Mapping information from photographic images to volumetric medical imaging scans is essential for linking spaces with physical environments, such as in image-guided surgery. Current methods of accurate photographic image to Computed Tomography (CT) image mapping can be computationally intensive and/or require specialized hardware. For general purpose 3-D mapping of bulk specimens in histological processing, a cost-effective solution is necessary. Here, we compare the integration of a commercial 3-D camera and cell phone imaging with a surface registration pipeline. Using surgical implants and chuck-eye steak as phantom tests, we obtain 3-D CT reconstruction and sets of photographic images from two sources: Canfield Imaging's H1 camera and an iPhone 14 Pro. We perform surface reconstruction from the photographic images using commercial tools and open-source code for Neural Radiance Fields (NeRF) respectively. We complete surface registration of the reconstructed surfaces with the Iterative Closest Point (ICP) method. Manually placed landmarks were identified at three locations on each of the surfaces. Registration of the Canfield surfaces for three objects yields landmark distance errors of 1.747, 3.932, and 1.692 mm, while registration of the respective iPhone camera surfaces yields errors of 1.222, 2.061, and 5.155-mm. Photographic imaging of an organ sample prior to tissue sectioning provides a low-cost alternative to establish correspondence between histological samples and 3-D anatomical samples.
Lesion count is a key outcome to assess disease progression and the efficacy of new treatments for mpox. Accurately counting the hundreds to thousands of skin lesions typical of clade I mpox in endemic areas poses considerable human resource challenges. To document lesion appearance, evolution, and resolution during the PALM007 randomized controlled clinical trial of tecovirimat for mpox, we established smartphone-based protocols for standardized total body photography at remote sites in the Democratic Republic of the Congo. The trial is supported by the Institut National de la Recherche Biomédicale, NIAID, and funded by NCI Contract No.75N910D00024, Task Order No.75N91020F00025.
To evaluate the effect of illumination, camera orientation, and camera distance on color consistency of different skin tones, several swatches from a Pantone SkinTone Guide deck were selected and photographed at varying distances, illuminations, and angles. The RGB values from each selected swatch from each image were converted to LAB units and compared with the converted “true” values provided by Pantone. The ability of various color references to correct different skin tones was tested by comparing color values from a subject’s forearm skin to “true” color values of the closest Pantone SkinTone swatch found by visual comparison.
Through vertex component analysis of hyperspectral imaging data in the visible spectral range, we differentiated erythematous and pigmented areas in patients with cutaneous chronic graft-versus-host disease. We explored the feasibility of hyperspectral imaging in combination with unsupervised learning algorithms to differentiate active disease from inactive post-inflammatory skin changes, a fundamental practice gap in caring for these patients. We compared erythema and pigment maps to the visual assessment by a dermatologist as the ground truth.
Through noninvasive monitoring of leukocyte motion in skin capillaries of patients after hematopoietic cell transplantation, we found increased leukocyte rolling and adhesion prior to clinical signs of disease. In this longitudinal pilot study, we explored the feasibility to detect changes in leukocyte-endothelial interactions that precede acute graft-versus-host disease in patients after hematopoietic cell transplantation. We present the pattern of change in leukocyte rolling and adhesion in three patients over the course of the first 100 days post-transplant. Our preliminary data show increased leukocyte-endothelial interactions prior to clinical signs of any organ acute graft-versus-host disease.
Inflammatory tissue response is one of the first and most common manifestations of acute graft-versus-host disease (aGVHD), a potentially deadly immune-mediated disease that occurs in 30-60% of patients after stem cell transplantation. A fundamental challenge in developing effective treatment strategies for aGVHD is the lack of tools to study disease biology in real-time in post-transplant patients. The inflammatory tissue response causes increased expression of specialized endothelial proteins on vessel walls making leukocytes to roll, adhere and eventually extravasate into the tissue at a higher rate than in normal conditions. Although the importance of leukocyte-endothelial interactions to detect and track inflammation has been well shown in murine models, there are no published clinical studies in humans. In this study, we explore the feasibility to detect presence of aGVHD in post-transplant patients through the imaging of in vivo leukocyte motion. We used a clinical confocal microscope (Vivascope 1500) to acquire videos of 5 aGVHD patients and 5 controls (no aGVHD) within 50±30 days post-transplant. The microscope is capable of real-time imaging of individual cells in the postcapillary vessels at 9 frames per second. Through video analysis, we extracted five quantitative parameters: number and velocity of rolling leukocytes, number of adherent leukocytes (stationary >30 s), blood flow velocity, and number of vessels. In a limited number of subjects, we show that parameters characteristic of the dynamic motion in skin capillaries can be observed noninvasively in post-transplant patients. Further studies are needed to test the diagnostic potential of these parameters.
