Occlusal discoloration due to staining frequently occurs on the pits and fissures of teeth. Noncariogenic discoloration (non-CD) refers to the attachment of staining chromogens to sound surfaces, whereas cariogenic discoloration (CD) represents the discoloration of porous structures due to bacterial metabolites and mineral loss from the enamel surface. This study evaluated whether it is possible to distinguish between non-CD and CD on stained occlusal surfaces with fluorescence assessed by the quantitative light-induced fluorescence (QLF) technology. Sixty-two extracted human permanent teeth with suspected discolorations on the pit and fissure were examined. The maximum values of fluorescence loss (ΔFmax) and red fluorescence gain (ΔRmax) were calculated using QLF images. Using histology as the gold standard, it was found that 12 teeth were sound (non-CD), while 50 teeth had enamel and dentine caries (CD). The validity tests at the enamel histological caries level, ΔRmax (ρ  =  0.80) were strongly correlated with the histology (P  <  0.001). At the optimum threshold (105.0) of ΔRmax, it showed high levels of sensitivity and specificity (0.96 and 0.83, respectively). Therefore, QLF can be used to distinguish non-CD from CD on occlusal surfaces using red fluorescence values with high validity.

Label-free microscopy is a very powerful technique that can be applied to study samples with no need for exogenous fluorescent probes, keeping the main benefits of multiphoton microscopy, such as longer penetration depths and intrinsic optical sectioning while enabling serial multitechniques examinations on the same specimen. Among the many label-free microscopy methods, harmonic generation (HG) is one of the most intriguing methods due to its generally low photo-toxicity and relative ease of implementation. Today, HG and common two-photon microscopy (TPM) are well-established techniques, and are routinely used in several research fields. However, they require a significant amount of fine-tuning to be fully exploited, making them quite difficult to perform in parallel. Here, we present our designed multimodal microscope, capable of performing simultaneously TPM and HG without any kind of compromise thanks to two, separate, individually optimized laser sources with axial chromatic aberration compensation. We also apply our setup to the examination of a plethora of ex vivo samples to prove its capabilities and the significant advantages of a multimodal approach.

The research and development for biomedical applications are recently focused on multifunctional nanoparticles. To integrate various functionalities, different methods of modifying the particle’s physical properties are developed. Among the considered, nanodiamond (ND) is a promising candidate for the development of multifunctional complex due to its variable features in size, structure, surface chemistry, physical properties, and biocompatibility. In addition to its well-studied structural, surface, electrochemical and photonic properties, strong magnetism of ND can be observed. In the present work, magnetically modified ND is introduced in terms of its bioapplications. Along with the soft ferromagnetism of ND, the increased fluorescence at one- and two-photon excitation is realized. Utilizing the combined magnetic and fluorescence properties of the magnetically modified ND, fluorescence imaging, fluorescence lifetime imaging and manipulation of cells by magnetic field are demonstrated. The perspectives to use the magnetic ND for drug delivery, cells magnetic separation and filtration, in bioengineering to control the cell distribution combined with imaging and treatment are discussed.

We have developed a compact hollow core fiber (HCF)-based imaging platform capable of simultaneous in vivo confocal reflectance and two-photon imaging through the mouse pupil. We demonstrate the performance of this platform by imaging retinal ganglion cells (RGCs) in which the fluorophores YFP and GCaMP3 are expressed in Thy1-YFP-16 and Thy1-GCaMP3 transgenic mice, respectively. Confocal reflectance images of the mouse retina served as a reference for the simultaneous acquisition of the two-photon signals that clearly showed RGCs with single-cell resolution. The use of an HCF platform makes the system compact with future application in the longitudinal investigation into the structure and function of healthy and diseased RGCs.

