Wide-field Fluorescence Imaging Research Articles

Dual-color multiple-particle tracking at 50-nm localization and over 100-µm range in 3D with temporal focusing two-photon microscopy.

Nanoscale particle tracking in three dimensions is crucial to directly observe dynamics of molecules and nanoparticles in living cells. Here we present a three-dimensional particle tracking method based on temporally focused two-photon excitation. Multiple particles are imaged at 30 frames/s in volume up to 180 × 180 × 100 µm3. The spatial localization precision can reach 50 nm. We demonstrate its capability of tracking fast swimming microbes at speed of ~200 µm/s. Two-photon dual-color tracking is achieved by simultaneously exciting two kinds of fluorescent beads at 800 nm to demonstrate its potential in molecular interaction studies. Our method provides a simple wide-field fluorescence imaging approach for deep multiple-particle tracking.

Monte Carlo modelling of photodynamic therapy treatments comparing clustered three dimensional tumour structures with homogeneous tissue structures

We explore the effects of three dimensional (3D) tumour structures on depth dependent fluence rates, photodynamic doses (PDD) and fluorescence images through Monte Carlo radiation transfer modelling of photodynamic therapy. The aim with this work was to compare the commonly used uniform tumour densities with non-uniform densities to determine the importance of including 3D models in theoretical investigations. It was found that fractal 3D models resulted in deeper penetration on average of therapeutic radiation and higher PDD. An increase in effective treatment depth of 1 mm was observed for one of the investigated fractal structures, when comparing to the equivalent smooth model. Wide field fluorescence images were simulated, revealing information about the relationship between tumour structure and the appearance of the fluorescence intensity. Our models indicate that the 3D tumour structure strongly affects the spatial distribution of therapeutic light, the PDD and the wide field appearance of surface fluorescence images.

A Clinical Wide-Field Fluorescence Endoscopic Device for Molecular Imaging Demonstrating Cathepsin Protease Activity in Colon Cancer.

Early and effective detection of cancers of the gastrointestinal tract will require novel molecular probes and advances in instrumentation that can reveal functional changes in dysplastic and malignant tissues. Here, we describe adaptation of a wide-field clinical fiberscope to perform wide-field fluorescence imaging while preserving its white-light capability for the purpose of providing wide-field fluorescence imaging capability to point-of-care microscopes. We developed andused a fluorescent fiberscope to detect signals from a quenched probe, BMV109, that becomes fluorescent when cleaved by, and covalently bound to, active cathepsin proteases. Cathepsins are expressed in inflammation- and tumor-associated macrophages as well as directly from tumor cells and are a promising target for cancer imaging. The fiberscope has a 1-mm outer diameter enabling validation via endoscopic exams in mice, and therefore we evaluated topically applied BMV109 for the ability to detect colon polyps in an azoxymethane-induced colon tumor model in mice. This wide-field endoscopic imaging device revealed consistent and clear fluorescence signals from BMV109 that specifically localized to the polypoid regions as opposed to the normal adjacent colon tissue (p < 0.004) in the murine colon carcinoma model. The sensitivity of detection of BMV109 with the fluorescence fiberscope suggested utility of these tools for early detection at hard-to-reach sites. The fiberscope was designed to be used in conjunction with miniature, endoscope-compatible fluorescence microscopes for dual wide-field and microscopic cancer detection.

Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids.

Absolute, precise quantification methods expand the scope of nucleic acids research and have many practical applications. Digital polymerase chain reaction (dPCR) is a powerful method for nucleic acid detection and absolute quantification. However, it requires thermal cycling and accurate temperature control, which are difficult in resource-limited conditions. Accordingly, isothermal methods, such as recombinase polymerase amplification (RPA), are more attractive. We developed a picoliter well array (PWA) chip with 27,000 consistently sized picoliter reactions (314 pL) for isothermal DNA quantification using digital RPA (dRPA) at 39°C. Sample loading using a scraping liquid blade was simple, fast, and required small reagent volumes (i.e., <20 μL). Passivating the chip surface using a methoxy-PEG-silane agent effectively eliminated cross-contamination during dRPA. Our creative optical design enabled wide-field fluorescence imaging in situ and both end-point and real-time analyses of picoliter wells in a 6-cm2 area. It was not necessary to use scan shooting and stitch serial small images together. Using this method, we quantified serial dilutions of a Listeria monocytogenes gDNA stock solution from 9 × 10-1 to 4 × 10-3 copies per well with an average error of less than 11% (N = 15). Overall dRPA-on-chip processing required less than 30 min, which was a 4-fold decrease compared to dPCR, requiring approximately 2 h. dRPA on the PWA chip provides a simple and highly sensitive method to quantify nucleic acids without thermal cycling or precise micropump/microvalve control. It has applications in fast field analysis and critical clinical diagnostics under resource-limited settings.

