Multimodal Optical Microscopy Research Articles

Exploring the structure, metabolism, and biochemistry of the neuronal microenvironment label-free using fast simultaneous multimodal optical microscopy

The technologies to examine the neuronal microenvironment label free remain critically underexplored. There is a gap in our knowledge of underlying metabolic, biochemical, and electrophysiological mechanisms behind several neurological processes at a cellular level, which can be traced to the lack of versatile and high-throughput tools to investigate neural networks. In this paper, four label-free contrasts were explored as mechanisms to study neuronal activity, namely, scattering, birefringence, autofluorescence from metabolic cofactors and molecules, and local biochemistry. To overcome challenges of observing neuronal activity spanning three orders of magnitude in space and time, microscopes had to be developed to simultaneously capture these contrasts quickly, with high resolution, and over a large FOV. We developed versatile autofluorescence lifetime, multiharmonic generation, polarization-sensitive interferometry, and Raman imaging in epi-detection (VAMPIRE) microscopy to simultaneously observe multiple facets of neuronal structure and dynamics. The accelerated computational-imaging-driven acquisition speeds, the utilization of a single light source to evoke all contrasts, the simultaneous acquisition that provides an otherwise impossible multimodal dynamic imaging capability, and the real-time processing of the data enable VAMPIRE microscopy as a powerful imaging platform for neurophotonics and beyond.

Multimodal imaging of a liver-on-a-chip model using labelled and label-free optical microscopy techniques.

A liver-on-a-chip model is an advanced complex in vitro model (CIVM) that incorporates different cell types and extracellular matrix to mimic the microenvironment of the human liver in a laboratory setting. Given the heterogenous and complex nature of liver-on-a-chip models, brightfield and fluorescence-based imaging techniques are widely utilized for assessing the changes occurring in these models with different treatment and environmental conditions. However, the utilization of optical microscopy techniques for structural and functional evaluation of the liver CIVMs have been limited by the reduced light penetration depth and lack of 3D information obtained using these imaging techniques. In this study, the potential of both labelled as well as label-free multimodal optical imaging techniques for visualization and characterization of the cellular and sub-cellular features of a liver-on-a-chip model was investigated. (1) Cellular uptake and distribution of Alexa 488 (A488)-labelled non-targeted and targeted antisense oligonucleotides (ASO and ASO-GalNAc) in the liver-on-a-chip model was determined using multiphoton microscopy. (2) Hyperspectral stimulated Raman scattering (SRS) microscopy of the C-H region was used to determine the heterogeneity of chemical composition of circular and cuboidal hepatocytes in the liver-on-a-chip model in a label-free manner. Additionally, the spatial overlap between the intracellular localization of ASO and lipid droplets was explored using simultaneous hyperspectral SRS and fluorescence microscopy. (3) The capability of light sheet fluorescence microscopy (LSFM) for full-depth 3D visualization of sub-cellular distribution of A488-ASO and cellular phenotypes in the liver-on-a-chip model was demonstrated. In summary, multimodal optical microscopy is a promising platform that can be utilized for visualization and quantification of 3D cellular organization, drug distribution and functional changes occurring in liver-on-a-chip models, and can provide valuable insights into liver biology and drug uptake mechanisms by enabling better characterization of these liver models.

Optical nanoscopy challenges in the metaverse era. The case of multimodal imaging of chromatin in the nucleus.

Optical Nanoscopy deals with designing and developing optical microscopes able to produce images that are rich sources of quantitative information at the nanoscale. This fact offers an unprecedented insight into the molecular mechanisms that govern and determine the fate of living cells, coupled with a large amount of data to be structured. In this scenario, multimodal optical microscopy is a growing attitude boosted by artificial intelligence. Metaverse environments offer potential advantages in analysing data in terms of interactivity in a kind of “infinite” space to visualise multimodal data. In the era of super-resolved fluorescence microscopy, the photochemical parameters, from brightness to lifetime, can be merged with the ones coming from label-free approaches like Mueller matrix microscopy. Here, we report on the potential of convolutional neural networks (CNN) and independent component analysis (ICA) algorithms in the domain of machine learning and deep learning with the ambitious target to create a robust virtual environment “to see” what we could not perceive before. Is the metaverse an helpful approach to elucidating the still open universal question in biology about how chromatin organisation in the nucleus rules the compaction and function of the human genome in the interphase of cells and mitotic chromosomes? We start with the development of a multimodal optical microscope reinforced by the artificial intelligence component.

