Introduction to the Optical Molecular Probes, Imaging and Drug Delivery 2023 feature issue.

A feature issue is being presented by a team of guest editors containing papers based on studies presented at the Optical Molecular Probes, Imaging and Drug Delivery conference as part of the Optica Biophotonics Congress in Vancouver, Canada from April 24-27, 2023.

Two-color diffuse flow cytometer.

Significance: Hematogenous metastasis is mediated by circulating tumor cells (CTCs) and CTC clusters (CTCCs). We recently developed "diffuse flow cytometry" (DiFC) to detect fluorescent protein (FP) expressing CTCs in small animals. Extending DiFC to allow detection of two FPs simultaneously would allow concurrent study of different CTC sub-populations or heterogeneous CTCCs in the same animal.

Lipid nanoparticle encapsulated large peritoneal macrophages migrate to the lungs via the systemic circulation in a model of clodronate-mediated lung-resident macrophage depletion.

A mature tissue resident macrophage (TRM) population residing in the peritoneal cavity has been known for its unique ability to migrate to peritoneally located injured tissues and impart wound healing properties. Here, we sought to expand on this unique ability of large peritoneal macrophages (LPMs) by investigating whether these GATA6+ LPMs could also intravasate into systemic circulation and migrate to extra-peritoneally located lungs upon ablating lung-resident alveolar macrophages (AMs) by intranasally administered clodronate liposomes in mice. C12-200 cationic lipidoid-based nanoparticles were employed to selectively deliver a small interfering RNA (siRNA)-targeting CD-45 labeled with a cyanine 5.

In Vivo Labeling and Detection of Circulating Tumor Cells in Mice Using OTL38.

Purpose: We recently developed an optical instrument to non-invasively detect fluorescently labeled circulating tumor cells (CTCs) in mice called 'Diffuse in vivo Flow Cytometry' (DiFC). OTL38 is a folate receptor (FR) targeted near-infrared (NIR) contrast agent that is FDA approved for use in fluorescence guided surgery of ovarian and lung cancer. In this work, we investigated the use OTL38 for in vivo labeling and detection of FR + CTCs with DiFC.

Ratiometric fluorescence sensing and quantification of circulating blood sodium sensors in mice .

In this work, we introduce ratiometric diffuse flow cytometry (R-DiFC) for quantitative measurement of circulating fluorescent red blood cell (fRBC) sensors for systemic blood sodium levels. Unlike in our previous work in measuring circulating fRBC sensors, R-DiFC allows simultaneous measurement of two fluorophores encapsulated in the sensor, the ratio of which enables self-calibration of the fluorescence signal with different fRBC depths in biological tissue. We show that the R-DiFC signal varies significantly less than either fluorescence signal alone.

Special Section Guest Editorial: Seeing Inside Tissue with Optical Molecular Probes.

The editorial introduces the Special Section on Seeing Inside Tissue with Optical Molecular Probes.

Dual-ratio approach for detection of point fluorophores in biological tissue.

Significance: Diffuse flow cytometry (DiFC) is an emerging fluorescence sensing method to non-invasively detect labeled circulating cells . However, due to signal-to-noise ratio (SNR) constraints largely attributed to background tissue autofluorescence (AF), DiFC's measurement depth is limited.

Aim: The dual ratio (DR)/dual slope is an optical measurement method that aims to suppress noise and enhance SNR to deep tissue regions.

Dual-ratio approach for detection of point fluorophores in biological tissue.

Significance: Diffuse in-vivo Flow Cytometry (DiFC) is an emerging fluorescence sensing method to non-invasively detect labeled circulating cells in-vivo. However, due to Signal-to-Noise Ratio (SNR) constraints largely attributed to background tissue autofluorescence, DiFC's measurement depth is limited. multiplies Aim: The Dual-Ratio (DR) / dual-slope is a new optical measurement method that aims to suppress noise and enhance SNR to deep tissue regions.

Near-infrared diffuse in vivo flow cytometry.