We present the nanoparticle-enabled experimentally trained wavelet-domain denoising method for optical coherence tomography (OCT). It employs an experimental training algorithm based on imaging of a test-object, made of the colloidal suspension of the monodisperse nanoparticles and contains the microscale inclusions. The geometry and the scattering properties of the test-object are known a priori allowing us to set the criteria for the training algorithm. Using a wide set of the wavelet kernels and the wavelet-domain filtration approaches, the appropriate filter is constructed based on the test-object imaging. We apply the proposed approach and chose an efficient wavelet denoising procedure by considering the combinations of the decomposition basis from five wavelet families with eight types of the filtration threshold. We demonstrate applicability of the wavelet-filtering for the in vitro OCT image of human brain meningioma. The observed results prove high efficiency of the proposed OCT image denoising technique.

We used intravital multiphoton microscopy to study the recovery of hepatobiliary metabolism following carbon tetrachloride (CCl4) induced hepatotoxicity in mice. The acquired images were processed by a first order kinetic model to generate rate constant resolved images of the mouse liver. We found that with progression of hepatotoxicity, the spatial gradient of hepatic function disappeared. A CCl4-induced damage mechanism involves the compromise of membrane functions, resulting in accumulation of processed 6-carboxyfluorescein molecules. At day 14 following induction, a restoration of the mouse hepatobiliary function was found. Our approach allows the study of the response of hepatic functions to chemical agents in real time and is useful for studying pharmacokinetics of drug molecules through optical microscopic imaging.

We provide direct experimental comparison of the optoacoustic imaging performance of two different 64-element linear detector array (LDA) units based on polyvinylidene difluoride (PVDF) films. The first LDA unit was based on traditional flexible circuit (FC) technology and consisted of an FC glued to the nonmetalized signal surface of a 28-μm-thick PVDF film providing 300  /  80-μm axial resolution/lateral resolution (AR/LR) and 0.4-kPa noise equivalent pressure of its single element. The other LDA unit was manufactured using a technology of low-temperature photolithographic etching (PE) of a signal electrode onto a 25-μm-thick PVDF film providing 300  /  40-μm AR/LR and 1 kPa noise equivalent pressure. As compared with a previously reported LDA unit based on a 100-μm PVDF thick film, the main advantage of using the thinner PVDF films was 10-fold improvement in axial resolution, whereas the main drawback was 10-fold increased noise equivalent pressure. In terms of in vivo imaging performance, higher bandwidth of PE LDA probe was more important than the higher sensitivity of FC LDA unit.

We address the automatic differentiation of human tissue using multispectral imaging with promising potential for automatic visualization during surgery. Currently, tissue types have to be continuously differentiated based on the surgeon’s knowledge only. Further, automatic methods based on optical in vivo properties of human tissue do not yet exist, as these properties have not been sufficiently examined. To overcome this, we developed a hyperspectral camera setup to monitor the different optical behavior of tissue types in vivo. The aim of this work is to collect and analyze these behaviors to open up optical opportunities during surgery. Our setup uses a digital camera and several bandpass filters in front of the light source to illuminate different tissue types with 16 specific wavelength ranges. We analyzed the different intensities of eight healthy tissue types over the visible spectrum (400 to 700 nm). Using our setup and sophisticated postprocessing in order to handle motion during capturing, we are able to find tissue characteristics not visible for the human eye to differentiate tissue types in the 16-dimensional wavelength domain. Our analysis shows that this approach has the potential to support the surgeon’s decisions during treatment.

