Video-rate two-photon microendoscopy using second harmonic resonance fiber scanning.

We present a 2.5-mm-diameter resonant fiber scanning two-photon microendoscope with a 30-mm long forward-viewing rigid probe tip that enables video-rate imaging (20 Hz frame rate) suitable for hand-held imaging of tissues without motion artifacts. Higher-order harmonic oscillation scanning techniques are developed to significantly increase the frame rate compared to prior published fiber scanning microendoscopy designs while maintaining the field-of-view (∼125 µm), the optical resolution (1.

Fractionated photoimmunotherapy stimulates an anti-tumour immune response: an integrated mathematical and in vitro study.

Background: Advanced epithelial ovarian cancer (EOC) has high recurrence rates due to disseminated initial disease presentation. Cytotoxic phototherapies, such as photodynamic therapy (PDT) and photoimmunotherapy (PIT, cell-targeted PDT), have the potential to treat disseminated malignancies due to safe intraperitoneal delivery.

Methods: We use in vitro measurements of EOC tumour cell and T cell responses to chemotherapy, PDT, and epidermal growth factor receptor targeted PIT as inputs to a mathematical model of non-linear tumour and immune effector cell interaction.

High-speed forward-viewing optical coherence tomography probe based on Lissajous sampling and sparse reconstruction.

We present a novel endoscopy probe using optical coherence tomography (OCT) that combines sparse Lissajous scanning and compressed sensing (CS) for faster data collection. This compact probe is only 4 mm in diameter and achieves a large field of view (FOV) of 2.25 mm and a 10 mm working distance.

Red light-activated depletion of drug-refractory glioblastoma stem cells and chemosensitization of an acquired-resistant mesenchymal phenotype.

Glioblastoma stem cells (GSCs) are potent tumor initiators resistant to radiochemotherapy, and this subpopulation is hypothesized to re-populate the tumor milieu due to selection following conventional therapies. Here, we show that 5-aminolevulinic acid (ALA) treatment-a pro-fluorophore used for fluorescence-guided cancer surgery-leads to elevated levels of fluorophore conversion in patient-derived GSC cultures, and subsequent red light-activation induces apoptosis in both intrinsically temozolomide chemotherapy-sensitive and -resistant GSC phenotypes. Red light irradiation of ALA-treated cultures also exhibits the ability to target mesenchymal GSCs (Mes-GSCs) with induced temozolomide resistance.

Methods for assessing and removing non-specific photoimmunotherapy damage in patient-derived tumor cell culture models.

Tumor-targeted, activatable photoimmunotherapy (taPIT) has been shown to selectively destroy tumor in a metastatic mouse model. However, the photoimmunoconjugate (PIC) used for taPIT includes a small fraction of non-covalently associated (free) benzoporphyrin derivative (BPD), which leads to non-specific killing in vitro. Here, we report a new treatment protocol for patient-derived primary tumor cell cultures ultrasensitive to BPD photodynamic therapy (BPD-PDT).

Site-Specific Conjugation of Native Antibody: Transglutaminase-Mediated Modification of a Conserved Glutamine While Maintaining the Primary Sequence and Core Fc Glycan via Trimming with an Endoglycosidase.

A versatile chemo-enzymatic tool to site-specifically modify native (nonengineered) antibodies is using transglutaminase (TGase, E.C. 2.

Multi-objective optimization of custom compound prism arrays for multiplexed optical imaging.

Compound prism arrays are a powerful, yet underutilized, solution for producing high transmission and customized chromatic dispersion profiles over broad bandwidths, the quality of which is unobtainable with commercially available prisms or diffraction gratings. However, the computational complexity associated with designing these prism arrays presents a barrier to the widespread adoption of their use. Here we introduce customizable prism designer software that facilitates high-speed optimization of compound arrays guided by target specifications for chromatic dispersion linearity and detector geometry.

An open-source LED array illumination system for automated multiwell plate cell culture photodynamic therapy experiments.

Photodynamic therapy (PDT) research would benefit from an automated, low-cost, and easy-to-use cell culture light treatment setup capable of illuminating multiple well replicates within standard multiwell plate formats. We present an LED-array suitable for performing high-throughput cell culture PDT experiments. The setup features a water-cooling loop to keep the LED-array temperature nearly constant, thus stabilizing the output power and spectrum.

Denoising multiplexed microscopy images in n-dimensional spectral space.