Label-free metabolic and structural profiling of dynamic biological samples using multimodal optical microscopy with sensorless adaptive optics

Label-free optical microscopy has matured as a noninvasive tool for biological imaging; yet, it is criticized for its lack of specificity, slow acquisition and processing times, and weak and noisy optical signals that lead to inaccuracies in quantification. We introduce FOCALS (Fast Optical Coherence, Autofluorescence Lifetime imaging, and Second harmonic generation) microscopy capable of generating NAD(P)H fluorescence lifetime, second harmonic generation (SHG), and polarization-sensitive optical coherence microscopy (OCM) images simultaneously. Multimodal imaging generates quantitative metabolic and morphological profiles of biological samples in vitro, ex vivo, and in vivo. Fast analog detection of fluorescence lifetime and real-time processing on a graphical processing unit enables longitudinal imaging of biological dynamics. We detail the effect of optical aberrations on the accuracy of FLIM beyond the context of undistorting image features. To compensate for the sample-induced aberrations, we implemented a closed-loop single-shot sensorless adaptive optics solution, which uses computational adaptive optics of OCM for wavefront estimation within 2 s and improves the quality of quantitative fluorescence imaging in thick tissues. Multimodal imaging with complementary contrasts improves the specificity and enables multidimensional quantification of the optical signatures in vitro, ex vivo, and in vivo, fast acquisition and real-time processing improve imaging speed by 4–40 × while maintaining enough signal for quantitative nonlinear microscopy, and adaptive optics improves the overall versatility, which enable FOCALS microscopy to overcome the limits of traditional label-free imaging techniques.

Abstract P3-18-10: Current options and future perspectives for breast margin assessment in clinical practice

Abstract Background: Ensuring negative margins at the time of breast conserving surgery for invasive breast cancer or ductal carcinoma in situ is necessary to minimize the chance of local recurrence and avoid re-excisions that cause patient concern and may delay adjuvant therapy. However, about 25% of women who have lumpectomy have a second surgery to remove residual disease, suggesting an ongoing need for improved methods of intraoperative margin assessment. This study aimed to perform a contemporary review of methods of intraoperative margin assessment during breast conservation surgery. Methods: A review of the scientific literature from 2009 to 2021 comprising 62 manuscripts of the current and proposed. intraoperative techniques for breast margin assessment during breast conservation surgery was conducted via PubMed and Google Scholar. The methods assessed were grouped into 10 categories based on the technology employed: bioimpedance/radiofrequency, high-resolution imaging, optical imaging, mass spectrometry, magnetic-resonance imaging, 2D/3D specimen CT, X-ray, multimodal optical microscopy, pharmacologic and pathological margin assessment. All technologies were reviewed for overall effectiveness in lowering re-excision rates derived from their respective advantages, limitations, sensitivity, and specificity. Results: Overall, 8 current and 7 technologies under development were assessed (Table 1). Frozen section and cytology yielded the highest diagnostic accuracy; however, these can be time-consuming, resource-intensive, and have limited sampling points. Conversely, 2D specimen CT provides rapid results but is limited by relatively low sensitivity. Future technologies such as optical coherence tomography showed promising results in demonstrating higher diagnostic accuracy for lumpectomy and margin shaves, but remain to be proven in clinical practice. Conclusion: Intraoperative margin assessments can lower final positive margins and subsequent re-excisions rates. A number of current and upcoming technologies that assess margin status range in their respective efficacies and limitations. There remains demand for improved margin assessment at the time of breast conserving surgery, but which technologies will become standard of care remains, at present, unclear. Table 1.Methods of intraoperative margin assessmentMethod:Brand:Sensitivity:Specificity:Source:Bioimpedence• ClearEdge™87.3%75.6%Dixon et al. 2016• MarginProbe75.2%70-87%Schanabel et al., 2014Cytology:• Imprint72%97%Esbona & Zhanhai 2012; Qui et al., 2018Frozen Section–83%95%Schmidt et al, 2020; Schwarz & Schmidt, 2020Mass Spectrometry• iKnife93.4-94.7%94.7-96.2%St. John et al., 2017• MasSpec Pen83-95%95-100%Garza et al., 2020Multimodal optical microscopy• fluorescence imaging combination withRCM/OCT subsystemTBDTBDScimone et al., 2021MRI• ClearSight™93%92%Moshe et al., 2016Optical Coherence Tomography (OCT)• OTIS™96%92%Mojahed et al., 2020; Schmidt et al. 2020Pharmacology• Bevacizumab98%79%Koch et al., 2017• Lumicell2-3D specimen CT• Mozart®93%78%Black et al., [poster]• Faxitron78.6-85.6%100%Emmadi & Wiley 2012; Bathla et al. 2011• MicroCT56%100%Qiu et al. 2018X-ray• XPCI83%83%Massimi et al. 2021 Citation Format: Huma Javaid, Ivan Marin, Jessica Montalvan, Logan Healy, Brian Menegaz, Cary Hsu, Eric Silberfein, Elizabeth Bonefas, Stacey A. Carter, Alastair M. Thompson. Current options and future perspectives for breast margin assessment in clinical practice [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-18-10.