The ability for noninvasive visualization of functional changes of a tumor’s oxygenation and circulatory system offers new advantages for prognosis and monitoring of the treatment efficacy. The results of breast cancer oxygen state study under chemotherapy action obtained by diffuse optical spectroscopy (DOS) in combination with Doppler ultrasonic imaging are presented. Complex use of optical and ultrasound methods gives complementary information about the size of the tumor node, peculiarities of its vascular bed, rate of its blood flow as well as oxygenation, and provide a picture of the tumor response to treatment. Comparison with tumor pathologic response allowed to identify differences in the changes of these parameters depending on the degree of pathological tumor response to chemotherapy. It was demonstrated that fourth and fifth degrees of therapeutic pathomorphism may be predicted by the increase of oxygen saturation level after the first cycle of chemotherapy. If the reduction or absence of the oxygen saturation dynamics is observed, first or second degree of pathological tumor response can be expected. Additional ultrasound investigation of the tumors may be useful for observation of the dynamics of tumor blood flow thereby for understanding the reasons of induced chemotherapy oxygenation changes. The proposed approach based on DOS and ultrasonography may be applied for monitoring of breast tumors under therapy and prediction of their sensitivity.

Using multiphoton microscopy (MPM), we demonstrated that effective inducing of two-photon excited luminescence and second-harmonic generation signals in nano/microparticles of clinoptilolite type of zeolite (CZ) by femtosecond near-infrared laser excitation can be successfully utilized in multiphoton imaging of the drug adsorption processes. Adsorption of photodynamic active dyes (hypericin, chlorin e6, methylene blue, and fluorescein) and their release from CZ pores in the presence of biomolecules, such as collagen from bovine Achilles tendon, albumin, and hemoglobin, were investigated by absorption and fluorescence spectrometry. To quantify the experimental results on hypericin release, here we use a kinetic curves fitting approach and calculate hypericin release rates in different environments. This approach allows to compare various mathematical models and uses more parameters to better characterize drug release profiles. In addition, magnetic CZ particles were fabricated and proposed as a promising material for drug delivery and controlled release in biological systems. Optical spectrometry and MPM are effective approaches that may reveal potential of natural zeolites in controlled drug delivery and biomedical imaging.

Employment of chlorin-based photosensitizers (PSs) provides additional advantages to photodynamic therapy (PDT) due to absorption peak around 405 nm allowing for superficial impact and efficient antimicrobial therapy. We report on the morphological and clinical study of the efficiency of PDT at 405 nm employing chlorin-based PS. Numerical studies demonstrated difference in the distribution of absorbed dose at 405 nm in comparison with traditionally employed wavelength of 660 nm and difference in the in-depth absorbed dose distribution for skin and mucous tissues. Morphological study was performed at the inner surface of rabbit ear with histological examinations at different periods after PDT procedure. Animal study revealed tissue reaction to PDT consisting in edema manifested most in 3 days after the procedure and neoangiogenesis. OCT diagnostics was confirmed by histological examination. Clinical study included antimicrobial PDT of pharynx chronic inflammatory diseases. It revealed no side effects or complications of the PDT procedure. Pharyngoscopy indicated reduction of inflammatory manifestations, and, in particular cases, hypervascularization was observed. Morphological changes were also detected in the course of monitoring, which are in agreement with pharyngoscopy results. Microbiologic study after PDT revealed no pathogenic bacteria; however, in particular cases, saprophytic flora was detected.

Several algorithms exist to solve the photoacoustic image reconstruction problem depending on the expected reconstructed image features. These reconstruction algorithms promote typically one feature, such as being smooth or sharp, in the output image. Combining these features using a guided filtering approach was attempted in this work, which requires an input and guiding image. This approach act as a postprocessing step to improve commonly used Tikhonov or total variational regularization method. The result obtained from linear backprojection was used as a guiding image to improve these results. Using both numerical and experimental phantom cases, it was shown that the proposed guided filtering approach was able to improve (as high as 11.23 dB) the signal-to-noise ratio of the reconstructed images with the added advantage being computationally efficient. This approach was compared with state-of-the-art basis pursuit deconvolution as well as standard denoising methods and shown to outperform them.