Hyperspectral fluorescence microscopy images of biological specimens frequently contain multiple observations of a sparse set of spectral features spread in space with varying intensity. Here, we introduce a spectral vector denoising algorithm that filters out noise without sacrificing spatial information by leveraging redundant observations of spectral signatures. The algorithm applies an n-dimensional Chebyshev or Fourier transform to cluster pixels based on spectral similarity independent of pixel intensity or location, and a denoising convolution filter is then applied in this spectral space.

Rethinking the immunotherapy numbers game.

Immunotherapies are a major breakthrough in oncology, yielding unprecedented response rates for some cancers. Especially in combination with conventional treatments or targeted agents, immunotherapeutics offer invaluable tools to improve outcomes for many patients. However, why not all patients have a favorable response remains unclear.

Photodynamic Treatments for Disseminated Cancer Metastases Using Fiber-Optic Technologies.

Tumor-targeted and -activatable photosensitizer delivery platforms are creating new opportunities to develop photodynamic therapy (PDT) of metastatic disease. This is possible by confining the activity of the photosensitizing chemical (i.e.

Two-photon peak molecular brightness spectra reveal long-wavelength enhancements of multiplexed imaging depth and photostability.

The broad use of two-photon microscopy has been enabled in part by Ti:Sapphire femtosecond lasers, which offer a wavelength-tunable source of pulsed excitation. Action spectra have thus been primarily reported for the tunable range of Ti:Sapphire lasers (∼700-1000 nm). However, longer wavelengths offer deeper imaging in tissue via reduced scattering and spectral dips in water absorption, and new generations of pulsed lasers offer wider tunable ranges.

High-power light-emitting diode array design and assembly for practical photodynamic therapy research.

Significance: Commercial lasers, lamps, and light-emitting diode (LED) light sources have stimulated the clinical translation of photodynamic therapy (PDT). Yet, the continued exploration of new photosensitizers (PSs) for PDT often requires separate activation wavelengths for each agent being investigated. Customized light sources for such research frequently come at significant financial or technical cost, especially when compounded over many agents and wavelengths.

Site-specific Bioconjugation and Convergent Click Chemistry Enhances Antibody-Chromophore Conjugate Binding Efficiency.

Photosensitizer (PS)-antibody conjugates (photoimmunoconjugates, PICs) enable cancer cell-targeted photodynamic therapy (PDT). Nonspecific chemical bioconjugation is widely used to synthesize PICs but gives rise to several shortcomings. The conjugates are heterogeneous, and the process is not easily reproducible.

Multichannel correlation improves the noise tolerance of real-time hyperspectral microimage mosaicking.

Live-subject microscopies, including microendoscopy and other related technologies, offer promise for basic biology research as well as the optical biopsy of disease in the clinic. However, cellular resolution generally comes with the trade-off of a microscopic field-of-view. Microimage mosaicking enables stitching many small scenes together to aid visualization, quantitative interpretation, and mapping of microscale features, for example, to guide surgical intervention.

Low-cost Custom Fabrication and Mode-locked Operation of an All-normal-dispersion Femtosecond Fiber Laser for Multiphoton Microscopy.

A protocol is presented to build a custom low-cost yet high-performance femtosecond (fs) fiber laser. This all-normal-dispersion (ANDi) ytterbium-doped fiber laser is built completely using commercially available parts, including $8,000 in fiber optic and pump laser components, plus $4,800 in standard optical components and extra-cavity accessories. Researchers new to fiber optic device fabrication may also consider investing in basic fiber splicing and laser pulse characterization equipment (~$63,000).

Cancer Cell-targeted and Activatable Photoimmunotherapy Spares T Cells in a 3D Coculture Model.

Photodynamic therapy (PDT) is an established therapeutic modality that uses nonionizing near-infrared light to activate photocytotoxicity of endogenous or exogenous photosensitizers (PSs). An ongoing avenue of cancer research involves leveraging PDT to stimulate antitumor immune responses; however, these effects appear to be best elicited in low-dose regimens that do not provide significant tumor reduction using conventional, nonspecific PSs. The loss of immune enhancement at higher PDT doses may arise in part from indiscriminate damage to local immune cell populations, including tumor-infiltrating T cells.

Illuminating the Numbers: Integrating Mathematical Models to Optimize Photomedicine Dosimetry and Combination Therapies.