Improved sensitivity to fluorescence for cancer detection in wide-field image-guided neurosurgery.

In glioma surgery, Protoporphyrin IX (PpIX) fluorescence may identify residual tumor that could be resected while minimizing damage to normal brain. We demonstrate that improved sensitivity for wide-field spectroscopic fluorescence imaging is achieved with minimal disruption to the neurosurgical workflow using an electron-multiplying charge-coupled device (EMCCD) relative to a state-of-the-art CMOS system. In phantom experiments the EMCCD system can detect at least two orders-of-magnitude lower PpIX. Ex vivo tissue imaging on a rat glioma model demonstrates improved fluorescence contrast compared with neurosurgical fluorescence microscope technology, and the fluorescence detection is confirmed with measurements from a clinically-validated spectroscopic probe. Greater PpIX sensitivity in wide-field fluorescence imaging may improve the residual tumor detection during surgery with consequent impact on survival.

Quantitative spatial frequency fluorescence imaging in the sub-diffusive domain for image-guided glioma resection.

Intraoperative 5- aminolevulinic acid induced-Protoporphyrin IX (PpIX) fluorescence guidance enables maximum safe resection of glioblastomas by providing surgeons with real-time tumor optical contrast. However, visual assessment of PpIX fluorescence is subjective and limited by the distorting effects of light attenuation and tissue autofluorescence. We have previously shown that non-invasive point measurements of absolute PpIX concentration identifies residual tumor that is otherwise non-detectable. Here, we extend this approach to wide-field quantitative fluorescence imaging by implementing spatial frequency domain imaging to recover tissue optical properties across the field-of-view in phantoms and ex vivo tissue.

Stereoscopic Integrated Imaging Goggles for Multimodal Intraoperative Image Guidance.

We have developed novel stereoscopic wearable multimodal intraoperative imaging and display systems entitled Integrated Imaging Goggles for guiding surgeries. The prototype systems offer real time stereoscopic fluorescence imaging and color reflectance imaging capacity, along with in vivo handheld microscopy and ultrasound imaging. With the Integrated Imaging Goggle, both wide-field fluorescence imaging and in vivo microscopy are provided. The real time ultrasound images can also be presented in the goggle display. Furthermore, real time goggle-to-goggle stereoscopic video sharing is demonstrated, which can greatly facilitate telemedicine. In this paper, the prototype systems are described, characterized and tested in surgeries in biological tissues ex vivo. We have found that the system can detect fluorescent targets with as low as 60 nM indocyanine green and can resolve structures down to 0.25 mm with large FOV stereoscopic imaging. The system has successfully guided simulated cancer surgeries in chicken. The Integrated Imaging Goggle is novel in 4 aspects: it is (a) the first wearable stereoscopic wide-field intraoperative fluorescence imaging and display system, (b) the first wearable system offering both large FOV and microscopic imaging simultaneously, (c) the first wearable system that offers both ultrasound imaging and fluorescence imaging capacities, and (d) the first demonstration of goggle-to-goggle communication to share stereoscopic views for medical guidance.