Assessment of breast cancer surgical margins with multimodal optical microscopy: A feasibility clinical study.

Providing surgical margin information during breast cancer surgery is crucial for the success of the procedure. The margin is defined as the distance from the tumor to the cut surface of the resection specimen. The consensus among surgeons and radiation oncologists is that there should be no tumor left within 1 to maximum 2 mm from the surface of the surgical specimen. If a positive margin remains, there is substantial risk for tumor recurrence, which may also result in potentially reduced cosmesis and eventual need for mastectomy. In this paper we report a novel multimodal optical imaging instrument based on combined high-resolution confocal microscopy-optical coherence tomography imaging for assessing the presence of potential positive margins on surgical specimens. Since rapid specimen analysis is critical during surgery, this instrument also includes a fluorescence imaging channel to enable rapid identification of the areas of the specimen that have potential positive margins. This is possible by specimen incubation with a cancer specific agent prior to imaging. In this study we used a quenched contrast agent, which is activated by cancer specific enzymes, such as urokinase plasminogen activators (uPA). Using this agent or a similar one, one may limit the use of high-resolution optical imaging to only fluorescence-highlighted areas for visualizing tissue morphology at the sub-cellular scale and confirming or ruling out cancer presence. Preliminary evaluation of this technology was performed on 20 surgical specimens and testing of the optical imaging findings was performed against histopathology. The combination of the three imaging modes allowed for high correlation between optical image analysis and histological ground-truth. The initial results are encouraging, showing instrument capability to assess margins on clinical specimens with a positive predictive value of 1.0 and a negative predictive value of 0.83.

Multimodal Microscopy: Fast Acquisition of Quantitative Phase and Fluorescence Imaging in 3D Space

Multimodal optical microscopy combines different optical and physiological principles working together to obtain rich perspectives from a single biological event. In this paper, the recent advances in multimodal microscopy systems based on off-axis digital holography and transport-of-intensity equation are reviewed. Both methods used the common-path configuration, and realized simultaneous quantitative phase and fluorescence imaging in 3D space. Feasibility checks with fluorescent micro-beads and live plant cells are presented. The results show that both methods are capable of fast acquisition of the quantitative phase and fluorescence information with digital refocusing ability.

Non-invasive monitoring of pharmacodynamics during the skin wound healing process using multimodal optical microscopy

ObjectiveImpaired diabetic wound healing is one of the serious complications associated with diabetes. In patients with diabetes, this impairment is characterized by several physiological abnormalities such as metabolic changes, reduced...