Cancer photomedicine offers unique mechanisms for inducing local tumor damage with the potential to stimulate local and systemic anti-tumor immunity. Optically-active nanomedicine offers these features as well as spatiotemporal control of tumor-focused drug release to realize synergistic combination therapies. Achieving quantitative dosimetry is a major challenge, and dosimetry is fundamental to photomedicine for personalizing and tailoring therapeutic regimens to specific patients and anatomical locations.

Custom fabrication and mode-locked operation of a femtosecond fiber laser for multiphoton microscopy.

Solid-state femtosecond lasers have stimulated the broad adoption of multiphoton microscopy in the modern laboratory. However, these devices remain costly. Fiber lasers offer promise as a means to inexpensively produce ultrashort pulses of light suitable for nonlinear microscopy in compact, robust and portable devices.

3D Culture Models of Malignant Mesothelioma Reveal a Powerful Interplay Between Photodynamic Therapy and Kinase Suppression Offering Hope to Reduce Tumor Recurrence.

In this issue, Cramer et al. introduce 3D culture models of metastatic mesothelioma to investigate basic cancer biology and new combination therapies for combating this complex and lethal disease. The results suggest that erlotinib-enhanced photodynamic therapy could further improve the efficacy of intraoperative light-activation to mop up residual tumor deposits in the clinic following surgical removal of macroscopic mesothelioma metastases.

Activatable clinical fluorophore-quencher antibody pairs as dual molecular probes for the enhanced specificity of image-guided surgery.

The emergence of fluorescently labeled therapeutic antibodies has given rise to molecular probes for image-guided surgery. However, the extraneous interstitial presence of an unbound and nonspecifically accumulated probe gives rise to false-positive detection of tumor tissue and margins. Thus, the concept of tumor-cell activation of smart probes provides a potentially superior mechanism of delineating tumor margins as well as small tumor deposits.

A photoactivable multi-inhibitor nanoliposome for tumour control and simultaneous inhibition of treatment escape pathways.

Nanoscale drug delivery vehicles can facilitate multimodal therapies of cancer by promoting tumour-selective drug release. However, few are effective because cancer cells develop ways to resist and evade treatment. Here, we introduce a photoactivable multi-inhibitor nanoliposome (PMIL) that imparts light-induced cytotoxicity in synchrony with a photoinitiated and sustained release of inhibitors that suppress tumour regrowth and treatment escape signalling pathways.

Simultaneous delivery of cytotoxic and biologic therapeutics using nanophotoactivatable liposomes enhances treatment efficacy in a mouse model of pancreatic cancer.

A lack of intracellular delivery systems has limited the use of biologics such as monoclonal antibodies (mAb) that abrogate molecular signaling pathways activated to promote escape from cancer treatment. We hypothesized that intracellular co-delivery of the photocytotoxic chromophore benzoporphyrin derivative monoacid A (BPD) and the anti-VEGF mAb bevacizumab in a nanophotoactivatable liposome (nanoPAL) might enhance the efficacy of photodynamic therapy (PDT) combined with suppression of VEGF-mediated signaling pathways. As a proof-of-concept we found that nanoPAL-PDT induced enhanced extra- and intracellular bevacizumab delivery and enhanced acute cytotoxicity in vitro.

Optical Imaging, Photodynamic Therapy and Optically Triggered Combination Treatments.

Optical imaging is becoming increasingly promising for real-time image-guided resections, and combined with photodynamic therapy (PDT), a photochemistry-based treatment modality, optical approaches can be intrinsically "theranostic." Challenges in PDT include precise light delivery, dosimetry, and photosensitizer tumor localization to establish tumor selectivity, and like all other modalities, incomplete treatment and subsequent activation of molecular escape pathways are often attributable to tumor heterogeneity. Key advances in molecular imaging, target-activatable photosensitizers, and optically active nanoparticles that provide both cytotoxicity and a drug release mechanism have opened exciting avenues to meet these challenges.

The role of photodynamic therapy in overcoming cancer drug resistance.

Many modalities of cancer therapy induce mechanisms of treatment resistance and escape pathways during chronic treatments, including photodynamic therapy (PDT). It is conceivable that resistance induced by one treatment might be overcome by another treatment. Emerging evidence suggests that the unique mechanisms of tumor cell and microenvironment damage produced by PDT could be utilized to overcome cancer drug resistance, to mitigate the compensatory induction of survival pathways and even to re-sensitize resistant cells to standard therapies.