The Prospective Instruments MPX-series is a turnkey compact multimodal microscope enabling advanced multiphoton imaging without requiring a laboratory or optical bench. One of the key design features is the ultrafast fiber laser engine integrated into the free moving scan head, which is lightweight, rugged, and allows the ultimate imaging flexibility. A modular design concept allows the user to explore a wide variety of biological applications and measurements, without compromising performance, in any indoor working space. In contrast, standard setups are non-transportable, utility-demanding, and the complexity can be complicated, therefore degrading the value and adding significant initial, short- and long-term expenses. Every clinical and biological researcher should have access to high-quality reliable optical microscopy modalities without being hardware experts. In this article we outline the demand and design of the MPX-microscope and present multiphoton imaging results from experiments in various configurations. There are no existing instruments on the market that are portable and combine easily switchable modes in one composite industry-ready device for life science, clinical, and pharmacological research.

In this work, we investigated the labeling efficiency of hydrophilic (DAPI) and hydrophobic (Nile Red) fluorescent probes using uni- and bi-directional diffusion on porcine liver tissue sections on the order of a few hundred microns in thickness. Bi-directional diffusion significantly enhanced the penetration of probes. Although diffusion most accurately describes the labeling process, we found that after removal of the labeling molecules residual probe molecules in the tissue sections continued to be transported deeper into the tissue resulting in additional tissue constituents being labeled. Our study shows that bi-directional labeling can significantly enhance the labeling of thick tissues for applications in which structural information in three dimensions is needed.

Metabolism and mitochondrial dysfunction are known to be involved in many different disease states. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, fluorescence intensity has commonly been used as an indicator of the NADH concentration in cells and tissues. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. Since NADH binding changes with metabolic state, this approach presents a new opportunity to track the cellular metabolic state.

Ligament healing of a grade III injury (i.e., a complete tear) involves a multifaceted chain of events that forms a neoligament, which is more scar-like in character than the native tissue. The remodeling process may last months or even years with the injured ligament never fully recovering pre-injury mechanical properties. With tissue engineering and regenerative medicine, understanding the normal healing process in ligament and quantifying it provide a basis to create and assess innovative treatments. Ligament fibroblasts produce a number of extracellular matrix (ECM) components, including collagen types I and III, decorin and fibromodulin. Using a combination of advanced histology, molecular biology, and nonlinear optical imaging approaches, the early ECM events during ligament healing have been better characterized and defined. First, the dynamic changes in ECM factors after injury are shown. Second, the factors associated with creeping substitution are identified. Finally, a method to quantify collagen organization is developed and used. Each ECM factor described herein as well as the temporal quantification of fiber organization helps elucidate the complexity of ligament healing.

Detection and tracking of stem cell state are difficult due to insufficient means for rapidly screening cell state in a noninvasive manner. This challenge is compounded when stem cells are cultured in aggregates or three-dimensional (3D) constructs because living cells in this form are difficult to analyze without disrupting cellular contacts. Multiphoton laser scanning microscopy is uniquely suited to analyze 3D structures due to the broad tunability of excitation sources, deep sectioning capacity, and minimal phototoxicity but is throughput limited. A novel multiphoton fluorescence excitation flow cytometry (MPFC) instrument could be used to accurately probe cells in the interior of multicell aggregates or tissue constructs in an enhanced-throughput manner and measure corresponding fluorescent properties. By exciting endogenous fluorophores as intrinsic biomarkers or exciting extrinsic reporter molecules, the properties of cells in aggregates can be understood while the viable cellular aggregates are maintained. Here we introduce a first generation MPFC system and show appropriate speed and accuracy of image capture and measured fluorescence intensity, including intrinsic fluorescence intensity. Thus, this novel instrument enables rapid characterization of stem cells and corresponding aggregates in a noninvasive manner and could dramatically transform how stem cells are studied in the laboratory and utilized in the clinic.

We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage—in the same embryo—thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.