Significance: Diffuse flow cytometry (DiFC) is an emerging technique for enumerating rare fluorescently labeled circulating cells noninvasively in the bloodstream. Thus far, we have reported red and blue-green versions of DiFC. Use of near-infrared (NIR) fluorescent light would in principle allow use of DiFC in deeper tissues and would be compatible with emerging NIR fluorescence molecular contrast agents.

Signal and measurement considerations for human translation of diffuse in vivo flow cytometry.

Significance: "Diffuse in vivo flow cytometry" (DiFC) is an emerging technology for fluorescence detection of rare circulating cells directly in large deep-seated blood vessels in mice. Because DiFC uses highly scattered light, in principle, it could be translated to human use. However, an open question is whether fluorescent signals from single cells would be detectable in human-scale anatomies.

Prospects for Fluorescence Molecular Liquid Biopsy of Circulating Tumor Cells in Humans.

Our team recently developed "Diffuse in vivo Flow Cytometry" (DiFC) for detection and enumeration rare circulating tumor cells (CTCs) in mice with highly-scattered fluorescent light. We have used DiFC to study dissemination of CTCs in a number of mouse models of metastasis with fluorescent protein expressing cells. Because DiFC uses diffuse light and interrogates large blood vessels in relatively deep tissue, in principle it could be translated to larger limbs, species, and even humans clinically.

Short-Term Circulating Tumor Cell Dynamics in Mouse Xenograft Models and Implications for Liquid Biopsy.

Motivation: Circulating tumor cells (CTCs) are widely studied using liquid biopsy methods that analyze fractionally-small peripheral blood (PB) samples. However, little is known about natural fluctuations in CTC numbers that may occur over short timescales , and how these may affect detection and enumeration of rare CTCs from small blood samples.

Methods: We recently developed an optical instrument called "diffuse flow cytometry" (DiFC) that uniquely allows continuous, non-invasive counting of rare, green fluorescent protein expressing CTCs in large blood vessels in mice.

Heterogeneity of circulating tumor cell dissemination and lung metastases in a subcutaneous Lewis lung carcinoma model.

Subcutaneous () tumor models are widely used in pre-clinical cancer metastasis research. Despite this, the dynamics and natural progression of circulating tumor cells (CTCs) and CTC clusters (CTCCs) in peripheral blood are poorly understood in these models. In this work, we used a new technique called 'diffuse flow cytometry' (DiFC) to study CTC and CTCC dissemination in an Lewis lung carcinoma (LLC) model in mice.

Fluorescence Labeling of Circulating Tumor Cells with a Folate Receptor-Targeted Molecular Probe for Diffuse In Vivo Flow Cytometry.

Purpose: We recently developed a new instrument called "diffuse in vivo flow cytometry" (DiFC) for enumeration of rare fluorescently labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins or were pre-labeled with a fluorescent dye ex vivo. In this work, we investigated the use of a folate receptor (FR)-targeted fluorescence molecular probe for in vivo labeling of FR+ CTCs for DiFC.

Prospects for the Use of Upconverting Nanoparticles as a Contrast Agent for Enumeration of Circulating Cells in vivo.

Purpose: We recently developed a new fluorescence-based technique called "diffuse in vivo flow cytometry" (DiFC) for enumerating rare circulating tumor cells (CTCs) directly in the bloodstream. Non-specific tissue autofluorescence is a persistent problem, as it creates a background which may obscure signals from weakly-labeled CTCs. Here we investigated the use of upconverting nanoparticles (UCNPs) as a contrast agent for DiFC, which in principle could significantly reduce the autofluorescence background and allow more sensitive detection of rare CTCs.

Flow-regulated endothelial glycocalyx determines metastatic cancer cell activity.

Cancer metastasis and secondary tumor initiation largely depend on circulating tumor cell (CTC) and vascular endothelial cell (EC) interactions by incompletely understood mechanisms. Endothelial glycocalyx (GCX) dysfunction may play a significant role in this process. GCX structure depends on vascular flow patterns, which are irregular in tumor environments.

Real-time particle-by-particle detection of erythrocyte-camouflaged microsensor with extended circulation time in the bloodstream.

