Application of FLIM-FIDSAM for the in vivo analysis of hormone competence of different cell types

Background fluorescence derived from subcellular compartments is a major drawback in high-resolution live imaging, especially of plant cells. A novel technique for contrast enhancement of fluorescence images of living cells expressing fluorescent fusion proteins termed fluorescence intensity decay shape analysis microscopy (FIDSAM) has been recently published and is applied here to plant cells expressing wild-type levels of a low-abundant membrane protein (BRI1-EGFP), demonstrating the applicability of FIDSAM to samples exhibiting about 80% autofluorescence. Furthermore, the combination of FIDSAM and fluorescence lifetime imaging microscopy enables the simultaneous determination and quantification of different ligand-specific responses in living cells with high spatial and temporal resolution even in samples with high autofluorescence background. Correlation of different responses can be used to determine the hormone ligand competence of different cell types as demonstrated here in BRI1-EGFP-expressing root and hypocotyl cells.     

In vivo Laser-induced Fluorescence Imaging of a Rat Pancreatic Cancer with Pheophorbide-a

Laser-induced fluorescence (LIF) of pheophorbide-a (Ph-a) was used for imaging of a rat pancreatic tumor. Using a dimensionless function (the ratio of Ph-a fluorescence by bluish autofluorescence), the fluorescence contrasts between excised tumors and their paired pancreas were investigated up to 48 h after a 9 mg kg^-1 Ph-a intravenous administration. Among five tested excitation wavelengths, 355 and 610 nm excitations gave the best distinctive contrasts, both 48 h after dye injection. The LIF imaging of six intrapancreatic tumors and six healthy pancreas was carried out in vivo using two laser excitations: 355 nm (Nd:YAG + tripling) for bluish autofluorescence and 610 nm (rhodamine 6G dye) for reddish autofluorescence and dye emission. Images were recorded through bandpass filters at 470 and 640 nm (autofluorescence) and at 680 nm (dye + autofluorescence) with an intensified charged-coupled device camera. Autofluorescence as Ph-a fluorescence images did not allow accurate LIF diagnosis of pancreatic carcinoma. An image processing, including for each pixel a computed division of Ph-a fluorescence (after subtraction of reddish autofluorescence) by bluish autofluorescence intensity generated poorly contrasted tumor images in five of six and false tumor localization in one of three of the tumor-bearing pancreas. A fitting of the digital 640 nm autofluorescence up to the mean 680 nm fluorescence intensity in pancreas prior to subtraction allowed a safe diagnosis to be made with well-contrasted tumor images. To assess automation ability of the processing, a same fitting coefficient (mean of individual values) was applied. In this way, false-negative (one of six) and false-positive (two of six) images were present in tumor-bearing animals as false-positive in one-half of the controls. A successful standardized procedure was then applied with a normalization of 640 and 680 nm pancreas intensities to a same set threshold prior processing. In opposition to thin-layered hollow organs, such as bronchial tube or digestive tract, LIF imaging of carcinoma inserted in a compact organ is exhausting. The use of a dye excitable in the red wavelength range (610 nm for Ph-a) may partly solve this problem, rendering LIF imaging more accurate and potentially automated.     

Photodynamic Imaging of a Rat Pancreatic Cancer with Pheophorbide a

Laser-induced fluorescence of pheophorbide a (Ph- a ) was used for in vitro photodynamic imaging (PDI) of a rat pancreatic acinar tumor. A 400 nm excitation induced a 470 nm autofluorescence and a 678 nm dye fluorescence in tumors and their surrounding pancreas 24 h after a 9 mg kg⁻¹ body weight Ph- a intravenous administration. With lower intensities in these blood-rich tumors than in pancreas, Ph- a fluorescence signals are unable to provide tumor images. A dimensionless function (the ratio of Ph- a fluorescence by autofluorescence, called R_t for the tumor and R_p for the pancreas) was used for fluorescence contrast calculation (C = R_t/R_p) between six tumors and their paired pancreas. Among five available laser excitation wavelengths, only the 355 nm excitation gave a distinctive contrast (C = 1.5). The PDI of six intrapancreatic tumors and their intraperitoneal metastasis and of two control normal pancreas was thus performed ex vivo using a 355 nm excitation source delivered by a tripled Nd: YAG laser and a charged-coupled device camera. Fluorescence images were recorded at 680 nm (dye), 640 nm (background) and 470 nm (autofluorescence) through three corresponding 10 nm width bandpass filters. Computed division for each pixel of Ph- a fluorescence values by autofluorescence generated false color image. In this way, contrasted tumor images were obtained. But in five out of six animals false-positive images were present due to an autofluorescence decrease in some normal pancreatic areas. A 470 nm autofluorescence imaging on the same tumors gave in all cases false-positive image and false-negative in half of the cases. These observations suggest that autofluorescence alone is unable to achieve accurate PDI of pancreatic carcinoma and that using Ph- a as a PDI dye needs strong improvements.     

