Fluorescence microscopy is an invaluable technique for investigating structural and biochemical changes in cells and tissues. While it is preferable to study these changes in living specimens, such studies are often compromised by the destructive properties of light which can cause cellular damage either directly (photoablation) or indirectly by generating toxic by-products (phototoxicity). To minimize these problems, new methods of illuminating cells are being developed. In particular, ultrafast infrared lasers have been employed to excite fluorophores at one-half and one- third the wavelength of the laser by a process called multiphoton excitation. This process limits excitation to a small volume of indicator which, together with fast scanning of the sample, may reduce photodamage. One source of photodamage is light-induced stimulation of H2O2 in cells. In this report, we tested whether scanning with an ultrafast Ti:sapphire laser could stimulate H2O2 production in cultured human and monkey cells measured with the fluorescent indicator dichlorodihydrofluorescein. We demonstrate that illumination at 800 - 900 nm induced H2O2 production in cells when laser power was increased above 10 mW (at the specimen plane). The frequency of scanning (duty cycle) also influenced H2O2 production indicating that a trade-off between power and exposure time may be an appropriate way to control this type of toxicity. Alternatively, high power and increased exposure time could provide an effective means for controlling H2O2 production and subsequent damage to cellular structures.
There is growing evidence that generation of reactive oxygen molecules (e.g., hydrogen peroxide, superoxide, hydroxyl and nitric oxide radicals) plays an important role in cell death. In this report we evaluated the effectiveness of the membrane permeable probe carboxy-dichlorodihydrofluorescein diacetate acetoxymethyl ester (C-DCDHF-DA-AM) for imaging the production of H2O2 in cultured cells. We examined the properties of three derivatives of the ester in saline droplets and compared the results with responses recorded from cells loaded with the ester. Results indicated that fluorescence was generated in cells and droplets by a photo-oxidative process involving H2O2. Videomicroscopy demonstrated that the cellular responses originated in small vesicles (presumably peroxisomes), with large responses filling the cytosol and enveloping the nucleus. We interpreted these responses as due to light-induced activation of flavin-containing oxidases, which generate H2O2 in peroxisomes, followed by diffusion of H2O2 throughout the cell. Escape of H2O2 from peroxisomes into Fe2+-containing compartments could have dire consequences on cell viability due to the production of hydroxyl free radicals. Such a mechanism could underly the phototoxic effects of visible light on cultured cells.
We have used digital imaging microscopy to evaluate possible guidance mechanisms of mouse neuroblastoma cells cultured on microfabricated glass substrates. Substrates were bound with either small charged amine or uncharged alkane molecules using silane-coupling chemistry or covered with proteins (laminin, collagen, fibronectin, albumin) by adsorption. Photolithographic procedures were used to pattern substrates with amine - alkane, amine - protein and alkane - protein combinations. Interference reflection microscopy (IRM) was used to visualize sites of cellular attachment on substrates (focal and close contacts), and image processing techniques were used to quantify the results in three different ways: total cellular area in contact with a substrate, area corresponding to focal and close contacts, and the percentage of focal and close contacts on each substrate. Cells attached to and extended neurites on each of the substrates were tested. IRM images of growth cones displayed similar gray levels on amine, alkane, fibronectin, and albumin substrates, whereas images on laminin and collagen were brighter. Brightness on laminin substrates was correlated with less area of focal contact and greater area with no contact. When cells were provided with choices on patterned substrates, they displayed the following preference of attachment: laminin, fibronectin, collagen>amine>alkane>albumin. This hierarchy reflected greater total surface area on preferred substrates. There was, however, no correlation between the hierarchy and the area corresponding to focal and close contacts or the percentage of contacts on different substrates. Our results support several recent observations which demonstrated that guidance was not correlated with differential adhesivity. These results are more in line with the theory that guidance is controlled by a signal transduction mechanism that couples locomotion with activation of membrane receptors.
Conference Committee Involvement (2)
Manipulation and Analysis of Biomolecules, Cells and Tissues
28 January 2003 | San Jose, CA, United States
Functional Imaging and Optical Manipulation of Living Cells and Tissues
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.