We propose a hybrid approach to image enhancement in acoustic resolution photoacoustic microscopy. The developed technique is based on compensation for nonuniform spatial sensitivity of the optoacoustic (OA) system in both optical and acoustic domains. Spatial distribution of optical fluence is derived from full three-dimensional Monte Carlo simulations accounting for conical geometry of tissue laser illumination at the wavelength of 532 nm. Approximate nonuniform spatial response of acoustic detector with numerical aperture of 0.6 is derived from the two-dimensional k-Wave modeling. Application of the developed technique allows to improve the spatial resolution and to balance in-depth signal-level distribution in OA images of phantom and in-vivo objects.

Autofluorescence-based imaging techniques have become very important in the ophthalmological field. Being noninvasive and very sensitive, they are broadly used in clinical routines. Conventional autofluorescence intensity imaging is largely influenced by the strong fluorescence of lipofuscin, a fluorophore that can be found at the level of the retinal pigment epithelium. However, different endogenous retinal fluorophores can be altered in various diseases. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an imaging modality to investigate the autofluorescence of the human fundus in vivo. It expands the level of information, as an addition to investigating the fluorescence intensity, and autofluorescence lifetimes are captured. The Heidelberg Engineering Spectralis-based fluorescence lifetime imaging ophthalmoscope is used to investigate a 30-deg retinal field centered at the fovea. It detects FAF decays in short [498 to 560 nm, short spectral channel (SSC) and long (560 to 720 nm, long spectral channel (LSC)] spectral channels, the mean fluorescence lifetimes (τm) are calculated using bi- or triexponential approaches. These are meant to be relatively independent of the fluorophore’s intensity; therefore, fluorophores with less intense fluorescence can be detected. As an example, FLIO detects the fluorescence of macular pigment, retinal carotenoids that help protect the human fundus from light damages. Furthermore, FLIO is able to detect changes related to various retinal diseases, such as age-related macular degeneration, albinism, Alzheimer’s disease, diabetic retinopathy, macular telangiectasia type 2, retinitis pigmentosa, and Stargardt disease. Some of these changes can already be found in healthy eyes and may indicate a risk to developing such diseases. Other changes in already affected eyes seem to indicate disease progression. This review article focuses on providing detailed information on the clinical findings of FLIO. This technique detects not only structural changes at very early stages but also metabolic and disease-related alterations. Therefore, it is a very promising tool that might soon be used for early diagnostics.

Nowadays, dynamically developing optical (photonic) technologies play an ever-increasing role in medicine. Their adequate and effective implementation in diagnostics, surgery, and therapy needs reliable data on optical properties of human tissues, including skin. This paper presents an overview of recent results on the measurements and control of tissue optical properties. The issues reported comprise a brief review of optical properties of biological tissues and efficacy of optical clearing (OC) method in application to monitoring of diabetic complications and visualization of blood vessels and microcirculation using a number of optical imaging technologies, including spectroscopic, optical coherence tomography, and polarization- and speckle-based ones. Molecular modeling of immersion OC of skin and specific technique of OC of adipose tissue by its heating and photodynamic treatment are also discussed.

The objective of the study is the quantitative analysis of the dose-time dependences of changes occurring in collagen of bladder and rectum after gamma-irradiation using optical methods [nonlinear microscopy in a second harmonic generation (SHG) detection regime and cross-polarization optical coherence tomography (CP OCT)]. For quantitative assessment of the collagen structure, regions of interest on the SHG-images of two-dimensional (2-D) distribution of SHG signal intensity of collagen were chosen in the submucosa. The mean SHG signal intensity and its standard deviation were calculated by ImageJ 1.39p (NIH). For quantitative analysis of CP OCT data, an integral depolarization factor (IDF) was calculated. Quantitative calculation of the SHG signal intensity and the IDF can provide additional information about the processes of the collagen radiation-induced degradation and subsequent remodeling. High positive correlation between the mean SHG signal intensity and the mean IDF of bladder and rectum demonstrates that CP OCT can be used as an “optical biopsy” in the grading of collagen radiation damage.

