Direct frequency domain fluorescence lifetime imaging using simultaneous ultraviolet and visible excitation.

Due to the complexity, limited practicality, and cost of conventional fluorescence lifetime imaging/microscopy (FLIM) instrumentation, FLIM adoption has been mostly limited to academic settings. We present a novel point scanning frequency-domain (FD) FLIM instrumentation design capable of simultaneous multi-wavelength excitation, simultaneous multispectral detection, and sub-nanosecond to nanosecond fluorescence lifetime estimation. Fluorescence excitation is implemented using intensity-modulated CW diode lasers that are available in a selection of wavelengths spanning the UV-VI-NIR range (375-1064 nm). Digital laser intensity modulation was adopted to enable simultaneous frequency interrogation at the fundamental frequency and corresponding harmonics. Time-resolved fluorescence detection is implemented using low-cost, fixed-gain, narrow bandwidth (100 MHz) avalanche photodiodes, thus, enabling cost-effective fluorescence lifetime measurements at multiple emission spectral bands simultaneously. Synchronized laser modulation and fluorescence signal digitization (250 MHz) is implemented using a common field-programmable gate array (FPGA). This synchronization reduces temporal jitter, which simplifies instrumentation, system calibration, and data processing. The FPGA also allows for the implementation of the real-time processing of the fluorescence emission phase and modulation at up to 13 modulation frequencies (processing rate matching the sampling rate of 250 MHz). Rigorous validation experiments have demonstrated the capabilities of this novel FD-FLIM implementation to accurately measure fluorescence lifetimes in the range of 0.5-12 ns. In vivo endogenous, dual-excitation (375nm/445nm), multispectral (four bands) FD-FLIM imaging of human skin and oral mucosa at 12.5 kHz pixel rate and room-light conditions was also successfully demonstrated. This versatile, simple, compact, and cost-effective FD-FLIM implementation will facilitate the clinical translation of FLIM imaging and microscopy.

Direct frequency domain fluorescence lifetime imaging using field programmable gate arrays for real time processing.

Frequency domain (FD) fluorescence lifetime imaging (FLIM) involves the excitation of the sample of interest with a modulated light source and digitization of the fluorescence emission for further analysis. Traditional FD-FLIM systems use heterodyne or homodyne detection, where the excitation light source and detector are modulated at specific frequency(s). More recently, FD-FLIM systems that use reflection of the light source as a trigger or phase reference for lifetime calculations have been developed. These detection schemes, however, require extra components that increase the cost and complexity of the FD-FLIM system. Here, we report a novel FD-FLIM detection scheme whereby the light source modulation and emission digitization are implemented using Field Programmable Gate Arrays (FPGAs), and fixed gain avalanche photodiodes are used for fluorescence detection. The reported FD-FLIM system was designed for probing nanosecond lifetime fluorophores (2-10 ns) at three emission bands simultaneously. The system utilizes a 375 nm diode laser for excitation at multiple simultaneous modulation frequencies (between 1 MHz and 83 MHz, bandwidth limited intentionally by using a lowpass filter) and three fixed gain avalanche photodiodes for simultaneous detection of three emission bands: 405/20 nm, 440/40 nm, and 525/50 nm (center/FWHM). Real-time computation of the modulation and phase lifetimes is simply performed by direct application of the discrete Fourier transform (max. of 10 frequencies) to the digitized fluorescence emission signals. The accuracy and sensitivity of this novel FD-FLIM detection scheme was demonstrated by imaging standard fluorophores and ex vivo unfixed human coronary artery tissue samples.

Automated detection of superficial macrophages in atherosclerotic plaques using autofluorescence lifetime imaging.

BACKGROUND AND AIMS: Macrophages play an important role in the development and destabilization of advanced atherosclerotic plaques. Hence, the clinical imaging of macrophage content in advanced plaques could potentially aid in identifying patients most at risk of future clinical events. The lifetime of the autofluorescence emission from atherosclerotic plaques has been correlated with lipids and macrophage accumulation in ex vivo human coronary arteries, suggesting the potential of intravascular endogenous fluorescence or autofluorescence lifetime imaging (FLIM) for macrophage imaging. The aim of this study was to quantify the accuracy of the coronary intima autofluorescence lifetime to detect superficial macrophage accumulation in atherosclerotic plaques. METHODS: Endogenous FLIM imaging was performed on 80 fresh postmortem coronary segments from 23 subjects. The plaque autofluorescence lifetime at an emission spectral band of 494 ±â€¯20.5 nm was used as a discriminatory feature to detect superficial macrophage accumulation in atherosclerotic plaques. Detection of superficial macrophage accumulation in the imaged coronary segments based on immunohistochemistry (CD68 staining) evaluation was taken as the gold standard. Receiver Operating Characteristic (ROC) curve analysis was applied to select an autofluorescence lifetime threshold value to detect superficial macrophages accumulation. RESULTS: A threshold of 6 ns in the plaque autofluorescence lifetime at the emission spectral band of 494 ±â€¯20.5 nm was applied to detect plaque superficial macrophages accumulation, resulting in ∼91.5% accuracy. CONCLUSIONS: This study demonstrates the capability of endogenous FLIM imaging to accurately identify superficial macrophages accumulation in human atherosclerotic plaques, a key biomarker of atherosclerotic plaque vulnerability.