In vivo brain imaging with multimodal optical coherence microscopy in a mouse model of thromboembolic photochemical stroke

.We used a new multimodal imaging system that combines optical coherence microscopy and brightfield microscopy. Using this in vivo brain monitoring approach and cranial window implantation, we three-dimensionally visualized the vascular network during thrombosis, with high temporal (18 s) and spatial (axial, ; lateral, ) resolution. We used a modified mouse model of photochemical thromboembolic stroke in order to more accurately parallel human stroke. Specifically, we applied green laser illumination to focally occlude a branch of the middle cerebral artery. Despite the recanalization of the superficial arteries at 24 h after stroke, no blood flow was detected in the small vessels within deeper regions. Moreover, after 24 h of stroke progression, scattering signal enhancement was observed within the stroke region. We also evaluated the infarct extent and shape histologically. In summary, we present a novel approach for real-time mouse brain monitoring and ischemic variability analysis. This multimodal imaging method permits the analysis of thrombosis progression and reperfusion. Additionally and importantly, the system could be used to study the effect of poststroke drug treatments on blood flow in small arteries and capillaries of the brain.

Label-Free Non-linear Multimodal Optical Microscopy—Basics, Development, and Applications

Nonlinear optical (NLO) microscopy has proven to be a powerful tool especially for tissue imaging with sub-cellular resolution, high penetration depth, endogenous contrast specificity, pinhole-less optical sectioning capability. In this review, we discuss label-free nonlinear optical microscopes including the two-photon fluorescence (TPF), fluorescence lifetime imaging microscopy (FLIM), polarization-resolved second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) techniques with various samples. The nonlinear signals are generated from collagen in tissue (SHG), amylopectin from starch granules (SHG), sarcomere structure of fresh muscle (SHG), elastin in skin (TPF), nicotinamide adenine dinucleotide (NADH) in cells (TPF) and lipid droplets in cells (CARS). Again, the nonlinear signals are very specific to the molecular structure of the sample and its relative orientation to the polarization of the incident light. Thus, polarization-resolved nonlinear optical microscopy provides high image contrast and quantitative estimate of sample orientation. An overview of the advancements on polarization-resolved SHG microscopy including Stokes vector based polarimetry, circular dichroism, and susceptibility are also presented in this review article. The working principles and corresponding implements of above-mentioned microscopy techniques are elucidated. The potential of time-resolved TPF lifetime imaging microscopy (TP-FLIM) is explored by imaging endogenous fluorescence of NAD(P)H, a key coenzyme in cellular metabolic processes. We also discuss single laser source time-resolved multimodal CARS-FLIM microscopy using time-correlated single-photon counting (TCSPC) in combination with continuum generation from photonic crystal fiber (PCF). Using examples, we demonstrate that the multimodal NLO microscopy is a powerful tool to assess the molecular specificity with high resolution.

Revealing brain pathologies with multimodal visible light optical coherence microscopy and fluorescence imaging.

.We present a multimodal visible light optical coherence microscopy (OCM) and fluorescence imaging (FI) setup. Specification and phantom measurements were performed to characterize the system. Two applications in neuroimaging were investigated. First, curcumin-stained brain slices of a mouse model of Alzheimer’s disease were examined. Amyloid-beta plaques were identified based on the fluorescence of curcumin, and coregistered morphological images of the brain tissue were provided by the OCM channel. Second, human brain tumor biopsies retrieved intraoperatively were imaged prior to conventional neuropathologic work-up. OCM revealed the three-dimensional structure of the brain parenchyma, and FI added the tumor tissue-specific contrast. Attenuation coefficients computed from the OCM data and the florescence intensity values were analyzed and showed a statistically significant difference for 5-aminolevulinic acid (5-ALA)-positive and -negative brain tissues. OCM findings correlated well with malignant hot spots within brain tumor biopsies upon histopathology. The combination of OCM and FI seems to be a promising optical imaging modality providing complementary contrast for applications in the field of neuroimaging.

