Multiphoton laser-scanning microscopy is still developing rapidly, both technologically and by broadening its range of application. Technical progress has been made in the optimization of fluorophores, in increasing the imaging depth of multiphoton microscopy, and in microscope miniaturization. These advances further facilitate the study of neuronal structure and signaling in living and even in behaving animals, in particular in combination with the expression of fluorescent proteins. In addition, nonlinear optical contrast mechanisms other than multiphoton excitation of fluorescence are being explored.
"While this method is very straightforward and has the advantage that very fast frame rates can be achieved, the ability to attain high-spatial-resolution images deep within scattering tissues is greatly reduced –. In highly scattering media, signal photons that are scattered appear to originate from a position other than that of the focal volume, and thus appear out of focus , . This is not, however, an issue in weakly scattering tissue, or at shallow depths within strongly scattering specimens. "
[Show abstract][Hide abstract] ABSTRACT: Multifocal multiphoton laser scanning microscopy (mfMPLSM) in the biological and medical sciences has the potential to become a ubiquitous tool for obtaining high-resolution images at video rates. While current implementations of mfMPLSM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for mfMPLSM in which whole-field detection with a single detector, rather than detection with a matrix of detectors, such as a charge-coupled device (CCD) camera, is implemented. This advance makes mfMPLSM fully compatible for use in imaging through scattering media. Further, we demonstrate that this method makes it possible to simultaneously obtain multiple images and view differences in excitation parameters in a single scan of the specimen.
IEEE Journal of Selected Topics in Quantum Electronics 03/2012; 18(1-18):14 - 28. DOI:10.1109/JSTQE.2010.2077622 · 2.83 Impact Factor
"Quantitative methods for imaging [Ca 2+ ] i in vivo in the striatum have been technically challenging. Thus while confocal and multiphoton microscopy provide superior cellular resolution for [Ca 2+ ] i fluorescence imaging, they have limited imaging depth (Helmchen and Denk, 2002; Zipfel et al., 2003)., Recent advances in microprobe techniques (e.g., the gradual refractive index lens (Jung et al., 2004; Levene et al., 2004) and micro-prisms (Chia and Levene, 2009, 2010)) now allow for dynamic [Ca 2+ ] i measurements in subcortical brain regions in vivo. "
[Show abstract][Hide abstract] ABSTRACT: Cocaine induces fast dopamine increases in brain striatal regions, which are recognized to underlie its rewarding effects. Both dopamine D1 and D2 receptors are involved in cocaine's reward but the dynamic downstream consequences of cocaine effects in striatum are not fully understood. Here we used transgenic mice expressing EGFP under the control of either the D1 receptor (D1R) or the D2 receptor (D2R) gene and microprobe optical imaging to assess the dynamic changes in intracellular calcium ([Ca(2+)](i)) responses (used as marker of neuronal activation) to acute cocaine in vivo separately for D1R- versus D2R-expressing neurons in striatum. Acute cocaine (8 mg/kg, i.p.) rapidly increased [Ca(2+)](i) in D1R-expressing neurons (10.6 ± 3.2%) in striatum within 8.3 ± 2.3 min after cocaine administration after which the increases plateaued; these fast [Ca(2+)](i) increases were blocked by pretreatment with a D1R antagonist (SCH23390). In contrast, cocaine induced progressive decreases in [Ca(2+)](i) in D2R-expressing neurons (10.4 ± 5.8%) continuously throughout the 30 min that followed cocaine administration; these slower [Ca(2+)](i) decreases were blocked by pretreatment with a D2R antagonist (raclopride). Since activation of striatal D1R-expressing neurons (direct-pathway) enhances cocaine reward, whereas activation of D2R-expressing neurons suppresses it (indirect-pathway) (Lobo et al., 2010), this suggests that cocaine's rewarding effects entail both its fast stimulation of D1R (resulting in abrupt activation of direct-pathway neurons) and a slower stimulation of D2R (resulting in longer-lasting deactivation of indirect-pathway neurons). We also provide direct in vivo evidence of D2R and D1R interactions in the striatal responses to acute cocaine administration.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 09/2011; 31(37):13180-90. DOI:10.1523/JNEUROSCI.2369-11.2011 · 6.34 Impact Factor
"At the same time, the imaging depth of two-photon microscopy is much larger because of the reduced scattering of infrared light that is used to excite the sample. Therefore , two-photon microscopy is an ideal tool to study deep tissue and even living animals (Helmchen and Denk, 2002). Two main concepts that were capable to effectively improve the axial resolution have been introduced independently, i.e. 4Pi microscopy (Hell and Stelzer, 1992) and I 5 M microscopy (Gustafsson et al., 1999). "
[Show abstract][Hide abstract] ABSTRACT: In the recent past, a variety of fluorescence microscopy methods emerged that proved to bypass a fundamental limit in light microscopy, the diffraction barrier. Among diverse methods that provide subdiffraction spatial resolution, far-field microscopic techniques are in particular important as they can be operated in complex biological samples such as cells or tissue. Valuable new insights into biomolecular structure, organization and even dynamic processes in living cells have been gained with these novel microscopic techniques. In the present review, the most important concepts of far-field microscopy with subdiffraction resolution are introduced. The underlying physical concepts are discussed, and practical considerations for the application of these methods are made.
Journal of Biotechnology 03/2010; 149(4):243-51. DOI:10.1016/j.jbiotec.2010.03.012 · 2.87 Impact Factor
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