Interstitial photodynamic therapy: Light fractionation effects on a preclinical model of glioblastoma

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 347 natural (sunlight) light sources. Fluorescence imageswere taken to locate the distribution of the curcumin in themosquito larvae. Both curcumins derivative exhibited photoactivity, reaching 100% larval mortality after exposure to different light sources shown above. http://dx.doi.org/10.1016/j.pdpdt.2015.07.087 Study of ALA andMALmixtures in pig skin: PpIX production Alessandra Keiko Lima Fujita, Priscila Fernanda Campos de Menezes, Phamilla Gracielli Sousa Rodrigues, Michelle Barreto Requena, Cristina Kurachi, Andre Escobar, Rozana Wendler da Rocha, Andrigo Barboza de Nardi, Vanderlei S. Bagnato Sao Carlos Institute of Physics, University of Sao Paulo, Brazil Topical PDT using ALA (aminolevulinic acid) or methyl-ALA (MAL) as precursor of protoporphyrin IX (PpIX) shows some limitations. ALA is more hydrophilic than MAL, resulting in differences at the molecule interaction with the cells. The goal of study is evaluated the optimization of topical PDTusingALA andMAL in differentmixtures. This studywas done in vivo in porcine skinmodels using 7 categories of ALA andMALmixtures. The cream in different mixtures was applied and an occlusive dressing was placed. PpIX production in a porcine skin model was monitored using widefield fluorescence imaging and fluorescence spectroscopy collected at skin surface until 5 h of treatment. Seven types of mixture were tested, from 0 to 100% combinations, varying on 20%. The imaging analyses, showed the best results in PpIX productionwithmixtures from 40% to 80% of ALA. Both fluorescence techniques showed that the higher relative production of PpIX was present when the ALA concentration was of 40–80%. The investigation of the PpIX production as a function of the tissue depth is being performed. These results can be useful in Clinical Applications of PDT using topical ALA and M-ALA (Financial support: CNPq). http://dx.doi.org/10.1016/j.pdpdt.2015.07.088 Interstitial photodynamic therapy: Light fractionation effects on a preclinical model of glioblastoma Henri-Arthur Leroy, Maximilien Vermandel, Bertrand Leroux, Serge Mordon, Nicolas Reyns INSERM U1189 – ONCO-THAI Lille, Department of Neurosurgery/University Hospital, University of Lille,

Analysis using fluorescence of different photosensitizers in tumor model on the chorioallantoic membrane to application of photodynamic therapy

a commercial needle free injection system. The Protoporphyrin IX (PPIX) production was monitored using widefield fluorescence imaging at skin surface until 3 h of treatment. The visual damage after 48h of PDT was evaluated and the biopsies were removed from skin to perform the histological analyses. The superficial damage extension and the threshold dose were calculated. The results found to threshold dose is the smallest for application using needle free injection followed by ALA cream and light. These results prove the efficiency of the needle free injection to induce skin damage in the lowest light dose. The damage induce only by light can be suggested as a thermal damage. However, this damage is lower when compared toother conditions after PDT. It is possible to assume that the IPL can be useful to perform topical PDT using ALA. However, the thermal damage needs to be controlled carefully.

Influence of absorption and scattering coefficients on singlet oxygen luminescence during photosensitization in skin-stimulating phantom

a commercial needle free injection system. The Protoporphyrin IX (PPIX) production was monitored using widefield fluorescence imaging at skin surface until 3 h of treatment. The visual damage after 48h of PDT was evaluated and the biopsies were removed from skin to perform the histological analyses. The superficial damage extension and the threshold dose were calculated. The results found to threshold dose is the smallest for application using needle free injection followed by ALA cream and light. These results prove the efficiency of the needle free injection to induce skin damage in the lowest light dose. The damage induce only by light can be suggested as a thermal damage. However, this damage is lower when compared toother conditions after PDT. It is possible to assume that the IPL can be useful to perform topical PDT using ALA. However, the thermal damage needs to be controlled carefully.