Direct mineralogical imaging of economic ore and rock samples with multi-modal nonlinear optical microscopy

Multi-modal nonlinear optical (NLO) microscopy, including stimulated Raman scattering (SRS) and second harmonic generation (SHG), was used to directly image mineralogical features of economic ore and rock samples. In SRS/SHG imaging, ore samples generally require minimal preparation and may be rapidly imaged, even in their wet state. 3D structural details, at submicron resolution, are revealed tens of microns deep within samples. Standard mineral imaging based on scanning electron microscopy (SEM), with elemental analysis via energy dispersive X-Ray spectroscopy, was used to independently validate the mineral composition of the samples. Spatially-resolved SRS from dominant Raman-resonant bands precisely maps the locations of specific minerals contained within the samples. SHG imaging reveals locally non-centrosymmetric structures, such as quartz grains. Competing absorption and nonlinear scattering processes, however, can reduce contrast in SRS imaging. Importantly, the correlation between standard electron microscopy and multi-modal NLO optical microscopy shows that the latter offers rapid image contrast based on the mineral content of the sample.

In Vivo Assessment of Engineered Skin Cell Delivery with Multimodal Optical Microscopy.

The healing process is often significantly impaired under conditions of chronic or large area wounds, which are often treated clinically using autologous split-thickness skin grafts. However, in many cases, harvesting of donor tissue presents a serious problem such as in the case of very large area burns. In response to this, engineered biomaterials have emerged that attempt to mimic the natural skin environment or deliver a suitable therapy to assist in the healing process. In this study, a custom-built multimodal optical microscope capable of noninvasive structural and functional imaging is used to investigate both the engineered tissue microenvironment and the in vivo wound healing process. Investigation of various engineered scaffolds show the strong relationship among the microenvironment of the scaffold, the organization of the cells within the scaffold, and the delivery pattern of these cells onto the healing wound. Through noninvasive tracking of these processes and parameters, multimodal optical microscopy provides an important tool in the assessment of engineered scaffolds both in vitro and in vivo.

A quantitative framework for the analysis of multimodal optical microscopy images.

Multimodal optical microscopy, a set of imaging techniques based on unique, yet complementary contrast mechanisms and spatially and temporally co-registered data acquisition, has emerged as a powerful biomedical tool. However, the analysis of the dense, high-dimensional datasets acquired by these instruments remains mostly qualitative and restricted to analysis of each modality individually. Using a custom-built multimodal nonlinear optical microscope, high dimensional datasets were acquired for automated classification of functional cell states as well as identification of histopathological features in tissues slices. Supervised classification of cell death modes was performed through support vector machines (SVM) and semi-supervised classification of tissue slices was performed through the use of the expectation maximization (EM) algorithm. Applications of these techniques to the automated classification of cell death modes as well as to the identification of tissue components in fixed ex vivo tissue slices are presented. The analysis techniques developed provide a direct link between multimodal image contrast and biological structure and function, resulting in highly accurate classification in both settings. Quantification of multimodal optical microscopy images through statistical modeling of the high dimensional data acquired gives a strong correlation between biological structure and function and image contrast. These methods are sensitive to the identification of diagnostic, cellular-level features important in a variety of clinical settings.

Multimodal Optical Resolution Photoacoustic and Fluorescence Microscopy in the Frequency Domain

Multimodal Optical Resolution Photoacoustic and Fluorescence Microscopy in the Frequency Domain

Precise Spatially Selective Photothermolysis Using Modulated Femtosecond Lasers and Real-time Multimodal Microscopy Monitoring.

The successful application of lasers in the treatment of skin diseases and cosmetic surgery is largely based on the principle of conventional selective photothermolysis which relies strongly on the difference in the absorption between the therapeutic target and its surroundings. However, when the differentiation in absorption is not sufficient, collateral damage would occur due to indiscriminate and nonspecific tissue heating. To deal with such cases, we introduce a novel spatially selective photothermolysis method based on multiphoton absorption in which the radiant energy of a tightly focused near-infrared femtosecond laser beam can be directed spatially by aiming the laser focal point to the target of interest. We construct a multimodal optical microscope to perform and monitor the spatially selective photothermolysis. We demonstrate that precise alteration of the targeted tissue is achieved while leaving surrounding tissue intact by choosing appropriate femtosecond laser exposure with multimodal optical microscopy monitoring in real time.

