Marcus T. Cicerone

CASCADE is a novel peak fitting tool for biological Raman spectra which shows high true positive peak recovery.

Significance: Broadband coherent anti-Stokes Raman scattering (BCARS) enables label-free hyperspectral chemical imaging, but many signal components are small compared to the accompanying heteroscedastic noise, making optimal denoising crucial. Aim: We seek to identify the highest-performing hyperspectral image denoising methods for BCARS. Approach: We derive a physics-based, variance stabilization transform that yields nearly intensity-independent BCARS noise variance while preserving the dispersive Raman response. Within this framework, we compare six denoisers using a shared phase-retrieval pipeline: singular value decomposition, BM4D, Noise2Noise, deep image prior, self-supervised diffusion, and a cross-covariance-based singular value decomposition (CCV) introduced here. Evaluation uses five simulated cubes with exact ground truth and experimental measurements of glycerol, polystyrene beads, and Caenorhabditis elegans . Results: In simulations, the variance stabilization transform reduces the ratio of maximum to minimum channel noise variance from approximately 72 to 1.2. Deep image prior and self-supervised diffusion denoisers achieve the highest mean foreground peak signal-to-noise ratios. CCV matches the mean foreground structural similarity of deep image prior with substantially shorter runtime and no per-cube rank tuning. Experimental comparisons reveal sample-dependent performance. Conclusions: Physics-based variance stabilization provides a common framework for adapting and evaluating hyper-spectral denoisers for BCARS. CCV offers a practical balance of reconstruction fidelity, computational speed, and automated operation.

Mitochondrial dysfunction is a hallmark of aging, yet how mitochondrial states are remodeled across tissues and subcellular compartments in vivo remains elusive. Progress has been limited, in part, because mitochondrial physiology is highly sensitive to experimental perturbations, underscoring the need for minimally disruptive measurement strategies. Here, we establish a tissue–resolved, in vivo framework for the quantitative analysis of mitochondrial states in live, intact Caenorhabditis elegans without confounding effects from mounting–induced hypoxia. This platform couples two–photon fluorescence lifetime imaging microscopy (2p–FLIM) with a custom segmentation pipeline, MitoSLIT, to track functional and structural features across multiple tissues and single neurons. By integrating membrane potential-associated TMRM intensity, lifetime–based microenvironmental metrics, and morphological descriptors, we uncover localized metabolic heterogeneity masked by conventional intensity analysis. Leveraging this framework, we mapped physiological aging against mitochondrial shifts induced by acute stress and fission–fusion mutations. Our analyses reveal that mitochondrial aging is highly tissue-specific, executing distinct trajectories across cell types. Extending the framework to genetically identified neurons revealed age–dependent divergence between somatic and axonal mitochondrial states, accompanied by structural remodeling and a late shift in optical redox ratio. Together, our findings demonstrate that mitochondrial populations do not converge on a uniform bioenergetic endpoint during aging, but rather follow highly compartmentalized, tissue–specific spatiotemporal trajectories in vivo .

Broadband coherent anti‐Stokes Raman scattering (BCARS) microscopy is a powerful label‐free biological imaging technique, but the raw signal requires careful processing. The vibrationally resonant (Raman) fingerprint signal is usually small compared with instrumental noise sources and the nonresonant background (NRB) inherent in the BCARS signal. Fortunately, the NRB exhibits a systematic phase relationship with the coherent Raman response, acting as a heterodyne amplifier for the weak fingerprint signal. Due to this heterodyne effect, the Raman response can be recovered quantitatively and invariantly across different instruments, provided the NRB shape is known. Even with heterodyne amplification, the amplitudes of fingerprint signal components are often comparable to system noise. Singular value decomposition (SVD), which utilizes spatial information, is often employed for additional noise filtering. Consequently, finding optimal processing parameters to properly distinguish the NRB and Raman responses and suppress noise in the complex BCARS signal requires a reference system that realistically represents the spectral and spatial properties of BCARS signals obtained from biological samples. We present a digital tissue phantom that meets these criteria as a tool for testing candidate signal processing pipelines. The digital phantom is generated with simulated hyperspectral Raman images having system‐specific noise and background characteristics. We analyze phantom datasets with differing background and signal‐to‐noise conditions to evaluate their impact on the performance of multiple signal processing pipelines. Specifically, we investigate the application of a Butterworth filter‐based routine to directly estimate the NRB from the BCARS signal. Additionally, we evaluate a Lorentzian wavelet transform as an alternative to the Hilbert transform for extracting the Raman spectrum from the BCARS signal. While we demonstrate this phantom for BCARS, it can be used for any spectroscopic Raman imaging approach.

