system included pinhole diameter, laser power, PMT gain and for the widefield system included arc lamp intensity, and camera gain. The signal-to-noise ratio (SNR) and the root-mean-square error (RMS error) were measured to assess system performance. Photobleaching dynamics were studied. Finally, theoretical sensitivity studies were performed to estimate the incremental response (sensitivity) and false-positive detection rates (specificity). Results indicate that hyperspectral imaging assays are highly dependent on system parameters and experimental conditions. For detection of green fluorescent protein (GFP)-expressing cells in fixed lung tissues, a confocal pinhole of five airy disk units, high excitation intensity and low detector gain were optimal. The theoretical sensitivity studies revealed that widefield hyperspectral microscopy was able to detect GFP with fewer false positive occurrences than confocal microscopy, even though confocal microscopy offered improved signal and noise characteristics. These studies provide a framework for optimization that can be applied to a variety of hyperspectral imaging systems.

Hyperspectral imaging fluorescence excitation scanning for colon cancer detection

Abstract. Optical spectroscopy and hyperspectral imaging have shown the potential to discriminate between cancerous and noncancerous tissue with high sensitivity and specificity. However, to date, these techniques have not been effectively translated to real-time endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents new technology that may be well suited for endoscopic implementation. However, the feasibility of detecting differences between normal and cancerous mucosa using fluorescence excitation-scanning hyperspectral imaging has not been evaluated. The goal of this study was to evaluate the initial feasibility of using fluorescence excitation-scanning hyperspectral imaging for measuring changes in fluorescence excitation spectrum concurrent with colonic adenocarcinoma using a small pre-pilot-scale sample size. Ex vivo analysis was performed using resected pairs of colorectal adenocarcinoma and normal mucosa. Adenocarcinoma was confirmed by histologic evaluation of hematoxylin and eosin (H&E) permanent sections. Specimens were imaged using a custom hyperspectral imaging fluorescence excitation-scanning microscope system. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation range of 390 to 450 nm that could be the basis for wavelength-dependent detection of colorectal cancers. Hence, excitation-scanning hyperspectral imaging may offer an alternative approach for discriminating adenocarcinoma from surrounding normal colonic mucosa, but further studies will be required to evaluate the accuracy of this approach using a larger patient cohort.

Tunable thin-film optical filters for hyperspectral microscopy

Hyperspectral imaging was originally developed for use in remote sensing applications. More recently, it has been applied to biological imaging systems, such as fluorescence microscopes. The ability to distinguish molecules based on spectral differences has been especially advantageous for identifying fluorophores in highly autofluorescent tissues. A key component of hyperspectral imaging systems is wavelength filtering. Each filtering technology used for hyperspectral imaging has corresponding advantages and disadvantages. Recently, a new optical filtering technology has been developed that uses multi-layered thin-film optical filters that can be rotated, with respect to incident light, to control the center wavelength of the pass-band. Compared to the majority of tunable filter technologies, these filters have superior optical performance including greater than 90% transmission, steep spectral edges and high out-of-band blocking. Hence, tunable thin-film optical filters present optical characteristics that may make them well-suited for many biological spectral imaging applications. An array of tunable thin-film filters was implemented on an inverted fluorescence microscope (TE 2000, Nikon Instruments) to cover the full visible wavelength range. Images of a previously published model, GFP-expressing endothelial cells in the lung, were acquired using a charge-coupled device camera (Rolera EM-C2, Q-Imaging). This model sample presents fluorescently-labeled cells in a highly autofluorescent environment. Linear unmixing of hyperspectral images indicates that thin-film tunable filters provide equivalent spectral discrimination to our previous acousto-optic tunable filter–based approach, with increased signal-to-noise characteristics. Hence, tunable multi-layered thin film optical filters may provide greatly improved spectral filtering characteristics and therefore enable wider acceptance of hyperspectral widefield microscopy.

Hyperspectral imaging fluorescence excitation scanning for detecting colorectal cancer: pilot study

Optical spectroscopy and hyperspectral imaging have shown the theoretical potential to discriminate between cancerous and non-cancerous tissue with high sensitivity and specificity. To date, these techniques have not been able to be effectively translated to endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents a new technology that may be well-suited for endoscopic implementation. However, the feasibility of detecting differences between normal and cancerous mucosa using fluorescence excitation-scanning hyperspectral imaging has not been evaluated. The objective of this pilot study was to evaluate the changes in the fluorescence excitation spectrum of resected specimen pairs of colorectal adenocarcinoma and normal colorectal mucosa. Patients being treated for colorectal adenocarcinoma were enrolled. Representative adenocarcinoma and normal colonic mucosa specimens were collected from each case. Specimens were flash frozen in liquid nitrogen. Adenocarcinoma was confirmed by histologic evaluation of H&E permanent sections. Hyperspectral image data of the fluorescence excitation of adenocarcinoma and surrounding normal tissue were acquired using a custom microscope configuration previously developed in our lab. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation spectral range of 390-450 nm. We conclude that fluorescence excitation-scanning hyperspectral imaging may offer an alternative approach for differentiating adenocarcinoma and surrounding normal mucosa of the colon. Future work will focus on expanding the number of specimen pairs analyzed and will utilize fresh tissues where possible, as flash freezing and reconstituting tissues may have altered the autofluorescence properties.