Fluorescent Nanodiamond–Gold Hybrid Particles for Multimodal Optical and Electron Microscopy Cellular Imaging

There is a continuous demand for imaging probes offering excellent performance in various microscopy techniques for comprehensive investigations of cellular processes by more than one technique. Fluorescent nanodiamond-gold nanoparticles (FND-Au) constitute a new class of "all-in-one" hybrid particles providing unique features for multimodal cellular imaging including optical imaging, electron microscopy, and, and potentially even quantum sensing. Confocal and optical coherence microscopy of the FND-Au allow fast investigations inside living cells via emission, scattering, and photothermal imaging techniques because the FND emission is not quenched by AuNPs. In electron microscopy, transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) analysis of FND-Au reveals greatly enhanced contrast due to the gold particles as well as an extraordinary flickering behavior in three-dimensional cellular environments originating from the nanodiamonds. The unique multimodal imaging characteristics of FND-Au enable detailed studies inside cells ranging from statistical distributions at the entire cellular level (micrometers) down to the tracking of individual particles in subcellular organelles (nanometers). Herein, the processes of endosomal membrane uptake and release of FNDs were elucidated for the first time by the imaging of individual FND-Au hybrid nanoparticles with single-particle resolution. Their convenient preparation, the availability of various surface groups, their flexible detection modalities, and their single-particle contrast in combination with the capability for endosomal penetration and low cytotoxicity make FND-Au unique candidates for multimodal optical-electronic imaging applications with great potential for emerging techniques, such as quantum sensing inside living cells.

Longitudinal in vivo tracking of adverse effects following topical steroid treatment.

Topical steroids are known for their anti-inflammatory properties and are commonly prescribed to treat many adverse skin conditions such as eczema and psoriasis. While these treatments are known to be effective, adverse effects including skin atrophy are common. In this study, the progression of these effects is investigated in an in vivo mouse model using multimodal optical microscopy. Utilizing a system capable of performing two-photon excitation fluorescence microscopy (TPEF) of reduced nicotinamide adenine dinucleotide (NADH) to visualize the epidermal cell layers and second harmonic generation (SHG) microscopy to identify collagen in the dermis, these processes can be studied at the cellular level. Fluorescence lifetime imaging microscopy (FLIM) is also utilized to image intracellular NADH levels to obtain molecular information regarding metabolic activity following steroid treatment. In this study, fluticasone propionate (FP)-treated, mometasone furoate (MF)-treated and untreated animals were imaged longitudinally using a custom-built multimodal optical microscope. Prolonged steroid treatment over the course of 21 days is shown to result in a significant increase in mean fluorescence lifetime of NADH, suggesting a faster rate of maturation of epidermal keratinocytes. Alterations to collagen organization and the structural microenvironment are also observed. These results give insight into the structural and biochemical processes of skin atrophy associated with prolonged steroid treatment.

Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations.

Liver sinusoidal endothelial cells (LSECs) act as a filter between blood and the hepatocytes. LSECs are highly fenestrated cells; they contain transcellular pores with diameters between 50 to 200 nm. The small sizes of the fenestrae have so far prohibited any functional analysis with standard and advanced light microscopy techniques. Only the advent of super-resolution optical fluorescence microscopy now permits the recording of such small cellular structures. Here, we demonstrate the complementary use of two different super-resolution optical microscopy modalities, 3D structured illumination microscopy (3D-SIM) and single molecule localization microscopy in a common optical platform to obtain new insights into the association between the cytoskeleton and the plasma membrane that supports the formation of fenestrations. We applied 3D-SIM to multi-color stained LSECs to acquire highly resolved overviews of large sample areas. We then further increased the spatial resolution for imaging fenestrations by single molecule localization microscopy applied to select small locations of interest in the same sample on the same microscope setup. We optimized the use of fluorescent membrane stains for these imaging conditions. The combination of these techniques offers a unique opportunity to significantly improve studies of subcellular ultrastructures such as LSEC fenestrations.

Multimodal optical microscopy methods reveal polyp tissue morphology and structure in Caribbean reef building corals.

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polypsand (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeterscale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,(2) superior optical sectioning ability,and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.