Multimodal optical coherence tomography and fluorescence lifetime imaging with interleaved excitation sources for simultaneous endogenous and exogenous fluorescence.

Multimodal imaging probes a variety of tissue properties in a single image acquisition by merging complimentary imaging technologies. Exploiting synergies amongst the data, algorithms can be developed that lead to better tissue characterization than could be accomplished by the constituent imaging modalities taken alone. The combination of optical coherence tomography (OCT) with fluorescence lifetime imaging microscopy (FLIM) provides access to detailed tissue morphology and local biochemistry. The optical system described here merges 1310 nm swept-source OCT with time-domain FLIM having excitation at 355 and 532 nm. The pulses from 355 and 532 nm lasers have been interleaved to enable simultaneous acquisition of endogenous and exogenous fluorescence signals, respectively. The multimodal imaging system was validated using tissue phantoms. Nonspecific tagging with Alexa Flour 532 in a Watanbe rabbit aorta and active tagging of the LOX-1 receptor in human coronary artery, demonstrate the capacity of the system for simultaneous acquisition of OCT, endogenous FLIM, and exogenous FLIM in tissues.

Simultaneous morphological and biochemical endogenous optical imaging of atherosclerosis.

AIMS: The aim of this study was to validate novel imaging technology for simultaneous morphological and biochemical endogenous optical imaging of coronary atherosclerotic plaque. METHODS AND RESULTS: Optical coherence tomography (OCT) generates high-resolution 3D images of plaque morphology and endogenous fluorescence lifetime imaging microscopy (FLIM) characterizes biochemical composition. Both imaging modalities rely on plaque's intrinsic optical characteristics, making contrast agents unnecessary. A multimodal OCT/FLIM system was utilized to generate luminal biochemical maps superimposed on high-resolution (7 µm axial and 13 µm lateral) structural volumetric images. Forty-seven fresh postmortem human coronary segments were imaged: pathological intimal thickening (PIT, n = 26), fibroatheroma (FA, n = 12), thin-cap FA (TCFA, n = 2), and fibrocalcific plaque (CA, n = 7), determined by histopathology. Multimodal images were evaluated, and each plaque identified as PIT, FA, TCFA, or CA based on expert OCT readers, and as having high-lipid (HL), high-collagen (HC), or low-collagen/low-lipid (LCL) luminal composition based on linear discriminant analysis of FLIM. Of 47 plaques, 89.4% (42/47) of the plaques were correctly identified based on OCT/FLIM evaluation using tissue histopathology and immunohistochemistry as the gold standard. Four of the misclassifications corresponded to confusing PIT with HL luminal composition for FA with HL cap. The other corresponded to confusing FA with a HC cap for FA with an LCL cap. CONCLUSION: We have demonstrated the feasibility of accurate simultaneous OCT/FLIM morphological and biochemical characterization of coronary plaques at spatial resolutions and acquisition speeds compatible with catheter-based intravascular imaging. The success of this pilot study sets up future development of a multimodal intravascular imaging system that will enable studies that could help improve our understanding of plaque pathogenesis.

Automated classification of optical coherence tomography images for the diagnosis of oral malignancy in the hamster cheek pouch.

Most studies evaluating the potential of optical coherence tomography (OCT) for the diagnosis of oral cancer are based on visual assessment of OCT B-scans by trained experts. Human interpretation of the large pool of data acquired by modern high-speed OCT systems, however, can be cumbersome and extremely time consuming. Development of image analysis methods for automated and quantitative OCT image analysis could therefore facilitate the evaluation of such a large volume of data. We report automated algorithms for quantifying structural features that are associated with the malignant transformation of the oral epithelium based on image processing of OCT data. The features extracted from the OCT images were used to design a statistical classification model to perform the automated tissue diagnosis. The sensitivity and specificity of distinguishing malignant lesions from benign lesions were found to be 90.2% and 76.3%, respectively. The results of the study demonstrate the feasibility of using quantitative image analysis algorithms for extracting morphological features from OCT images to perform the automated diagnosis of oral malignancies in a hamster cheek pouch model.