Deep neural networks have been used to map multi-modal, multi-photon microscopy measurements of a label-free tissue sample to its corresponding histologically stained brightfield microscope colour image. It is shown that the extra structural and functional contrasts provided by using two source modes, namely two-photon excitation microscopy and fluorescence lifetime imaging, result in a more faithful reconstruction of the target haematoxylin and eosin stained mode. This modal mapping procedure can aid histopathologists, since it provides access to unobserved imaging modalities, and translates the high-dimensional numerical data generated by multi-modal, multi-photon microscopy into traditionally accepted visual forms. Furthermore, by combining the strengths of traditional chemical staining and modern multi-photon microscopy techniques, modal mapping enables label-free, non-invasive studies of in vivo tissue samples or intravital microscopic imaging inside living animals. The results show that modal co-registration and the inclusion of spatial variations increase the visual accuracy of the mapped results.

Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of autofluorescent metabolic coenzymes has been widely used to investigate energetic perturbations in living cells and tissues in a label-free manner with subcellular resolution. While the currently used state-of-the-art instruments are highly sensitive to local molecular changes associated with these metabolic processes, they are inherently slow and limit the study of dynamic metabolic environments. Here, a sustained video-rate 2P-FLIM imaging system is demonstrated for time-lapse lifetime imaging of reduced nicotinamide adenine dinucleotide, an autofluorescent metabolic coenzyme involved in both aerobic and anaerobic processes. This system is sufficiently sensitive to differences in metabolic activity between aggressive and nonaggressive cancer cell lines and is demonstrated for both wide field-of-view autofluorescence imaging as well as sustained video-rate image acquisition of metabolic dynamics following induction of apoptosis. The unique capabilities of this imaging platform provide a powerful technological advance to further explore rapid metabolic dynamics in living cells.

In vivo imaging of human retinal pigment epithelial (RPE) cells has been demonstrated through multiple adaptive optics (AO)-based modalities. However, whether consistent and complete information regarding the cellular structure of the RPE mosaic is obtained across these modalities remains uncertain due to limited comparisons performed in the same eye. Here, an imaging platform combining multimodal AO-scanning light ophthalmoscopy (AO-SLO) with AO-optical coherence tomography (AO-OCT) is developed to make a side-by-side comparison of the same RPE cells imaged across four modalities: AO-darkfield, AO-enhanced indocyanine green (AO-ICG), AO-infrared autofluorescence (AO-IRAF), and AO-OCT. Co-registered images were acquired in five subjects, including one patient with choroideremia. Multimodal imaging provided multiple perspectives of the RPE mosaic that were used to explore variations in RPE cell contrast in a subject-, location-, and even cell-dependent manner. Estimated cell-to-cell spacing and density were found to be consistent both across modalities and with normative data. Multimodal images from a patient with choroideremia illustrate the benefit of using multiple modalities to infer the cellular structure of the RPE mosaic in an affected eye, in which disruptions to the RPE mosaic may locally alter the signal strength, visibility of individual RPE cells, or even source of contrast in unpredictable ways.

Adaptive optics scanning light ophthalmoscopy (AOSLO) allows non-invasive visualization of the living human eye at the microscopic scale; but even with correction of the ocular wavefront aberrations over a large pupil, the smallest cells in the photoreceptor mosaic cannot always be resolved. Here, we synergistically combine annular pupil illumination with sub-Airy disk confocal detection to demonstrate a 33% improvement in transverse resolution (from 2.36 to 1.58 µm) and a 13% axial resolution enhancement (from 37 to 32 µm), an important step towards the study of the complete photoreceptor mosaic in heath and disease. Interestingly, annular pupil illumination also enhanced the visualization of the photoreceptor mosaic in non-confocal detection schemes such as split detection AOSLO, providing a strategy for enhanced multimodal imaging of the cone and rod photoreceptor mosaic.

Programmed cell death, or apoptosis, is an essential process in development and homeostasis, and disruptions in associated pathways are responsible for a wide variety of diseases such as cancer, developmental abnormalities, and Alzheimer’s disease. On the other hand, cell death, in many cases, is the desired outcome of therapeutic treatments targeting diseases such as cancer. Recently, metabolic imaging based on two-photon fluorescence microscopy has been developed and shown to be highly sensitive to certain cell death processes, most notably apoptosis, thus having the potential as an advanced label-free screening tool. However, the typically low acquisition rates of this imaging technique have resulted in a limited throughput approach, allowing only a small population of cells to be tracked at well-separated time points. To address this limitation, a high-speed two-photon fluorescence lifetime imaging microscopy (2P-FLIM) platform capable of video-rate imaging is applied to study and further characterize the metabolic dynamics associated with cell death. Building upon previous work demonstrating the capabilities of this system, this microscope is utilized to study rapid metabolic changes during cell death induction, such as dose-dependency of metabolic response, response in invasive vs. noninvasive cancer cells, and response in an apoptosis-resistant cell line, which is further shown to undergo autophagy in response to toxic stimuli. Results from these experiments show that the early apoptosis-related metabolic dynamics are strongly correlated with important cellular parameters including responsiveness to apoptosis-inducing stimuli. The high speed and sensitivity of the presented imaging approach enables new investigations into this highly dynamic and complex process.

Two-photon fluorescence lifetime imaging microscopy (FLIM) is a widely used technique in biomedical optical imaging. Presently, many two-photon time-domain FLIM setups are limited by long acquisition and postprocessing times that decrease data throughput and inhibit the ability to image fast sub-second processes. Here, we present a versatile two-photon FLIM setup capable of video-rate (up to 25 fps) imaging with graphics processing unit (GPU)-accelerated pixelwise phasor analysis displayed and saved simultaneously with acquisition. The system uses an analog output photomultiplier tube in conjunction with 12-bit digitization at 3.2 GHz to overcome the limited maximum acceptable photon rate associated with the photon counting electronics in many FLIM systems. This allows for higher throughput FLIM acquisition and analysis, and additionally enables the user to assess sample fluorescence lifetime in real-time. We further explore the capabilities of the system to examine the kinetics of Rhodamine B uptake by human breast cancer cells and characterize the effect of pixel dwell time on the reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) autofluorescence lifetime estimation accuracy.