Mesoscopic Fluorescence Molecular Tomography for Evaluating Engineered Tissues

Abstract

Optimization of regenerative medicine strategies includes the design of biomaterials, development of cell-seeding methods, and control of cell-biomaterial interactions within the engineered tissues. Among these steps, one paramount challenge is to non-destructively image the engineered tissues in their entirety to assess structure, function, and molecular expression. It is especially important to be able to enable cell phenotyping and monitor the distribution and migration of cells throughout the bulk scaffold. Advanced fluorescence microscopic techniques are commonly employed to perform such tasks; however, they are limited to superficial examination of tissue constructs. Therefore, the field of tissue engineering and regenerative medicine would greatly benefit from the development of molecular imaging techniques which are capable of non-destructive imaging of three-dimensional cellular distribution and maturation within a tissue-engineered scaffold beyond the limited depth of current microscopic techniques. In this review, we focus on an emerging depth-resolved optical mesoscopic imaging technique, termed Laminar Optical Tomography (LOT) or Mesoscopic Fluorescence Molecular Tomography (MFMT), which enables longitudinal imaging of cellular distribution in thick tissue engineering constructs at depths of a few millimeters and with relatively high resolution. The physical principle, image formation, and instrumentation of LOT/MFMT systems are introduced. Representative applications in tissue engineering include imaging the distribution of human mesenchymal stem cells (hMSCs) embedded in hydrogels, imaging of bio-printed tissues, and in vivo applications.

High-dynamic-range fluorescence laminar optical tomography (HDR-FLOT)

“…Depth-resolved LOT/FLOT has been demonstrated in imaging tumors/fluorescencelabeled tumors [13,15,16,40], perfused rat hearts [1], and fluorescence-labeled cells in tissue engineering [11,12], as well as for visualizing neural activities in mouse brain and spinal cord [6,8,41]. In a realistic experiment, usually we do not know the fluorescence situation/distribution of the sample, which means that it is difficult to choose a proper excitation power or exposure time for the unknown inhomogeneous fluorescence distribution.…”

“…Soon after, LOT was rapidly adapted for fluorescent molecular imaging, which was termed either fluorescence laminar optical tomography (FLOT) [1,[5][6][7] or mesoscopic fluorescence molecular tomography (MFMT) [10,11], and several improvements or alternative system designs were investigated [5,6,[10][11][12][13][14]. FLOT has been reported to quantify depth-resolved distribution of fluorescence-labeled tumors [13,15,16], to image fluorescence-labeled cells in tissue engineering [11,12,16], and to visualize neural activities in mouse brains in vivo [6,17]. In the FLOT system configuration, a charge-coupled device (CCD) or electronmultiplying CCD (EMCCD) can be used as the array detector since either one has a higher sampling density in comparison to a photomultiplier tube (PMT) array or an avalanche photodiode (APD) array [6,16].…”

“…FLOT has been reported to quantify depth-resolved distribution of fluorescence-labeled tumors [13,15,16], to image fluorescence-labeled cells in tissue engineering [11,12,16], and to visualize neural activities in mouse brains in vivo [6,17]. In the FLOT system configuration, a charge-coupled device (CCD) or electronmultiplying CCD (EMCCD) can be used as the array detector since either one has a higher sampling density in comparison to a photomultiplier tube (PMT) array or an avalanche photodiode (APD) array [6,16]. During signal collection in FLOT with reflectance imaging mode, the photons collected at larger source-detector separations have a higher statistical probability of travelling through deeper tissues.…”

“…During signal collection in FLOT with reflectance imaging mode, the photons collected at larger source-detector separations have a higher statistical probability of travelling through deeper tissues. In general, the detector with a larger sourcedetector separation collects a lower signal compared to a detector closer to the illumination source, indicating that the deeper area will have a low signal-to-noise ratio (SNR), which will limit reconstruction accuracy [16]. We can increase either the excitation power or exposure time to enhance the collected signals from deeper regions.…”

Depth-resolved imaging of colon tumor using optical coherence tomography and fluorescence laminar optical tomography

“…LOT imaging of skin cancer based on absorption contrast has been demonstrated [46]. Fluorescence-based LOT (FLOT) has the potential to quantify depth-resolved distribution of fluorescence-labeled tumor [47][48][49][50].…”