Light microscopy of mammalian gametes and embryos: methods and applications.

In recent years, we have witnessed an unprecedented advancement of light microscopy techniques which has allowed us to better understand biological processes occurring during oogenesis and early embryonic development in mammals. In short, two modes of cellular imaging are now available: those involving fluorescent labels and those which are fluorophore-free. Fluorescence microscopy, in its various forms, is used predominantly in research, as it provides detailed information about cellular processes; however, it can involove an increased risk of photodamage. Fluorophore-free techniques provide, on the other hand, a smaller amount of biological data but they are safer for cells and therefore can be potentially used in a clinical setting. Here, we review various fluorescence and fluorophore-free visualisation approaches and discuss their applicability in developmental biology and reproductive medicine.

Enhanced radiation damage caused by iodinated contrast agents during CT examination.

OBJECTIVE: To access the effect of iodinate contrast agent (ICA) on DNA double-stand breaks (DSBs) in human peripheral blood lymphocytes during computed tomography (CT) examinations. MATERIALS AND METHODS: This present study was approved by the institutional ethics committee; written informed patient consent was obtained from 70 patients. A total of 48 patients underwent computed tomography urography (CTU), in which only one time CT scanning was examined after injecting ICA, and 22 patients received unenhanced whole abdominal CT, among them 10 patients were selected to get ICA injection immediately after irradiation. Blood samples were taken from all patients prior to and immediately after CT scan, as well as 8min after the injection of ICA. The lymphocytes in these blood samples were separated by using density-gradient centrifugation, fixed and immunostained with γH2AX antibody. The average number of phosphorylated histone H2AX (γH2AX) foci per lymphocyte was counted under a fluorescence microscopy. Differences in the number of γH2AX-foci were statistically analyzed using independent sample t test and one way ANOVA. RESULT: The three patient groups had no significant differences in the baseline foci numbers(P>0.05). The γH2AX-focus levels increased in both groups after CT scan. Patients who underwent CTU examinations had a greater DSBs level (mean±standard error of mean, 0.945±0.184 foci per cell) than those who received unenhanced whole abdominal CT scan (mean±standard error of mean, 0.700±0.112 foci per cell), increasing by about 37.9%; The ICA injected before CT scan itself had an effect on the DSBs, which increased DSBs level by approximately 90.3% (0.059±0.018vs 0.031±0.025, P<0.05), but no significant difference was found if added after irradiation, increasing DSBs level only by 3.2% approximately (0.711±0.091vs 0.689±0.108, P=0.499). CONCLUSION: The iodinated contrast agent itself can lead to an increase in the level of DSBs as assessed with γH2AX foci formation, and the application of ICA can amplify DNA damage induced by diagnostic x-ray procedures such as whole abdominal CT.

Live Cell Imaging: Assessing the Phototoxicity of 488 and 546 nm Light and Methods to Alleviate it.

In live cell imaging of fluorescent proteins, phototoxicity of the excitation light can be problematical. Cell death is obvious, but reduced cell viability can make the interpretation of observations error prone. We characterized the phototoxic consequences of 488 and 546 nm light on untransformed human cells and tested methods that have or could be used to alleviate photodamage. Unlabeled RPE1 cells were given single 0.5-2.5 min irradiations in early G1 from a mercury arc lamp on a fluorescence microscope. Four hundred eighty-eight nanometer light produced a dose-dependent decrease in the percentage of cells that progressed to mitosis, slowing of the cell cycle for some of those entering mitosis, and a ∼12% incidence of cell death for the highest dose. For 546 nm light we found a 10-15% reduction in the percentage of cells entering mitosis, no strong dose dependency, and a ∼2% incidence of cell death for the longest irradiations. For cells expressing GFP-centrin1 or mCherry-centrin1, fewer entered mitosis for each dose than unlabeled cells. For constant total dose 488 nm light irradiations of unlabeled cells, reducing the intensity 10-fold or spreading the exposures out as a series of 10 sec pulses at 1 min intervals produced a minor and not consistent improvement in the percentage of cells entering mitosis. Reducing oxidative processes, by culturing at ∼3% oxygen or adding the reducing agent Trolox noticeably increased the fraction of cells entering mitosis. Thus, for long-term imaging there can be value to using RFP constructs and for GFP-tagged proteins reducing oxidative processes. J. Cell. Physiol. 232: 2461-2468, 2017. © 2016 Wiley Periodicals, Inc.

Estimating the risk of squamous cell cancer induction in skin following nonlinear optical imaging.

High power femto-second (fs) laser pulses used for in-vivo nonlinear optical (NLO) imaging can form cyclobutane pyrimidine dimers (CPD) in DNA, which may lead to carcinogenesis via subsequent mutations. Since UV radiation from routine sun exposure is the primary source of CPD lesions, we evaluated the risk of CPD-related squamous cell carcinoma (SCC) in human skin due to NLO imaging relative to that from sun exposure. We developed a unique cancer risk model expanding previously published estimation of risk from exposure to continuous wave (CW) laser. This new model showed that the increase in CPD-related SCC in skin from NLO imaging is negligible above that due to regular sun exposure.

A quantitative method for measuring phototoxicity of a live cell imaging microscope.