Study of ALA and MAL mixtures in pig skin: PpIX production

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 347 natural (sunlight) light sources. Fluorescence imageswere taken to locate the distribution of the curcumin in themosquito larvae. Both curcumins derivative exhibited photoactivity, reaching 100% larval mortality after exposure to different light sources shown above. http://dx.doi.org/10.1016/j.pdpdt.2015.07.087 Study of ALA andMALmixtures in pig skin: PpIX production Alessandra Keiko Lima Fujita, Priscila Fernanda Campos de Menezes, Phamilla Gracielli Sousa Rodrigues, Michelle Barreto Requena, Cristina Kurachi, Andre Escobar, Rozana Wendler da Rocha, Andrigo Barboza de Nardi, Vanderlei S. Bagnato Sao Carlos Institute of Physics, University of Sao Paulo, Brazil Topical PDT using ALA (aminolevulinic acid) or methyl-ALA (MAL) as precursor of protoporphyrin IX (PpIX) shows some limitations. ALA is more hydrophilic than MAL, resulting in differences at the molecule interaction with the cells. The goal of study is evaluated the optimization of topical PDTusingALA andMAL in differentmixtures. This studywas done in vivo in porcine skinmodels using 7 categories of ALA andMALmixtures. The cream in different mixtures was applied and an occlusive dressing was placed. PpIX production in a porcine skin model was monitored using widefield fluorescence imaging and fluorescence spectroscopy collected at skin surface until 5 h of treatment. Seven types of mixture were tested, from 0 to 100% combinations, varying on 20%. The imaging analyses, showed the best results in PpIX productionwithmixtures from 40% to 80% of ALA. Both fluorescence techniques showed that the higher relative production of PpIX was present when the ALA concentration was of 40–80%. The investigation of the PpIX production as a function of the tissue depth is being performed. These results can be useful in Clinical Applications of PDT using topical ALA and M-ALA (Financial support: CNPq). http://dx.doi.org/10.1016/j.pdpdt.2015.07.088 Interstitial photodynamic therapy: Light fractionation effects on a preclinical model of glioblastoma Henri-Arthur Leroy, Maximilien Vermandel, Bertrand Leroux, Serge Mordon, Nicolas Reyns INSERM U1189 – ONCO-THAI Lille, Department of Neurosurgery/University Hospital, University of Lille,

The effectivity of ALA-PDT using IPL evaluated by image and histological analysis using porcine skin models in vivo

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 347 natural (sunlight) light sources. Fluorescence imageswere taken to locate the distribution of the curcumin in themosquito larvae. Both curcumins derivative exhibited photoactivity, reaching 100% larval mortality after exposure to different light sources shown above. http://dx.doi.org/10.1016/j.pdpdt.2015.07.087 Study of ALA andMALmixtures in pig skin: PpIX production Alessandra Keiko Lima Fujita, Priscila Fernanda Campos de Menezes, Phamilla Gracielli Sousa Rodrigues, Michelle Barreto Requena, Cristina Kurachi, Andre Escobar, Rozana Wendler da Rocha, Andrigo Barboza de Nardi, Vanderlei S. Bagnato Sao Carlos Institute of Physics, University of Sao Paulo, Brazil Topical PDT using ALA (aminolevulinic acid) or methyl-ALA (MAL) as precursor of protoporphyrin IX (PpIX) shows some limitations. ALA is more hydrophilic than MAL, resulting in differences at the molecule interaction with the cells. The goal of study is evaluated the optimization of topical PDTusingALA andMAL in differentmixtures. This studywas done in vivo in porcine skinmodels using 7 categories of ALA andMALmixtures. The cream in different mixtures was applied and an occlusive dressing was placed. PpIX production in a porcine skin model was monitored using widefield fluorescence imaging and fluorescence spectroscopy collected at skin surface until 5 h of treatment. Seven types of mixture were tested, from 0 to 100% combinations, varying on 20%. The imaging analyses, showed the best results in PpIX productionwithmixtures from 40% to 80% of ALA. Both fluorescence techniques showed that the higher relative production of PpIX was present when the ALA concentration was of 40–80%. The investigation of the PpIX production as a function of the tissue depth is being performed. These results can be useful in Clinical Applications of PDT using topical ALA and M-ALA (Financial support: CNPq). http://dx.doi.org/10.1016/j.pdpdt.2015.07.088 Interstitial photodynamic therapy: Light fractionation effects on a preclinical model of glioblastoma Henri-Arthur Leroy, Maximilien Vermandel, Bertrand Leroux, Serge Mordon, Nicolas Reyns INSERM U1189 – ONCO-THAI Lille, Department of Neurosurgery/University Hospital, University of Lille,