Leukocyte-endothelial interactions have been well-characterized by intravital microscopy in mice. Quantitative parameters descriptive of these dynamic processes, e.g. the level of leukocyte rolling, adhesion and extravasation, can detect and track inflammation. Despite technology available to study individual cell motion noninvasively in human skin, we are not aware of any published exploratory or clinical studies. In this preliminary study, we explore the feasibility to extract parameters characteristic of individual cell motion in the postcapillary vessels of healthy human skin from videos taken by a noninvasive clinical confocal microscope (Vivascope 1500). The microscope is capable of real-time imaging of individual cells at 9 frames per second. We took videos of ten cutaneous vessels per each of two body sites (volar forearm and upper anterior chest) of ten healthy subjects. We then characterized the dynamic motion of cells via subsequent video analysis by extracting the following parameters: blood flow velocity, number of adherent leukocytes (stationary <30 s), and number and diameter of vessels. We observed variation in blood flow velocity within 1 minute in the same vessel, between vessels within an 8x8 mm field of view, and within two different body sites. Leukocyte adhesion, more commonly associated with inflammatory conditions, can also be observed in healthy skin. Further studies are needed to test the potential of this approach to detect inflammation.
Chronic graft-versus-host disease (cGVHD) is a frequent and potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HCT) and commonly affects the skin, resulting in distressing patient morbidity. The percentage of involved body surface area (BSA) is commonly used for diagnosing and scoring the severity of cGVHD. However, the segmentation of the involved BSA from patient whole body serial photography is challenging because (1) it is difficult to design traditional segmentation method that rely on hand crafted features as the appearance of cGVHD lesions can be drastically different from patient to patient; (2) to the best of our knowledge, currently there is no publicavailable labelled image set of cGVHD skin for training deep networks to segment the involved BSA. In this preliminary study we create a small labelled image set of skin cGVHD, and we explore the possibility to use a fully convolutional neural network (FCN) to segment the skin lesion in the images. We use a commercial stereoscopic Vectra H1 camera (Canfield Scientific) to acquire ~400 3D photographs of 17 cGVHD patients aged between 22 and 72. A rotational data augmentation process is then applied, which rotates the 3D photos through 10 predefined angles, producing one 2D projection image at each position. This results in ~4000 2D images that constitute our cGVHD image set. A FCN model is trained and tested using our images. We show that our method achieves encouraging results for segmenting cGVHD skin lesion in photographic images.
KEYWORDS: Blood, In vivo imaging, Green fluorescent protein, Luminescence, Flow cytometry, Fiber optics, Photon counting, Signal detection, Tumors, Absorption
Circulating tumor cells in the bloodstream are sensitive indicators for metastasis and disease prognosis. Circulating cells have usually been monitored via extraction from blood, and more recently in vivo using free-space optics; however, long-term intravital monitoring of rare circulating cells remains a major challenge. We demonstrate the application of a two-photon-fluorescence optical fiber probe for the detection of cells in whole blood and in vivo. A double-clad fiber was used to enhance the detection sensitivity. Two-channel detection was employed to enable simultaneous measurement of multiple fluorescent markers. Because the fiber probe circumvents scattering and absorption from whole blood, the detected signal strength from fluorescent cells was found to be similar in phosphate-buffered saline (PBS) and in whole blood. The detection efficiency of cells labeled with the membrane-binding dye 1,1-dioctadecyl-3,3,3,3-tetramethylindoldicarbocyanine, 4-chlorobenzenesulfonate (DiD) was demonstrated to be the same in PBS and in whole blood. A high detection efficiency of green fluorescent protein (GFP)-expressing cells in whole blood was also demonstrated. To characterize in vivo detection, DiD-labeled untransfected and GFP-transfected cells were injected into live mice, and the cell circulation dynamics was monitored in real time. The detection efficiency of GFP-expressing cells in vivo was consistent with that observed ex vivo in whole blood.
KEYWORDS: Blood, Green fluorescent protein, In vivo imaging, Signal detection, Photon counting, Luminescence, Flow cytometry, Absorption, Scattering, Veins
We have demonstrated the use of a double-clad fiber probe to conduct two-photon excited flow cytometry in vitro and in
vivo. We conducted two-channel detection to measure fluorescence at two distinct wavelengths simultaneously. Because
the scattering and absorption problems from whole blood were circumvented by the fiber probe, the detected signal
strength from the cells were found to be similar in PBS and in whole blood. We achieved the same detection efficiency
of the membrane-binding lipophilic dye DiD labeled cells in PBS and in whole blood. High detection efficiency of green
fluorescent protein (GFP)-expressing cells in whole blood was demonstrated. DiD-labeled untransfected and GFP-transfected
cells were injected into live mice and the circulation dynamics of the externally injected cells were monitored.
The detection efficiency of GFP-expressing cells in vivo was consistent with that observed in whole blood.
