RESUMO
Multiphoton fluorescence microscopy (MPFM) has been a game-changer for optical imaging, particularly for studying biological tissues deep within living organisms. MPFM overcomes the strong scattering of light in heterogeneous tissue by utilizing nonlinear excitation that confines fluorescence emission mostly to the microscope focal volume. This enables high-resolution imaging deep within intact tissue and has opened new avenues for structural and functional studies. MPFM has found widespread applications and has led to numerous scientific discoveries and insights into complex biological processes. Today, MPFM is an indispensable tool in many research communities. Its versatility and effectiveness make it a go-to technique for researchers investigating biological phenomena at the cellular and subcellular levels in their native environments. In this Review, the principles, implementations, capabilities, and limitations of MPFM are presented. Three application areas of MPFM, neuroscience, cancer biology, and immunology, are reviewed in detail and serve as examples for applying MPFM to biological research.
Assuntos
Microscopia de Fluorescência por Excitação Multifotônica , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Humanos , Animais , Neoplasias/diagnóstico por imagem , Neoplasias/patologiaRESUMO
Recent progress in fluorescence imaging allows neuroscientists to observe the dynamics of thousands of individual neurons, identified genetically or by their connectivity, across multiple brain areas and for extended durations in awake behaving mammals. We discuss advances in fluorescent indicators of neural activity, viral and genetic methods to express these indicators, chronic animal preparations for long-term imaging studies, and microscopes to monitor and manipulate the activity of large neural ensembles. Ca2+ imaging studies of neural activity can track brain area interactions and distributed information processing at cellular resolution. Across smaller spatial scales, high-speed voltage imaging reveals the distinctive spiking patterns and coding properties of targeted neuron types. Collectively, these innovations will propel studies of brain function and dovetail with ongoing neuroscience initiatives to identify new neuron types and develop widely applicable, non-human primate models. The optical toolkit's growing sophistication also suggests that "brain observatory" facilities would be useful open resources for future brain-imaging studies.
Assuntos
Mapeamento Encefálico/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neocórtex/diagnóstico por imagem , Neocórtex/metabolismo , Neurônios/metabolismo , Imagem Óptica/métodos , Animais , Cálcio/metabolismo , Camundongos , Modelos Animais , Neurociências/métodosRESUMO
Neuronal cell types are the nodes of neural circuits that determine the flow of information within the brain. Neuronal morphology, especially the shape of the axonal arbor, provides an essential descriptor of cell type and reveals how individual neurons route their output across the brain. Despite the importance of morphology, few projection neurons in the mouse brain have been reconstructed in their entirety. Here we present a robust and efficient platform for imaging and reconstructing complete neuronal morphologies, including axonal arbors that span substantial portions of the brain. We used this platform to reconstruct more than 1,000 projection neurons in the motor cortex, thalamus, subiculum, and hypothalamus. Together, the reconstructed neurons constitute more than 85 meters of axonal length and are available in a searchable online database. Axonal shapes revealed previously unknown subtypes of projection neurons and suggest organizational principles of long-range connectivity.
Assuntos
Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Neuritos/fisiologia , Tratos Piramidais/fisiologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Software , TransfecçãoRESUMO
Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.
Assuntos
Encéfalo/fisiologia , Proteínas Ativadoras de GTPase/genética , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Optogenética/métodos , Ritmo Teta , Vigília , Potenciais de Ação , Animais , Encéfalo/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Ratos , Ratos Sprague-Dawley , CorridaRESUMO
Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm2 field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond.
Assuntos
Corantes , Microscopia de Fluorescência por Excitação Multifotônica , Animais , Microscopia de Fluorescência por Excitação Multifotônica/métodosRESUMO
Volumetric imaging of synaptic transmission in vivo requires high spatial and high temporal resolution. Shaping the wavefront of two-photon fluorescence excitation light, we developed Bessel-droplet foci for high-contrast and high-resolution volumetric imaging of synapses. Applying our method to imaging glutamate release, we demonstrated high-throughput mapping of excitatory inputs at >1,000 synapses per volume and >500 dendritic spines per neuron in vivo and unveiled previously unseen features of functional synaptic organization in the mouse primary visual cortex.