Photobiomodulation therapy (PBM) has shown positive effects on stem cell differentiation in monolayer cell culture model, but little is known about its effect on three-dimensional (3-D) agarose gel culture. This study evaluated the PBM effect of human dental pulp stem cells (hDPSCs) differentiation and phosphatase alkaline activity (ALP) using an agarose 3-D model under different nutritional conditions. hDPSCs were characterized and seeded on a 0.3% agarose gel layer with different media (osteogenic, adipogenic, chondrogenic) and were assigned into four groups: control 10% fetal bovine serum (FBS), control 5% FBS, PBM 10% FBS, and PBM 5% FBS. Irradiation was performed with continuous-wave InGaAlP laser, 660 nm, 100 mW, 3,3  J  /  cm2, spot size 0.3  cm2, 10 s of exposure time, and 1 J of energy per point with 6-h interval between sessions. All groups were evaluated at 7 and 14 days. ALP assay was performed to analyze the deposition of mineralized tissue. At 7 days, PBM 5% FBS group presented better stimulation in osteogenic and adipogenic differentiation compared with control. After 14 days, hDPSCs cultured in 3-D exhibited osteogenic, adipogenic, and chondrogenic differentiation; furthermore, compared to control, PBM significantly stimulated all differentiation processes (p  <  0.05). It can be concluded that hDPSCs cultured in 3-D agarose associated to PBM could be a promising tool for tissue engineering applications.

Recently, compression optical clearing (OC) was applied to detect dermal carotenoid using reflection spectroscopy. To enhance the precision and accuracy of reflection spectroscopy to better detect the spectral absorption of beta-carotene inside biological phantom, here, we simultaneously use compression and immersion OC using dimethyl sulfoxide. In addition, we analytically extract the absorption coefficient of beta-carotene using diffuse reflectance spectroscopy (as an analytical OC). Our results show that the presented analytical OC can be applied alone as a noninvasive method to measure cutaneous chromophores at deep tissues. Finally, we also improve the ability of the analytical clearing method mediated with experimental OC. Our result demonstrates that the combination of analytical and experimental clearing methods enhance the ability of diffuse reflection spectroscopy for extracting the absorption coefficient of beta-carotene as one of the chromospheres inside biological phantom.

This study explores photoacoustic (PA) speckle tracking to characterize flow as an alternative to ultrasound (US) speckle tracking or current PA flow imaging methods. In cases where tracking of submicrometer particles is required, the US signal-to-noise ratio and contrast might be low due to limited reflectivity of subwavelength size targets at low concentrations. However, it may be possible to perform more accurate velocimetry using PAs due to different contrast mechanisms utilized in PA imaging. Here, we introduce a PA-based speckle tracking method that overcomes the directional dependence of Doppler imaging and the limited field of view of current correlation-based methods used in PA flow imaging. The feasibility of this method is demonstrated in a potential application—minimally invasive diagnosis of ventricular shunt malfunction, where the velocity of optically absorbing particles was estimated in a shunt catheter using block matching of PA and US signals. Overall, our study demonstrates the potential of the PA-based motion tracking method under various flow rates where US imaging cannot be effectively used for specking tracking because of its low contrast and low signal-to-noise ratio.

Virus infection of a human cell was determined only 3 h after invagination. We used viral vector Ad-CMV-control (AdC), which lacks the E1 gene coding for early polypeptide 1 (E1). AdC can replicate in human embryonic kidney 293 (HEK293) cells into which the E1 gene has been transfected. According to partial least-square regression discriminant analysis, it was assumed that two kinds of reaction take place in the cell during viral invasion. The first response of the cell was determined 3 h after the virus invasion, and the second one was determined ∼9  h later. The first one seems to be due to compositional changes in DNA. Analysis of large-scale datasets strongly indicated that the second reaction can be attributed to a reduction in protein concentration or uptake of phenylalanine into the nucleus.

This errata corrects an error in “Multifunctional biomedical applications of magnetic nanodiamond.”