Raman fingerprint spectroscopy and fluorescence lifetime imaging are emerging tools for studying metabolic profiles of biological specimens. While Raman fingerprint spectroscopy detects intrinsic molecular vibrations that reflect the molecular composition and chemical environment of a sample, fluorescence lifetime imaging measures changes in the excited-state lifetime of fluorophores that are sensitive to their microenvironments. Here, we present a multimodal imaging platform combining broadband coherent anti-Stokes Raman scattering (BCARS) and two-photon fluorescence lifetime imaging (2p-FLIM) microscopy that can acquire biologically relevant Raman fingerprint spectra and fluorescence lifetime signals in vivo and simultaneously. The tremendous chemical information obtained from spatially co-registered BCARS and 2p-FLIM images allows us to characterize the subtle differences between sub-cellular compartments and verify the potential false-positive results generated by fluorescence imaging alone. This is demonstrated by directly comparing the BCARS, 2p-FLIM, and two-photon excitation fluorescence (TPEF) signals simultaneously obtained from the same dye-stained organelle in the live, intact C. elegans expressing a green fluorescent protein (GFP) marker. In this work, we introduce the BCARS/2p-FLIM/TPEF setup scheme, the image acquisition steps, data processing, and representative results showing that the cross-modality imaging method enables rigorous characterization and in vivo detection at sub-cellular resolution. This protocol provides a framework for simultaneous chemical and fluorescence lifetime imaging to improve the accuracy of biological interpretation in complex living systems.

Raman fingerprint spectroscopy and fluorescence lifetime imaging are emerging tools for studying metabolic profiles of biological specimens. While Raman fingerprint spectroscopy detects intrinsic molecular vibrations that reflect the molecular composition and chemical environment of a sample, fluorescence lifetime imaging measures changes in the excited-state lifetime of fluorophores that are sensitive to their microenvironments. Here, we present a multimodal imaging platform combining broadband coherent anti-Stokes Raman scattering (BCARS) and two-photon fluorescence lifetime imaging (2p-FLIM) microscopy that can acquire biologically relevant Raman fingerprint spectra and fluorescence lifetime signals in vivo and simultaneously. The tremendous chemical information obtained from spatially co-registered BCARS and 2p-FLIM images allows us to characterize the subtle differences between sub-cellular compartments and verify the potential false-positive results generated by fluorescence imaging alone. This is demonstrated by directly comparing the BCARS, 2p-FLIM, and two-photon excitation fluorescence (TPEF) signals simultaneously obtained from the same dye-stained organelle in the live, intact C. elegans expressing a green fluorescent protein (GFP) marker. In this work, we introduce the BCARS/2p-FLIM/TPEF setup scheme, the image acquisition steps, data processing, and representative results showing that the cross-modality imaging method enables rigorous characterization and in vivo detection at sub-cellular resolution. This protocol provides a framework for simultaneous chemical and fluorescence lifetime imaging to improve the accuracy of biological interpretation in complex living systems.

Purpose: Biomaterial scaffolds capable of controlled release of bioactive molecules hold significant potential in tissue engineering, offering a promising avenue to enhance tissue regeneration. They provide localized and sustained delivery of biological cues to direct stem cell differentiation while creating a three-dimensional microenvironment that supports cell adhesion and growth. Methods: In this study, we utilized reverse micelle sugar glass nanoparticles (SGnPs), previously developed by our team, to encapsulate the chondrogenic growth factor TGFB1. This approach aimed to preserve the bioactivity of these molecules before their release. The TGFB1-SGnPs were directly incorporated into electrospun fibrous scaffolds, engineered specifically to ensure the sustained release of the growth factor during the culture of human bone marrow-derived mesenchymal stem/stromal cells (BMSCs). Results: TGFB1 was released in a sustained manner over 39 days from TGFB1-SGnP-incorporated fibrous scaffolds, made from poly (ε-caprolactone), poly (d-lactic acid) (PLA), and poly (lactic-co-glycolic acid). Among these formulations, the PLA-based scaffolds demonstrated the highest cumulative TGFB1 release over the study period. In vitro cell studies demonstrated that TGFB1-SGnP-PLA fibrous scaffolds supported the proliferation of BMSCs and enhanced chondrogenic differentiation. Transcript expression analysis of BMSCs seeded on TGFB1-SGnP-PLA fibrous scaffolds induced for chondrogenesis revealed an upregulation of chondrocyte-associated markers, including SOX9 , ACAN , COL2A1 , and COL1A1 . Conclusion: This study demonstrates the potential of using SGnPs to protect and deliver chondrogenic induction molecules from electrospun fibrous scaffolds in a sustained manner, promoting the chondrogenic differentiation of BMSCs in cartilage tissue engineering. Lay Summary. Researchers have developed advanced biomaterial scaffolds that release bioactive molecules to enhance tissue regeneration. These “smart scaffolds” provide a three-dimensional environment for cell growth and localized cues to support biological functions. Utilizing sugar glass nanoparticles (SGnPs) to encapsulate growth factors like TGFB1, electrospun fibrous scaffolds incorporating TGFB1-SGnPs were crafted to assess their effectiveness in supporting the activity of human bone marrow-derived mesenchymal stem/stromal cells (BMSCs). Results showed that TGFB1, particularly from TGFB1-SGnP-PLA scaffolds, significantly promoted BMSC proliferation and chondrogenic differentiation, as evidenced by increased markers associated with cartilage cells. This innovative approach demonstrates considerable potential for advancing cartilage tissue engineering and offers a new therapeutic strategy for conditions such as osteoarthritis, enhancing tissue repair and regeneration.