Multispectral imaging autofluorescence microscopy for the analysis of lymph-node tissues

Although histochemical and immunohistochemical methods are the standard procedures in diagnosis of lymphoproliferative disorders, useful improvements in evidencing histopathologic manifestations can be obtained with the introduction of tissue autofluorescence analyses. We used microspectrofluorometry and a Multispectral Imaging Autofluorescence Microscopy (MIAM) technique to analyze lymph-node biopsies from patients with lymphoadenopathy of different origins. Images of tissue autofluorescence were obtained by excitation at 365 nm of lymph-node sections and sequential detection with interference filters (50 nm bandwidth) peaked at 450, 550 and 658 nm. Monochrome images were combined together in a single red-green-blue color image. Most of the fluorescence was observed within the blue spectral band because of large contributions from extracellular collagen and elastin fibers as well as from reduced form of intracellular nicotinamide adenine dinucleotide (phosphate). Autofluorescence imaging shows morphological differences between neoplastic and non-neoplastic tissues. The reactive hyperplasia samples show the typical lymph-node organization with weak fluorescent follicles separated by high fluorescent connective trabeculae. In the neoplastic lymph nodes the loss of follicle organization is observed. Consequently, MIAM permits to discriminate between non-neoplastic and neoplastic tissues on the basis of their autofluorescence pattern. Multispectral imaging of tissue autofluorescence may present some advantages with respect to standard histochemical microscopy since it (1) does not require any chemical manipulation of samples; (2) gives real-time results performing the analysis immediately upon specimen resection; and (3) supplies a representation of the biological structure organization linked to endogenous fluorophores.     

Dependence of fibroblast autofluorescence properties on normal and transformed conditions. Role of the metabolic activity

The dependence of autofluorescence properties on the metabolic and functional engagement and on the transformation condition was studied on single cells. Normal Galliera rat fibroblasts at low subculture passage (cell strain), at high subculture passage (stabilized cell line), and transformed cell line derived from a rat sarcoma were used as a cell model. The study was performed by microspectrofluorometric and fluorescence imaging technique. The autofluorescence properties of cells were studied by excitation at two wavelengths, namely 366 nm and 436 nm, that are known to favor the emission of different fluorophores. Spectral shape analysis indicated that under excitation at 366 nm autofluorescence is ascribable mainly to coenzyme molecules, particularly to reduced pyridine nucleotides, while under excitation at 436 nm, flavin and lipopigment emission is favored. The energetic metabolic engagement of the different cell lines was analyzed in terms both of parameters related to anaerobic-aerobic pathways (biochemical assay) and of mitochondrial features (supravital cytometry). The results showed that the cell strain and the stabilized and transformed cell lines can be distinguished from one another on the basis of both overall fluorescence intensity and the relative contributions of spectral components. These findings indicated a relationship between autofluorescence properties and energetic metabolism engagement of the cells that, in turn, is dependent on the proliferative activity and the transformed condition of the cells. In that it is a direct expression of the energetic metabolic engagement, autofluorescence can be assumed as an intrinsic parameter of the cell biological condition, suitable for diagnostic purposes.     

Ultra-Small Air-Stable Triplet-Triplet Annihilation Upconversion Nanoparticles for Anti-Stokes Time-Resolved Imaging

Image contrast is often limited by background autofluorescence in steady-state bioimaging microscopy. Upconversion bioimaging can overcome this by shifting the emission lifetime and wavelength beyond the autofluorescence window. Here we demonstrate the first example of triplet-triplet annihilation upconversion (TTA-UC) based lifetime imaging microscopy. A new class of ultra-small nanoparticle (NP) probes based on TTA-UC chromophores encapsulated in an organic–inorganic host has been synthesised. The NPs exhibit bright UC emission (400–500 nm) in aerated aqueous media with a UC lifetime of ≈1 μs, excellent colloidal stability and little cytotoxicity. Proof-of-concept demonstration of TTA-UC lifetime imaging using these NPs shows that the long-lived anti-Stokes emission is easily discriminable from typical autofluorescence. Moreover, fluctuations in the UC lifetime can be used to map local oxygen diffusion across the subcellular structure. Our TTA-UC NPs are highly promising stains for lifetime imaging microscopy, affording excellent image contrast and potential for oxygen mapping that is ripe for further exploitation.     