KEYWORDS: Green fluorescent protein, Blood, Signal detection, Flow cytometry, Capillaries, In vivo imaging, Optical spheres, Tumors, Luminescence, Mirrors
We demonstrate enhanced sensitivity in two-photon flow cytometry with an extended cavity laser excitation source. At low power, the home-built 20-MHz oscillator was able to detect a significantly larger fraction, in either phosphate buffered saline (PBS) or whole blood, of green fluorescent protein (GFP)–expressing MCA-207 cells cross-labeled with the membrane-binding lipophilic dye DiD. A geometrical model is used to explain unique features of the signals resulting from the different spatial distribution of DiD and GFP. These unique features include sub-square law scaling of unsaturated two-photon signal, a sigmoidal sensitivity curve for detection under varying powers for cell detection thresholds as low as a single photon, and uncorrelated signal strengths in two detection channels.
Flow cytometry is a powerful technique for quantitative characterization of fluorescence in cells. Quantitation is achieved by ensuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow. Two-photon excitation has the advantages that it enables simultaneous excitation of multiple dyes and achieves a very high SNR through simplified filtering and fluorescence background reduction. We demonstrate that two-photon excitation in conjunction with a targeted multidye labeling strategy enables quantitative flow cytometry even under conditions of nonuniform flow, such as may be encountered in simple capillary flow or in vivo. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling cells with targeted nanoparticles containing multiple fluorophores enables normalization of the fluorescence signal and thus quantitative measurements under nonuniform excitation. Flow cytometry using two-photon excitation is demonstrated for detection and differentiation of particles and cells both in vitro in a glass capillary and in vivo in the blood stream of live mice. The technique also enables us to monitor the fluorescent dye labeling dynamics in vivo. In addition, we present a unique two-beam scanning method to conduct cell size measurement in nonuniform flow.
KEYWORDS: Flow cytometry, In vivo imaging, Blood, Luminescence, Signal detection, Near infrared, Fluorescent proteins, Quantum dots, Green fluorescent protein, Breast cancer
We have developed a new two-photon system for in vivo flow cytometry, thereby allowing us to
simultaneously quantify different circulating populations in a single animal. The instrument was able to resolve
minute-by-minute depletion dynamics of injected fluorescent microspheres at finer time scales than conventional
flow cytometry. Also observed were the circulation dynamics of human MCF-7 and MDA-MB-435 breast cancer
cells, which have low and high metastatic potential, respectively. After co-injection of both cell types into mice,
markedly greater numbers of MCF-7 cells were present in the circulation at early time points. While low metastatic
MCF-7 cells were cleared from the vascular system within 24 hours, detectable numbers of metastatic MDA-MB-
435 cells in the circulation remained constant over time. When we replace the commercial (80-MHz) NIR
excitation laser with a reduced-repetition-rate (20-MHz) mode-locked oscillator, the signal is enhanced four-fold,
enabling superior detection in blood of cell lines expressing fluorescent proteins tdTomato and mPlum (crosslabeled
with DiI and DiD). Detection sensitivity versus incident laser power is understood in terms of detected
event photon count distribution, which can be predicted with simple fluorophore distribution assumptions. The
technique of two-color, two-photon flow cytometry greatly enhances the capabilities of ex vivo flow cytometry to
investigate dynamics of circulating cells in cancer and other important diseases.
Two-photon excitation fluorescence of complex solvated molecules (Rhodamine590, Fluorescein, and G5-dendrimer conjugated Fluorescein) was successfully controlled using adaptive pulse shaping. We were able to maximize and minimize the ratio of fluorescent yield to average incident power or second-harmonic generation (SHG) in a thin optical crystal. The optimal excitation pulse shape was found experimentally using a genetic learning algorithm and no a priori knowledge. Pulses were shaped with an acousto-optic programmable dispersive filter (Dazzler AOPDF) controlling phase and amplitude of 20 individual frequency components. Convergence occurred over the order of 100 generations of experiments from an original set of 50 random individual pulses. Femtosecond laser pulses (~75 fs, 76 MHz repetition, 800 nm center wavelength, 3nJ without shaping) selected to maximize fluorescence yield / SHG were found to be complementary to those minimizing this ratio when visualized with a SHG-frequency resolved optical gating (SHG-FROG) device. At these powers, linear chirp of the pulse was far less significant in establishing coherent control than the more complex pulse shape. Regeneratively amplified pulses (~150 fs, 20 kHz repetition, 795 nm center wavelength, 2 μJ before shaping) were selected for maximum efficiency of fluorescent yield relative to incident power. The peak intensity, as determined by SHG, did not change significantly for optimal pulses when compared to early generations. This indicates that the improved two-photon fluorescent signal was not the result of simple convergence to a transform limited pulse, and suggests that the dye molecule excited state population is being coherently controlled. We are currently investigating the application of this result to enhancing signal in flow-cytometry and improved discrimination for multi-photon microscopy.
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