Thin-film tunable filters for hyperspectral fluorescence microscopy

Abstract

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.

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

“…A 458 nm long-pass filter cube was utilized that consisted of a long-pass dichroic beamsplitter (FF-458-Di02, Semrock, Inc., Rochester, NY) and long-pass emission filter (BLP01-458R, Semrock, Inc.). The wavelength accuracy, spectral resolution, and wavelength-dependent response of this system have been previously documented [96,97]. In brief, this system provides accurate wavelength reproducibility and the ability to vary the bandwidth.…”

“…An array of TFTF VersaChrome filters was utilized, each with a tuning range of approximately 50-60 nm, so as to be able to tune through a wide range of wavelengths. A 495 nm long-pass filter cube was utilized that consisted of a long-pass dichroic beamsplitter (FF-495-Di03, Semrock, Inc., Rochester, NY) and long-pass emission filter (BLP01-495R, Semrock, Inc.) [97,104]. The wavelength accuracy, spectral resolution, and wavelength-dependent response of this system have been previously documented [97,104].…”

“…The cell and tissue preparation has been described in detail elsewhere [96,97]. In brief, pulmonary microvascular en- dothelial cells (PMVECs) were isolated from CD rats.…”

“…Both GFP-expressing and non-GFP expressing PMVECs were grown to confluence and suspended in phosphate-buffered saline (PBS) prior to use. All animal and tissue resection procedures were carried out in accordance with Institutional Animal Care and Use Committee (IACUC) approved protocols and have also been described elsewhere [96][97][98]. In brief, male CD rats were anaesthetized and placed on mechanical ventilation.…”

“…However, only a limited amount of research has been performed to apply many of the spectral image processing algorithms developed by the remote sensing community to biomedical applications [10]. Even less work has been done to quantitatively assess the effectiveness of spectral image analysis algorithms as applied to biological image data or to compare algorithms or spectral imaging systems to each other [10,[65][66][67]. Hence, it is not always clear which spectral imaging platform or analysis algorithm should be used for a particular biomedical imaging application.…”