In vivo pH MEASUREMENT AND IMAGING OF TUMOR TISSUE USING A pH-SENSITIVE FLUORESCENT PROBE (5,6–CARBOXYFLUORESCEIN): INSTRUMENTAL AND EXPERIMENTAL STUDIES

Abstract This study evaluated the effectiveness of dual-wavelength ratio fluorescence imaging using a pH-dependent indicator (5,6–carboxyfluorescein, 5,6–CF) for in vivo pH mapping of tissue. A prototype version of a highly sensitive fluorescence imaging device consisting of a modified xenon lamp, an image-intensified camera and a digital imageprocessing system has been developed. 5,6–Carboxyfluorescein was used because its fluorescence emission increases as a function of pH in the physiological (6.0–7.4) pH range. The ratio of fluorescence intensities obtained with the imaging system has been calibrated using aqueous 5,6–CF standards at various pH values. Because the pH of interstitial fluid of malignant tumors tends to be lower than that of normal tissue and can be depressed by glucose administration, experiments were performed on 10 CDF mice bearing lymphoid leukemia P388 grafted subcutaneously. The range of linearity of the calibration curve was obtained between 5.3 and 6.7 with a measured pK, value of 5.93. Consequently the maximum sensitivity was observed in this range. The calculated pH from ratio images was 6.21 ± 0.12 in tumorous tissue. This value was equivalent to those obtained at the same time using microelectrodes (6.2 ± 0.3).
These experiments showed that a dose of 5 mg/kg 5,6–CF and an excitation power density of 2.5 mW/cm² are sufficient to give a fluorescent pH image of tumors. The limitation of 5,6–CF for the in vivo mapping of tissue results from its low pK_a and consequent range of sensitivity. The advantages of this imaging technique compared to microelectrodes are that it (1) is noninvasive, (2) displays a two-dimensional pH image with high resolution (profile distribution of pH in tissue) and (3) can be used to monitor pH over a few hours.     

Confocal/TEM overlay microscopy: a simple method for correlating confocal and electron microscopy of cells expressing GFP/YFP fusion proteins.

Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-microm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.     

Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC)

Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer.     

Spectrally resolved fluorescence imaging of human colonic adenomas

Native fluorescence (autofluorescence) of human tissues can be a valuable source of diagnostic information for detecting premalignant and malignant lesions in the human body. Digital imaging of autofluorescence may be useful for localization of such lesions during endoscopic examinations. Tissue fluorescence of 31 adenomatous polyps obtained from 16 patients has been excited in vitro using the 325 nm line of a He-Cd laser. Digital images of the autofluorescence are recorded in six spectral bands. This study provides new data about the spatial distributions of autofluorescence intensities emitted in different spectral bands by colonic adenomatous lesions and normal colonic mucosa. Areas characterized by autofluorescence intensity lower than in normal mucosa are found for a majority of the polyps under study. The observed patterns of spatial distribution differ for the different spectral bands and for different polypoid lesions. No inverse correlation is found between the emission intensity and the thickness of colonic mucosa. The results indicate the spectral bands most useful for diagnostic applications and demonstrate the complexity of the optical processes involved in shaping both the spectra and intensities of the autofluorescence.     

Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines

We introduce high resolution clear native electrophoresis (CNE) as a powerful technique to resolve enzymatically active mitochondrial complexes from cultured human cell lines and skeletal muscle biopsy samples. Quantitative enzymatic assays can be performed using small amounts of cultured cells with low mitochondria content, for example, around 10 mg of sedimented osteosarcoma cells (wet weight) which is equivalent to around 10 million cells. High resolution CNE offers general advantages for in-gel catalytic activity assays compared to blue native electrophoresis. It seems especially suited for assaying mitochondrial ATP synthase and respiratory chain complexes I and II in cell models of human mitochondrial disorders and for detailed analyses of patient cells and tissues with defects in oxidative phosphorylation.     

Design strategy for a near-infrared fluorescence probe for matrix metalloproteinase utilizing highly cell permeable boron dipyrromethene