Assuntos
Sinapses , Transmissão Sináptica , Animais , Transmissão Sináptica/fisiologia , Camundongos , Sinapses/fisiologia , Ácido Glutâmico/metabolismo , Córtex Visual/fisiologia , Córtex Visual/citologia , Espinhas Dendríticas/fisiologia , Neurônios/fisiologia , Córtex Visual Primário/fisiologia , Córtex Visual Primário/diagnóstico por imagem , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica/métodosRESUMO
In deep-tissue multiphoton microscopy, diffusion and scattering of fluorescent photons, rather than ballistic emanation from the focal point, have been a confounding factor. Here we report on a 2.17-g miniature three-photon microscope (m3PM) with a configuration that maximizes fluorescence collection when imaging in highly scattering regimes. We demonstrate its capability by imaging calcium activity throughout the entire cortex and dorsal hippocampal CA1, up to 1.2 mm depth, at a safe laser power. It also enables the detection of sensorimotor behavior-correlated activities of layer 6 neurons in the posterior parietal cortex in freely moving mice during single-pellet reaching tasks. Thus, m3PM-empowered imaging allows the study of neural mechanisms in deep cortex and subcortical structures, like the dorsal hippocampus and dorsal striatum, in freely behaving animals.
Assuntos
Hipocampo , Microscopia de Fluorescência por Excitação Multifotônica , Camundongos , Animais , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Córtex Cerebral , Corantes , FótonsRESUMO
Helicobacter pylori (H. pylori) is a bacterial pathogen that exclusively colonizes the human gastric mucosa and can cause persistent infection. In this process, H. pylori employs various strategies to avoid recognition by the human immune system. These range from passive defense strategies (e.g., altered LPS or flagellin structures) that prevent recognition by pattern recognition receptors (PRRs) to more active approaches, such as inhibition of IL-2 secretion and proliferation of T cells via VacA. Despite the growing evidence that H. pylori actively manipulates the human immune system for its own benefit, the direct interaction of H. pylori with immune cells in situ is poorly studied. Here, we present a novel intravital imaging model of the murine stomach gastric mucosa and show for the first time the in situ recruitment of neutrophils during infection and a direct H. pylori-macrophage interaction. For this purpose, we applied multiphoton intravital microscopy adapted with live drift correction software (VivoFollow) on LysM-eGFP and CX3CR1-eGFP reporter mice strains in which specific subsets of leukocytes are fluorescently labeled. Multiphoton microscopy is proving to be an excellent tool for characterizing interactions between immune cells and pathogens in vivo.
Assuntos
Infecções por Helicobacter , Helicobacter pylori , Microscopia Intravital , Macrófagos , Microscopia de Fluorescência por Excitação Multifotônica , Neutrófilos , Animais , Helicobacter pylori/imunologia , Camundongos , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Microscopia Intravital/métodos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Macrófagos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Mucosa Gástrica/microbiologia , Mucosa Gástrica/imunologia , Camundongos Endogâmicos C57BL , Estômago/microbiologia , Estômago/imunologiaRESUMO
The germinal center (GC) reaction produces high-affinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors. The mechanism by which B cells are selected remains unclear, as does the role of the two anatomically defined areas of the GC, light zone (LZ) and dark zone (DZ). We combined a transgenic photoactivatable fluorescent protein tracer with multiphoton laser-scanning microscopy and flow cytometry to examine anatomically defined LZ and DZ B cells and GC selection. We find that B cell division is restricted to the DZ, with a net vector of B cell movement from the DZ to the LZ. The decision to return to the DZ and undergo clonal expansion is controlled by T helper cells in the GC LZ, which discern between LZ B cells based on the amount of antigen captured and presented. Thus, T cell help, and not direct competition for antigen, is the limiting factor in GC selection.