Fluorescence-based imaging regimes require exposure of living samples under study to high intensities of focused incident illumination. An often underestimated, overlooked, or simply ignored fact in the design of any experimental imaging protocol is that exposure of the specimen to these excitation light sources must itself always be considered a potential source of phototoxicity. This can be problematic, not just in terms of cell viability, but much more worrisome in its more subtle manifestation where phototoxicity causes anomalous behaviors that risk to be interpreted as significant, whereas they are mere artifacts. This is especially true in the case of microbial pathogenesis, where host-pathogen interactions can prove especially fragile to light exposure in a manner that can obscure the very processes we are trying to observe. For these reasons, it is important to be able to bring the parameter of phototoxicity into the equation that brings us to choose one fluorescent imaging modality, or setup, over another. Further, we need to be able to assess the risk that phototoxicity may occur during any specific imaging experiment. To achieve this, we describe here a methodological approach that allows meaningful measurement, and therefore relative comparison of phototoxicity, in most any variety of different imaging microscopes. In short, we propose a quantitative approach that uses microorganisms themselves to reveal the range over which any given fluorescent imaging microscope will yield valid results, providing a metrology of phototoxic damage, distinct from photobleaching, where a clear threshold for phototoxicity is identified. Our method is widely applicable and we show that it can be adapted to other paradigms, including mammalian cell models.

Light exposure and cell viability in fluorescence microscopy.

Test systems for measuring cell viability in optical microscopy (based on colony formation ability or lysosomal integrity) were established and applied to native cells as well as to cells incubated with fluorescence markers or transfected with genes encoding for fluorescent proteins. Human glioblastoma and Chinese hamster ovary cells were irradiated by various light doses, and maximum doses where at least 90% of the cells survived were determined. These tolerable light doses were in the range between 25 J cm⁻² and about 300 J cm⁻² for native cells (corresponding to about 250-3000 s of solar irradiance and depending on the wavelength as well as on the mode of illumination, e.g. epi- or total internal reflection illumination) and decreased to values between 50 J cm⁻² and less than 1 J cm⁻² upon application of fluorescent markers, fluorescent proteins or photosensitizers. In high-resolution wide field or laser scanning microscopy of single cells, typically 10-20 individual cell layers needed for reconstruction of a 3D image could be recorded with tolerable dose values. Tolerable light doses were also maintained in fluorescence microscopy of larger 3D samples, e.g. cell spheroids exposed to structured illumination, but may be exceeded in super-resolution microscopy based on single molecule detection.

Diagnosis of urothelial carcinoma of the bladder using fluorescence endoscopy.

Bladder cancer is a frequent disease and represents the second most common genitourinary neoplasm. Although many aspects of the management of superficial bladder cancer are now well established, significant challenges remain, which influences patient outcome. Early detection and treatment of recurrent disease is required to optimize bladder preservation, reduce patient morbidity, and increase quality of life and survival. Fluorescence endoscopy, often referred to as 'photodynamic diagnosis' (PDD), with intravesical application of photosensitizing agents, has been developed to enhance the early detection of bladder cancer. There is growing evidence that PDD using 5-aminolaevulinic acid (ALA), hexyl-ALA ester or hypericin enhances the detection of bladder cancer, particularly of high-grade flat lesions. Furthermore, transurethral resection of bladder tumour under fluorescence guidance reduces the risk of recurrent tumours. However, the impact on the progression of disease remains unclear and must be investigated in prospective randomized trials.

Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy.

The green fluorescent protein (GFP) and other intrinsically fluorescent proteins (IFPs) are popular reporters because they allow visualization of cellular constituents in living specimens. IFP technology makes it possible to view dynamic processes in living cells, but extended observation, using fluorescence microscopy (both wide-field and confocal), can result in significant light energy exposure. Therefore, it is possible that cells experience light-induced damage that alters cell physiology and confounds observations. To understand the impact that extended viewing has on cells, we obtained quantitative information about the effect of light energy dose and observation conditions on tobacco BY-2 cell physiology. Our results show a non-linear relationship between the excitation light intensity and mitotic arrest, and the frequency of mitotic arrest is dependent on the presence of an IFP that absorbs the excitation light. Moreover, fluorescence microscopy induces the production of reactive oxygen species (ROS), as assayed using BY-2 cells loaded with oxidation-sensitive dyes, and the level of ROS production increases if the cells express an IFP that absorbs the excitation light energy. The dye oxidation follows sigmoidal kinetics and is reversible if the cells are exposed to low irradiation levels. In addition, the dye oxidation rate shows a non-linear relationship to the excitation light intensity, and a good correlation exists between photobleaching, mitotic arrest, and dye oxidation. The data highlight the importance of ROS scavenging for normal mitotic progression, and provide a reference for judiciously choosing conditions that avoid photobleaching that can lead to ROS accumulation and physiological damage.

Intravital fluorescence microscopy and phototocicity: effects on leukocytes.

The purpose of this investigation was to clarify whether the light exposure of fluorescently marked leukocytes and/or plasma influences the leukocyte behavior (rolling and firm adhesion). Anaesthetized Balb/c mice were laparotomized to expose the mesentery of the terminal ileum. Animals were randomly assigned to 5 experimental groups. One group was studied in trans-illumination mode, the other four groups received iv-bolus injections of either FITC-dextran (FITC-dx) 150 kD, rhodamine 6 G (rh6G), a combination of FITC-dx and rh6G, or acridine orange, respectively, and were then studied in epi-illumination mode. In each animal, eight vessels (6 venules, 2 arterioles) were exposed five times for 60 sec. to continuous light of the appropriate excitation wavelength with a 20-min. interval between exposures. The vessel diameters and leukocyte-endothelial cell interactions were quantified using intravital fluorescence microscopy. The diameters of arterioles and postcapillary venules remained unchanged in all groups studied. Rolling and adherent leukocytes were observed in postcapillary venules only and there were no significant differences between all groups. Under these conditions, the exposure of fluorescently labeled leukocytes and/or plasma to standard light levels for up to 300 seconds has no significant impact on leukocyte-endothelial cell interactions in mesenteric postcapillary venules.