Personalized medicine offers great potential benefits for disease management but requires continuous monitoring of drugs and drug targets. For instance, the therapeutic window for lithium therapy of bipolar disorder is very narrow, and more frequent monitoring of sodium levels could avoid toxicity. In this work, we developed and validated a platform for long-term, continuous monitoring of systemic analyte concentrations in vivo.

Fluorescence monitoring of rare circulating tumor cell and cluster dissemination in a multiple myeloma xenograft model in vivo.

Circulating tumor cells (CTCs) are of great interest in cancer research because of their crucial role in hematogenous metastasis. We recently developed “diffuse in vivo flow cytometry” (DiFC), a preclinical research tool for enumerating extremely rare fluorescently labeled CTCs directly in vivo. In this work, we developed a green fluorescent protein (GFP)-compatible version of DiFC and used it to noninvasively monitor tumor cell numbers in circulation in a multiple myeloma (MM) disseminated xenograft mouse model.

Metastatic cancer cell attachment to endothelium is promoted by endothelial glycocalyx sialic acid degradation.

While it is known that cancer cell interactions with vascular endothelial cells (ECs) drive metastatic cancer cell extravasation from blood vessels into secondary tumor sites, the mechanisms of action are still poorly understood. Here, we tested the hypothesis that neuraminidase-induced degradation of EC surface glycocalyx (GCX), particularly the sialic acid (SA) residue components of the GCX, will substantially increase metastatic cancer cell attachment to ECs. To our knowledge, our study is the first to isolate the role of GCX SA residues in cancer cell attachment to the endothelium, which were found to be differentially affected by the presence of neuraminidase and to indeed regulate metastatic cancer cell homing to ECs.

In Vivo Flow Cytometry of Extremely Rare Circulating Cells.

Circulating tumor cells (CTCs) are of great interest in cancer research, but methods for their enumeration remain far from optimal. We developed a new small animal research tool called "Diffuse in vivo Flow Cytometry" (DiFC) for detecting extremely rare fluorescently-labeled circulating cells directly in the bloodstream. The technique exploits near-infrared diffuse photons to detect and count cells flowing in large superficial arteries and veins without drawing blood samples.

Fluorescence detection, enumeration and characterization of single circulating cells in vivo: technology, applications and future prospects.

There are many diseases and biological processes that involve circulating cells in the bloodstream, such as cancer metastasis, immunology, reproductive medicine, and stem cell therapies. This has driven significant interest in new technologies for the study of circulating cells in small animal research models and clinically. Most currently used methods require drawing and enriching blood samples from the body, but these suffer from a number of limitations.

Near infrared spectroscopy for measuring changes in bone hemoglobin content after exercise in individuals with spinal cord injury.

Bone blood perfusion has an essential role in maintaining a healthy bone. However, current methods for measuring bone blood perfusion are expensive and highly invasive. This study presents a custom built near-infrared spectroscopy (NIRS) instrument to measure changes in bone blood perfusion.

Diffuse fluorescence fiber probe for in vivo detection of circulating cells.

There has been significant recent interest in the development of technologies for enumeration of rare circulating cells directly in the bloodstream in many areas of research, for example, in small animal models of circulating tumor cell dissemination during cancer metastasis. We describe a fiber-based optical probe that allows fluorescence detection of labeled circulating cells in vivo in a diffuse reflectance configuration. We validated this probe in a tissue-mimicking flow phantom model in vitro and in nude mice injected with fluorescently labeled multiple myeloma cells in vivo.

Multiplexed fluorescence mediated tomography with temporal and spectral data.

We recently developed an algorithm for multiplexed fluorescence tomographic imaging of at least four fluorophores concurrently in the red and near-infrared wavelength region by jointly using spectral and temporal data. We report the design of a fluorescence tomography instrument that acquires spectral and temporal data, and validate its use in tissue-mimicking phantoms with four embedded fluorescent targets with highly overlapped spectral signatures. Critically, this requires measurement or computation of extended fluorophore signature libraries, which capture the variability in the measured signal due to the unknown position of the targets in the media.