Assuntos
Centro Germinativo/citologia , Centro Germinativo/imunologia , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Antígenos/imunologia , Linfócitos B/citologia , Feminino , Humanos , Imunidade Humoral , Linfonodos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Baço/citologia , Linfócitos T/citologiaRESUMO
Biocompatible fluorescent agents are key contributors to the theranostic paradigm by enabling real-time in vivo imaging. This study explores the optical properties of phenylenediamine carbon dots (CDs) and demonstrates their potential for fluorescence imaging in cells and brain blood vessels. The nonlinear absorption cross-section of the CDs was measured and achieved values near 50 Goeppert-Mayer (GM) units with efficient excitation in the 775-895 nm spectral range. Mesoporous vaterite nanoparticles were loaded with CDs to examine the possibility of a biocompatible imaging platform. Efficient one- and two-photon imaging of the CD-vaterite composites uptaken by diverse cells was demonstrated. For an in vivo scenario, CD-vaterite composites were injected into the bloodstream of a mouse, and their flow was monitored within the blood vessels of the brain through a cranial window. These results show the potential of the platform for high-brightness biocompatible imaging with the potential for both sensing and simultaneous drug delivery.
Assuntos
Encéfalo , Carbono , Pontos Quânticos , Animais , Carbono/química , Camundongos , Encéfalo/diagnóstico por imagem , Pontos Quânticos/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Carbonato de Cálcio/química , Humanos , Nanopartículas/química , Corantes Fluorescentes/químicaRESUMO
Three-photon fluorescence microscopy (3PFM) is a promising brain research tool with submicrometer spatial resolution and high imaging depth. However, only limited materials have been developed for 3PFM owing to the rigorous requirement of the three-photon fluorescence (3PF) process. Herein, under the guidance of a band gap engineering strategy, CdTe/CdSe/ZnS quantum dots (QDs) emitting in the near-infrared window are designed for constructing 3PF probes. The formation of type II structure significantly increased the three-photon absorption cross section of QDs and caused the delocalization of electron-hole wave functions. The time-resolved transient absorption spectroscopy confirmed that the decay of biexcitons was significantly suppressed due to the appropriate band gap alignment, which further enhanced the 3PF efficiency of QDs. By utilizing QD-based 3PF probes, high-resolution 3PFM imaging of cerebral vasculature was realized excited by a 1600 nm femtosecond laser, indicating the possibility of deep brain imaging with these 3PF probes.
Assuntos
Encéfalo , Pontos Quânticos , Pontos Quânticos/química , Encéfalo/diagnóstico por imagem , Fótons , Animais , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Compostos de Cádmio/química , Sulfetos/química , Camundongos , Compostos de Zinco/química , Telúrio/química , Compostos de Selênio/química , HumanosRESUMO
Meningiomas rank among the most common intracranial tumors, and surgery stands as the primary treatment modality for meningiomas. The precise subtyping and diagnosis of meningiomas, both before and during surgery, play a pivotal role in enabling neurosurgeons choose the optimal surgical program. In this study, we utilized multiphoton microscopy (MPM) based on 2-photon excited fluorescence and second-harmonic generation to identify 5 common meningioma subtypes. The morphological features of these subtypes were depicted using the MPM multichannel mode. Additionally, we developed 2 distinct programs to quantify collagen content and blood vessel density. Furthermore, the lambda mode of the MPM characterized architectural and spectral features, from which 3 quantitative indicators were extracted. Moreover, we employed machine learning to differentiate meningioma subtypes automatically, achieving high classification accuracy. These findings demonstrate the potential of MPM as a noninvasive diagnostic tool for meningioma subtyping and diagnosis, offering improved accuracy and resolution compared with traditional methods.
Assuntos
Neoplasias Meníngeas , Meningioma , Humanos , Meningioma/diagnóstico por imagem , Colágeno , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neoplasias Meníngeas/diagnóstico por imagem , ComputadoresRESUMO
Currently, in situ monitoring of the adenosine triphosphate (ATP) level in lysosomes is critical to understand their involvement in various biological processes, but it remains difficult due to the interferences of limited targeting and low resolution of fluorescent probes. Herein, we report a classic Mn(II) probe (FX2-MnCl2) with near-infrared (NIR) nonlinear (NLO) properties, accompanied by three-four photon transition and fivefold fluorescence enhancement in the presence of ATP. FX2-MnCl2 combines with ATP through dual recognition sites of diethoxy and manganese ions to reflect slightly fluorescence lifetime change. Through the synergy of multiphoton fluorescence imaging (MP-FI) and multiphoton fluorescence lifetime imaging microscopy (MP-FLIM), it is further demonstrated that FX2-MnCl2 displays lysosome-specific targeting behavior, which can monitor lysosome-related ATP migration under NIR laser light. This work provides a novel multiphoton transformation fluorescence complex, which might be a potential candidate as a simple and straightforward biomarker of lysosome ATP in vitro for clinical diagnosis.