High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method

We present an optical lock-in detection scheme, called the integrating-bucket technique, as a signal-to-background ratio enhancement method for wide-field fluorescence imaging of biological cells. The proposed method uses sinusoidally modulated illumination light and captures four frames of fluorescence images, one per each quarter of the modulation period, by integrating the fluorescence intensity signal. The capability of this technique is demonstrated by imaging fluorescent bead solutions as well as labeled cells. The results show that the method yields a 4–10dB higher signal contrast than conventional fluorescence microscopy, and a background-free fluorescence image can be extracted within a sub-second time scale. Our findings indicate that the proposed method could be advantageous for the long-term study of live cells.

Validation of optical voltage reporting by the genetically encoded voltage indicator VSFP-Butterfly from cortical layer 2/3 pyramidal neurons in mouse brain slices.

Understanding how behavior emerges from brain electrical activity is one of the ultimate goals of neuroscience. To achieve this goal we require methods for large-scale recording of the electrical activity of specific neuronal circuits. A very promising approach is to use optical reporting of membrane voltage transients, particularly if the voltage reporter is genetically targeted to specific neuronal populations. Targeting in this way allows population signals to be recorded and interpreted without blindness to neuronal diversity. Here, we evaluated the voltage-sensitive fluorescent protein, VSFP Butterfly 2.1, a genetically encoded voltage indicator (GEVI), for monitoring electrical activity of layer 2/3 cortical pyramidal neurons in mouse brain slices. Standard widefield fluorescence and two-photon imaging revealed robust, high signal-to-noise ratio read-outs of membrane voltage transients that are predominantly synaptic in nature and can be resolved as discrete areas of synaptically connected layer 2/3 neurons. We find that targeted expression of this GEVI in the cortex provides a flexible and promising tool for the analysis of L2/3 cortical network function.

Fluorescence guided PDT for optimization of the outcome of skin cancer treatment

The photodynamic therapy (PDT) is an alternative technique that can be indicated for superficial basal cell carcinoma (sBCC), Bowen’s disease and actinic keratosis with high efficiency. The objective of this study is to present the importance of fluorescence imaging for PDT guidance and monitoring in real time. Confirming that the lesion is well prepared and the photosensitizer shows a homogenous distribution, the outcome after few PDT sessions will be positive and the recurrence should be lower. Our proposition in this study is use the widefield fluorescence imaging to evaluate the PDT protocol in situ and in real time for each lesion. This evaluation procedure is performed in two steps: first with the monitoring of the production of protoporphyrin IX (PpIX) induced by methyl aminolevulinate (MAL), an derivative of 5-aminolevulinic acid (ALA) and second with the detection of PpIX photobleaching after illumination. The fluorescence images provide information correlated with distinct clinical features and with the treatment outcome. Eight BCC lesions are presented and discussed in this study. Different fluorescence patterns of PpIX production and photobleaching could be correlated with the treatment response. The presented results show the potential of using widefield fluorescence imaging as a guidance tool to customized PDT.

Speckle correlation resolution enhancement of wide-field fluorescence imaging

Fluorescence microscopy is indispensable in nanoscience and biological sciences. The versatility of labeling target structures with fluorescent dyes permits to visualize structure and function at a subcellular resolution with a wide field of view. Due to the diffraction limit, conventional optical microscopes are limited to resolving structures larger than 200 nm. The resolution can be enhanced by near-field and far-field super-resolution microscopy methods. Near-field methods typically have a limited field of view and far-field methods are limited by the involved conventional optics. Here, we introduce a combined high-resolution and wide-field fluorescence microscopy method that improves the resolution of a conventional optical microscope by exploiting correlations in speckle illumination through a randomly scattering high-index medium: Speckle correlation resolution enhancement (SCORE). As a test, we collect two-dimensional fluorescence images of 100-nm diameter dye-doped nanospheres. We demonstrate a deconvolved resolution of 130 nm with a field of view of 10 x 10 $\text{\mu m}^2$.

Open-frame system for single-molecule microscopy.