Near-infrared (NIR) fluorescence probes are especially useful for simple and noninvasive in vivo imaging inside the body because of low autofluorescence and high tissue transparency in the NIR region compared with other wavelength regions. However, existing NIR fluorescence probes for matrix metalloproteinases (MMPs), which are tumor, atherosclerosis, and inflammation markers, have various disadvantages, especially as regards sensitivity. Here, we report a novel design strategy to obtain a NIR fluorescence probe that is rapidly internalized by free diffusion and well retained intracellularly after activation by extracellular MMPs. We designed and synthesized four candidate probes, each consisting of a cell permeable or nonpermeable NIR fluorescent dye as a F?rster resonance energy transfer (FRET) donor linked to the NIR dark quencher BHQ-3 as a FRET acceptor via a MMP substrate peptide. We applied these probes for detection of the MMP activity of cultured HT-1080 cells, which express MMP2 and MT1-MMP, by fluorescence microscopy. Among them, the probe incorporating BODIPY650/665, BODIPY-MMP, clearly visualized the MMP activity as an increment of fluorescence inside the cells. We then applied this probe to a mouse xenograft tumor model prepared with HT-1080 cells. Following intratumoral injection of the probe, MMP activity could be visualized for much longer with BODIPY-MMP than with the probe containing SulfoCy5, which is cell impermeable and consequently readily washed out of the tissue. This simple design strategy should be applicable to develop a range of sensitive, rapidly responsive NIR fluorescence probes not only for MMP activity, but also for other proteases.     

Quantifying the diffusion of a fluid through membranes by double phase encoded remote detection magnetic resonance imaging

We demonstrate that a position correlation magnetic resonance imaging (MRI) experiment based on two phase encoding steps separated by a delay can be used for quantifying diffusion across a membrane. This method is noninvasive, and no tracer substance or concentration gradient across the membrane is required. Because, in typical membranes, the T1 relaxation time of the fluid spins is usually much longer than the T2 time, we developed and implemented a new position correlation experiment based on a stimulated spin-echo, in which the relaxation attenuation of the signal is dominated by T1 instead of T2. This enables using relatively long delays needed in the diffusion measurements. The sensitivity of the double encoded experiment detected in a conventional way is still low because of the low filling factor of the fluid inside the NMR coil around the sample. We circumvent this problem by using the remote detection technique, which significantly increases the sensitivity, making it possible to do the measurements with gaseous fluids that have a low spin-density compared to liquids. We derive a model that enables us to extract a diffusion constant characterizing the diffusion rate through the membrane from the obtained correlation images. The double phase encoded MRI method is advantageous in any kind of diffusion studies, because the propagator of fluid molecules can directly be seen from the correlation image.     

A FLUORESCENCE IMAGING DEVICE FOR ENDOSCOPIC DETECTION OF EARLY STAGE CANCER – INSTRUMENTAL and EXPERIMENTAL STUDIES

Abstract Visible detection of early stage cancer labelled with the fluorescing porphyrin mixture of dihematoporphyrin-ether and dihematoporphyrin-ester (DHE) is often limited by a high and inhomogeneously distributed level of autofluorescence. A new imaging method for reducing autofluo-rescence is described. The method uses alternating fluorescence excitation with laser light in the violet and blue spectral ranges. Subtraction of the corresponding fluorescence images results in contrast enhancement due to an effective reduction in the autofluorescence contribution. A prototype version of a highly sensitive fluorescence imaging device including a modified krypton ion laser, an image-intensified solid-state TV-camera, a digital image-processing system, and video recording and monitoring has been developed.
Experiments performed with this fluorescence imaging system on a tumorous dog bladder showed that a dose of 0.2 mg DHE kg⁻¹ body wt, which is far below the dose administered for photodynamic therapy (2-5 mg kg⁻'body wt), is sufficient to give a high-contrast fluorescence image. The corresponding excitation power density was about 1 W m⁻². Photobleaching of DHE was observed during fluorescence detection at excitation power densities exceeding 1 kW m⁻². At these high excitation levels DHE fades out nearly completely within 0.25 min.     

Near-infrared Fluorescence Spectroscopy Detects Alzheimer's Disease In Vitro

The purpose of this study was to investigate whether near-infrared (NIR) fluorescence spectroscopy could be used to detect Alzheimer's disease (AD) by brain tissue autofluorescence. Unfixed temporal cortex specimens from AD cases and age-matched, non-AD controls were frozen at autopsy and then thawed just prior to spectral measurement. Spectra of intrinsic tissue fluorescence induced by 647 nm light were recorded from 650 to 850 nm. We used principal component analysis of the tissue spectra from 17 AD cases and 5 non-AD control cases in a calibration study to establish a diagnostic algorithm. Retrospectively applied to the calibration set, the algorithm correctly classified 23 of 24 specimens. In a prospective study of 19 specimens from 5 AD brains and 2 non-AD control brains, 3 of the 4 control specimens and all AD specimens were correctly diagnosed. Both the excitation light used and the measured brain tissue autofluorescence are at NIR wavelengths that can propagate through skull and overlying tissue. Therefore, our results demonstrate an optical spectroscopic technique that carries direct molecular level information about disease. This is the first step toward a clinical tool that has the potential to be applied to the noninvasive diagnosis of AD in living patients.     