Assuntos
Corantes Fluorescentes , Lisossomos , Microscopia de Fluorescência/métodos , Imagem Óptica , Fótons , Microscopia de Fluorescência por Excitação Multifotônica/métodosRESUMO
Lipid droplets (LDs) store energy and supply fatty acids and cholesterol. LDs are a hallmark of chronic nonalcoholic fatty liver disease (NAFLD). Recently, studies have focused on the role of hepatic macrophages in NAFLD. Green fluorescent protein (GFP) is used for labeling the characteristic targets in bioimaging analysis. Cx3cr1-GFP mice are widely used in studying the liver macrophages such as the NAFLD model. Here, we have developed a tool for two-photon microscopic observation to study the interactions between LDs labeled with LD2 and liver capsule macrophages labeled with GFP in vivo. LD2, a small-molecule two-photon excitation fluorescent probe for LDs, exhibits deep-red (700 nm) fluorescence upon excitation at 880 nm, high cell staining ability and photostability, and low cytotoxicity. This probe can clearly observe LDs through two-photon microscopy (TPM) and enables the simultaneous imaging of GFP+ liver capsule macrophages (LCMs) in vivo in the liver capsule of Cx3cr1-GFP mice. In the NAFLD mouse model, Cx3cr1+ LCMs and LDs increased with the progress of fatty liver disease, and spatiotemporal changes in LCMs were observed through intravital 3D TPM images. LD2 will aid in studying the interactions and immunological roles of hepatic macrophages and LDs to better understand NAFLD.
Assuntos
Gotículas Lipídicas , Fígado , Macrófagos , Animais , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Camundongos , Macrófagos/metabolismo , Fígado/diagnóstico por imagem , Fígado/metabolismo , Fígado/patologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/química , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Corantes Fluorescentes/química , Camundongos Endogâmicos C57BLRESUMO
Increasing evidence indicates the role of mitochondrial and vascular dysfunction in aging and aging-associated pathologies; however, the exact mechanisms and chronological processes remain enigmatic. High-energy demand organs, such as the brain, depend on the health of their mitochondria and vasculature for the maintenance of normal functions, therefore representing vulnerable targets for aging. This methodology article describes an analysis pipeline for three-dimensional (3-D) mitochondria-associated signal geometry of two-photon image stacks of brain vasculature. The analysis methods allow the quantification of mitochondria-associated signals obtained in real time in their physiological environment. In addition, signal geometry results will allow the extrapolation of fission and fusion events under normal conditions, during aging, or in the presence of different pathological conditions, therefore contributing to our understanding of the role mitochondria play in a variety of aging-associated diseases with vascular etiology.NEW & NOTEWORTHY Analysis pipeline for 3-D mitochondria-associated signal geometry of two-photon image stacks of brain vasculature.
Assuntos
Imageamento Tridimensional , Mitocôndrias , Mitocôndrias/metabolismo , Animais , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Células Endoteliais/metabolismo , Dinâmica Mitocondrial , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Camundongos , Envelhecimento/metabolismoRESUMO
The pre-clinical validation of cell therapies requires monitoring the biodistribution of transplanted cells in tissues of host organisms. Real-time detection of these cells in the circulatory system and identification of their aggregation state is a crucial piece of information, but necessitates deep penetration and fast imaging with high selectivity, subcellular resolution, and high throughput. In this study, multiphoton-based in-flow detection of human stem cells in whole, unfiltered blood is demonstrated in a microfluidic channel. The approach relies on a multiphoton microscope with diffractive scanning in the direction perpendicular to the flow via a rapidly wavelength-swept laser. Stem cells are labeled with metal oxide harmonic nanoparticles. Thanks to their strong and quasi-instantaneous second harmonic generation (SHG), an imaging rate in excess of 10 000 frames per second is achieved with pixel dwell times of 1 ns, a duration shorter than typical fluorescence lifetimes yet compatible with SHG. Through automated cell identification and segmentation, morphological features of each individual detected event are extracted and cell aggregates are distinguished from isolated cells. This combination of high-speed multiphoton microscopy and high-sensitivity SHG nanoparticle labeling in turbid media promises the detection of rare cells in the bloodstream for assessing novel cell-based therapies.