A theoretical‐experimental methodology for assessing the sensitivity of biomedical spectral imaging platforms, assays, and analysis methods

Spectral imaging technologies have been used for many years by the remote sensing community. More recently, these approaches have been applied to biomedical problems, where they have shown great promise. However, biomedical spectral imaging has been complicated by the high variance of biological data and the reduced ability to construct test scenarios with fixed ground truths. Hence, it has been difficult to objectively assess and compare biomedical spectral imaging assays and technologies. Here, we present a standardized methodology that allows assessment of the performance of biomedical spectral imaging equipment, assays, and analysis algorithms. This methodology incorporates real experimental data and a theoretical sensitivity analysis, preserving the variability present in biomedical image data. We demonstrate that this approach can be applied in several ways: to compare the effectiveness of spectral analysis algorithms, to compare the response of different imaging platforms, and to assess the level of target signature required to achieve a desired performance. Results indicate that it is possible to compare even very different hardware platforms using this methodology. Future applications could include a range of optimization tasks, such as maximizing detection sensitivity or acquisition speed, providing high utility for investigators ranging from design engineers to biomedical scientists.

Feasibility for detection of autofluorescent signatures in rat organs using a novel excitation-scanning hyperspectral imaging system

The natural fluorescence (autofluorescence) of tissues has been noted as a biomarker for cancer for several decades. Autofluorescence contrast between tumors and healthy tissues has been of significant interest in endoscopy, leading to development of autofluorescence endoscopes capable of visualizing 2-3 fluorescence emission wavelengths to achieve maximal contrast. However, tumor detection with autofluorescence endoscopes is hindered by low fluorescence signal and limited quantitative information, resulting in prolonged endoscopic procedures, prohibitive acquisition times, and reduced specificity of detection. Our lab has designed a novel excitation-scanning hyperspectral imaging system with high fluorescence signal detection, low acquisition time, and enhanced spectral discrimination. In this study, we surveyed a comprehensive set of excised tissues to assess the feasibility of detecting tissue-specific pathologies using excitation-scanning. Fresh, untreated tissue specimens were imaged from 360 to 550 nm on an inverted fluorescence microscope equipped with a set of thin-film tunable filters (Semrock, A Unit of IDEX). Images were subdivided into training and test sets. Automated endmember extraction (ENVI 5.1, Exelis) with PCA identified endmembers within training images of autofluorescence. A spectral library was created from 9 endmembers. The library was used for identification of endmembers in test images. Our results suggest (1) spectral differentiation of multiple tissue types is possible using excitation scanning; (2) shared spectra between tissue types; and (3) the ability to identify unique morphological features in disparate tissues from shared autofluorescent components. Future work will focus on isolating specific molecular signatures present in tissue spectra, and elucidating the contribution of these signatures in pathologies.

Three-dimensional measurement of cAMP gradients using hyperspectral confocal microscopy

Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions over a wide range of timescales. Several lines of evidence have suggested that the distribution of cAMP within cells is not uniform, and that cAMP compartmentalization is largely responsible for signaling specificity within the cAMP signaling pathway. However, to date, no studies have experimentally measured three dimensional (3D) cAMP distributions within cells. Here we use both 2D and 3D hyperspectral microscopy to visualize cAMP gradients in endothelial cells from the pulmonary microvasculature (PMVECs). cAMP levels were measured using a FRETbased cAMP sensor comprised of a cAMP binding domain from EPAC sandwiched between FRET donors and acceptors — Turquoise and Venus fluorescent proteins. Data were acquired using either a Nikon A1R spectral confocal microscope or custom spectral microscopy system. Analysis of hyperspectral image stacks from a single confocal slice or from summed images of all slices (2D analysis) indicated little or no cAMP gradients were formed within PMVECs under basal conditions or following agonist treatment. However, analysis of hyperspectral image stacks from 3D cellular geometries (z stacks) demonstrate marked cAMP gradients from the apical to basolateral membrane of PMVECs. These results strongly suggest that 2D imaging studies of cAMP compartmentalization — whether epifluorescence or confocal microscopy — may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D studies are required to assess mechanisms of signaling specificity.

LED-based endoscopic light source for spectral imaging

Colorectal cancer is the United States 3rd leading cancer in death rates.1 The current screening for colorectal cancer is an endoscopic procedure using white light endoscopy (WLE). There are multiple new methods testing to replace WLE, for example narrow band imaging and autofluorescence imaging.2 However, these methods do not meet the need for a higher specificity or sensitivity. The goal for this project is to modify the presently used endoscope light source to house 16 narrow wavelength LEDs for spectral imaging in real time while increasing sensitivity and specificity. The process to do such was to take an Olympus CLK-4 light source, replace the light and electronics with 16 LEDs and new circuitry. This allows control of the power and intensity of the LEDs. This required a larger enclosure to house a bracket system for the solid light guide (lightpipe), three new circuit boards, a power source and National Instruments hardware/software for computer control. The results were a successfully designed retrofit with all the new features. The LED testing resulted in the ability to control each wavelength’s intensity. The measured intensity over the voltage range will provide the information needed to couple the camera for imaging. Overall the project was successful; the modifications to the light source added the controllable LEDs. This brings the research one step closer to the main goal of spectral imaging for early detection of colorectal cancer. Future goals will be to connect the camera and test the imaging process.