We present the design and construction of a versatile, open frame inverted microscope system for wide-field fluorescence and single molecule imaging. The microscope chassis and modular design allow for customization, expansion, and experimental flexibility. We present two components which are included with the microscope which extend its basic capabilities and together create a powerful microscopy system: A Convex Lens-induced Confinement device provides the system with single-molecule imaging capabilities, and a two-color imaging system provides the option of imaging multiple molecular species simultaneously. The flexibility of the open-framed chassis combined with accessible single-molecule, multi-species imaging technology supports a wide range of new measurements in the health, nanotechnology, and materials science research sectors.

Detection of Non-Melanoma Skin Cancer by in vivo Fluorescence Imaging with Fluorocoxib A

Non-melanoma skin cancer (NMSC) is the most common form of cancer in the US and its incidence is increasing. The current standard of care is visual inspection by physicians and/or dermatologists, followed by skin biopsy and pathologic confirmation. We have investigated the use of in vivo fluorescence imaging using fluorocoxib A as a molecular probe for early detection and assessment of skin tumors in mouse models of NMSC. Fluorocoxib A targets the cyclooxygenase-2 (COX-2) enzyme that is preferentially expressed by inflamed and tumor tissue, and therefore has potential to be an effective broadly active molecular biomarker for cancer detection. We tested the sensitivity of fluorocoxib A in a BCC allograft SCID hairless mouse model using a wide-field fluorescence imaging system. Subcutaneous allografts comprised of 1000 BCC cells were detectable above background. These BCC allograft mice were imaged over time and a linear correlation (R2 = 0.8) between tumor volume and fluorocoxib A signal levels was observed. We also tested fluorocoxib A in a genetically engineered spontaneous BCC mouse model (Ptch1+/− K14-Cre-ER2 p53fl/fl), where sequential imaging of the same animals over time demonstrated that early, microscopic lesions (100 μm size) developed into visible macroscopic tumor masses over 11 to 17 days. Overall, for macroscopic tumors, the sensitivity was 88% and the specificity was 100%. For microscopic tumors, the sensitivity was 85% and specificity was 56%. These results demonstrate the potential of fluorocoxib A as an in vivo imaging agent for early detection, margin delineation and guided biopsies of NMSCs.

Assessment of ALA-induced PpIX production in porcine skin pretreated with microneedles.

Photodynamic therapy (PDT) is used for skin treatments of premalignant and cancer lesions and recognized as a non-invasive technique that combines tissue photosensitization and subsequent exposure to light to induce cell death. However, it is limited to the treatment of superficial lesions, mainly due to the low cream penetration. Therefore, the improvement of transdermal distribution of aminolevulinic acid (ALA) is needed. In this study, the kinetics and homogeneity of production of ALA-induced PpIX after the skin pre-treatment with microneedles rollers of 0.5, 1.0 and 1.5mm length were investigated. An improvement in homogeneity and production of PpIX was shown in a porcine model. Widefield fluorescence imaging three hours after the topical application of ALA-cream in the combined treatment with microeedles rollers.

DNA combing on low-pressure oxygen plasma modified polysilsesquioxane substrates for single-molecule studies.

Molecular combing and flow-induced stretching are the most commonly used methods to immobilize and stretch DNA molecules. While both approaches require functionalization steps for the substrate surface and the molecules, conventionally the former does not take advantage of, as the latter, the versatility of microfluidics regarding robustness, buffer exchange capability, and molecule manipulation using external forces for single molecule studies. Here, we demonstrate a simple one-step combing process involving only low-pressure oxygen (O2) plasma modified polysilsesquioxane (PSQ) polymer layer to facilitate both room temperature microfluidic device bonding and immobilization of stretched single DNA molecules without molecular functionalization step. Atomic force microscopy and Kelvin probe force microscopy experiments revealed a significant increase in surface roughness and surface potential on low-pressure O2 plasma treated PSQ, in contrast to that with high-pressure O2 plasma treatment, which are proposed to be responsible for enabling effective DNA immobilization. We further demonstrate the use of our platform to observe DNA-RNA polymerase complexes and cancer drug cisplatin induced DNA condensation using wide-field fluorescence imaging.