Assuntos
Células-Tronco , Humanos , Células-Tronco/citologia , Microscopia de Fluorescência por Excitação Multifotônica/métodosRESUMO
We have developed a miniature two-photon microscope equipped with an axial scanning mechanism and a long-working-distance miniature objective to enable multi-plane imaging over a volume of 420 × 420 × 180 µm3 at a lateral resolution of ~1 µm. Together with the detachable design that permits long-term recurring imaging, our miniature two-photon microscope can help decipher neuronal mechanisms in freely behaving animals.
Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/fisiologia , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Miniaturização/métodos , Neuroimagem/métodos , Animais , Comportamento Animal , Encéfalo/citologia , Técnicas Citológicas , Locomoção , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Progress in many scientific disciplines is hindered by the presence of independent noise. Technologies for measuring neural activity (calcium imaging, extracellular electrophysiology and functional magnetic resonance imaging (fMRI)) operate in domains in which independent noise (shot noise and/or thermal noise) can overwhelm physiological signals. Here, we introduce DeepInterpolation, a general-purpose denoising algorithm that trains a spatiotemporal nonlinear interpolation model using only raw noisy samples. Applying DeepInterpolation to two-photon calcium imaging data yielded up to six times more neuronal segments than those computed from raw data with a 15-fold increase in the single-pixel signal-to-noise ratio (SNR), uncovering single-trial network dynamics that were previously obscured by noise. Extracellular electrophysiology recordings processed with DeepInterpolation yielded 25% more high-quality spiking units than those computed from raw data, while DeepInterpolation produced a 1.6-fold increase in the SNR of individual voxels in fMRI datasets. Denoising was attained without sacrificing spatial or temporal resolution and without access to ground truth training data. We anticipate that DeepInterpolation will provide similar benefits in other domains in which independent noise contaminates spatiotemporally structured datasets.
Assuntos
Potenciais de Ação , Algoritmos , Cálcio/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Neuroimagem/métodos , Neurônios/fisiologia , Razão Sinal-Ruído , Animais , Humanos , Imageamento por Ressonância Magnética/métodos , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Imagem Multimodal/métodos , Neurônios/citologiaRESUMO
Multiphoton microscopy has become a powerful tool with which to visualize the morphology and function of neural cells and circuits in the intact mammalian brain. However, tissue scattering, optical aberrations and motion artifacts degrade the imaging performance at depth. Here we describe a minimally invasive intravital imaging methodology based on three-photon excitation, indirect adaptive optics (AO) and active electrocardiogram gating to advance deep-tissue imaging. Our modal-based, sensorless AO approach is robust to low signal-to-noise ratios as commonly encountered in deep scattering tissues such as the mouse brain, and permits AO correction over large axial fields of view. We demonstrate near-diffraction-limited imaging of deep cortical spines and (sub)cortical dendrites up to a depth of 1.4 mm (the edge of the mouse CA1 hippocampus). In addition, we show applications to deep-layer calcium imaging of astrocytes, including fibrous astrocytes that reside in the highly scattering corpus callosum.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neuroimagem/métodos , Animais , Astrócitos/metabolismo , Sinalização do Cálcio , Feminino , Proteínas de Fluorescência Verde , Masculino , Camundongos , Camundongos Transgênicos , Software , Antígenos Thy-1RESUMO
Over the last half century, the autofluorescence of the metabolic cofactors NADH (reduced nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) has been quantified in a variety of cell types and disease states. With the spread of nonlinear optical microscopy techniques in biomedical research, NADH and FAD imaging has offered an attractive solution to noninvasively monitor cell and tissue status and elucidate dynamic changes in cell or tissue metabolism. Various tools and methods to measure the temporal, spectral, and spatial properties of NADH and FAD autofluorescence have been developed. Specifically, an optical redox ratio of cofactor fluorescence intensities and NADH fluorescence lifetime parameters have been used in numerous applications, but significant work remains to mature this technology for understanding dynamic changes in metabolism. This article describes the current understanding of our optical sensitivity to different metabolic pathways and highlights current challenges in the field. Recent progress in addressing these challenges and acquiring more quantitative information in faster and more metabolically relevant formats is also discussed.