Stationary Headband for Clinical Time-of-Flight Optical Imaging at the Bedside

Conventional brain-imaging modalities may be limited by high cost, difficulty of bedside use, noncontinuous operation, invasiveness or an inability to obtain measurements of tissue function, such as oxygenation during stroke. Our goal was to develop a bedside clinical device able to generate continuous, noninvasive, tomographic images of the brain using low-power nonionizing optical radiation. We modified an existing stage-based time-of-flight optical tomography system to allow imaging of patients under clinical conditions. First, a stationary headband consisting of thin, flexible optical fibers was constructed. The headband was then calibrated and tested, including an assessment of fiber lengths, the existing system software was modified to collect headband data and to perform simultaneous collection of data and image reconstruction, and the existing hardware was modified to scan optically using this headband. The headband was tested on resin models and allowed for the generation of tomographic images in vitro; the headband was tested on critically ill infants and allowed for optical tomographic images of the neonatal brain to be obtained in vivo.     

The application of a compact multispectral imaging system with integrated excitation source to in vivo monitoring of fluorescence during topical photodynamic therapy of superficial skin cancers

A novel, compact and low-cost multispectral fluorescence imaging system with an integrated excitation light source is described. Data are presented demonstrating the application of this method to in vivo monitoring of fluorescence before, during and after topical 5-aminolevulinic acid photodynamic therapy of superficial skin cancers. The excitation source comprised a fluorescent tube with the phosphor selected to emit broadband violet light centered at 394 nm. The camera system simultaneously captured spectrally specific images of the fluorescence of the photosensitizer, protoporphyrin IX, the illumination profile and the skin autofluorescence. Real-time processing enabled images to be manipulated to create a composite image of high contrast. The application and validation of this method will allow further detailed studies of the characteristics and time-course of protoporphyrin IX fluorescence, during topical photodynamic therapy in human skin in vivo.     

High-resolution transmission electron microscopy using negative spherical aberration.

A novel imaging mode for high-resolution transmission electron microscopy is described. It is based on the adjustment of a negative value of the spherical aberration C S of the objective lens of a transmission electron microscope equipped with a multipole aberration corrector system. Negative spherical aberration applied together with an overfocus yields high-resolution images with bright-atom contrast. Compared to all kinds of images taken in conventional transmission electron microscopes, where the then unavoidable positive spherical aberration is combined with an underfocus, the contrast is dramatically increased. This effect can only be understood on the basis of a full nonlinear imaging theory. Calculations show that the nonlinear contrast contributions diminish the image contrast relative to the linear image for a positive-C S setting whereas they reinforce the image contrast relative to the linear image for a negative-C S setting. The application of the new mode to the imaging of oxygen in SrTiO3 and YBa2Cu3O7 demonstrates the benefit to materials science investigations. It allows us to image directly, without further image processing, strongly scattering heavy-atom columns together with weakly scattering light-atom columns.     

Denaturation mapping of Saccharomyces cerevisiae

Optical mapping of DNA provides large-scale genomic information that can be used to assemble contigs from next-generation sequencing, and to detect rearrangements between single cells. A recent optical mapping technique called denaturation mapping has the advantage of using physical principles rather than the action of enzymes to probe genomic structure. Denaturation mapping uses fluorescence microscopy to image the pattern of partial melting along a DNA molecule extended in a channel of cross-section 120 nm at the heart of a nanofluidic device. We used denaturation mapping to locate single DNA molecules on the yeast genome (12.1 Mbp) by comparing images to a computationally predicted map for the entire genome sequence. By locating 84 molecules we assembled an optical map of the yeast genome with > 50% coverage.     

Optical coherence tomography��current technology and applications in clinical and biomedical research

Optical coherence tomography (OCT) is a noninvasive imaging technique that provides real-time two- and three-dimensional images of scattering samples with micrometer resolution. By mapping the local reflectivity, OCT visualizes the morphology of the sample. In addition, functional properties such as birefringence, motion, or the distributions of certain substances can be detected with high spatial resolution. Its main field of application is biomedical imaging and diagnostics. In ophthalmology, OCT is accepted as a clinical standard for diagnosing and monitoring the treatment of a number of retinal diseases, and OCT is becoming an important instrument for clinical cardiology. New applications are emerging in various medical fields, such as early-stage cancer detection, surgical guidance, and the early diagnosis of musculoskeletal diseases. OCT has also proven its value as a tool for developmental biology. The number of companies involved in manufacturing OCT systems has increased substantially during the last few years (especially due to its success in opthalmology), and this technology can be expected to continue to spread into various fields of application.