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Abstract
.We present a multicolor fluorescence microscope system, under a selective plane illumination microscopy (SPIM) configuration, using three continuous wave-lasers and a single-channel-detection camera. The laser intensities are modulated with three time-delayed pulse trains that operate synchronously at one third of the camera frame rate, allowing a sequential excitation and an image acquisition of up to three different biomarkers. The feasibility of this imaging acquisition mode is demonstrated by acquiring single-plane multicolor images of living hyphae of Neurospora crassa. This allows visualizing simultaneously the localization and dynamics of different cellular components involved in apical growth in living hyphae. The configuration presented represents a noncommercial, cost-effective alternative microscopy system for the rapid and simultaneous acquisition of multifluorescent images and can be potentially useful for three-dimensional imaging of large biological samples.
Highlights
Developmental biology studies require both two-dimensional (2-D) and three-dimensional (3-D) visualization of different dynamic microstructures, marked with specific fluorescent proteins, to elucidate their functionality in a determined biological process
Confocal laser scanning microscopy (CLSM) and/or multiphoton microscopy (MPM) have been successfully used to excite multiple fluorescent markers and visualize selectively different planes of the samples.[1,2,3,4,5]. These techniques are not suited to visualize large volumetric samples because the image is constructed sequentially point by point while the laser is raster scanning the sample, imposing a strong limit on the acquisition speed. Other systems, such as spinning disk confocal systems (SDCS), use a rotating disk with a pattern of pinholes installed in a microscope plane that is conjugated with the specimen.[6]
SDCS provide faster image acquisition, and are better suited to track the dynamics of microstructures that move within the scanning frame rate of a confocal laser scanning microscopy (CLSM), producing images without jagged edges and better definition
Summary
Developmental biology studies require both two-dimensional (2-D) and three-dimensional (3-D) visualization of different dynamic microstructures, marked with specific fluorescent proteins, to elucidate their functionality in a determined biological process For those purposes, confocal laser scanning microscopy (CLSM) and/or multiphoton microscopy (MPM) have been successfully used to excite multiple fluorescent markers and visualize selectively different planes of the samples (obtaining the so-called optically sectioned images).[1,2,3,4,5] these techniques are not suited to visualize large volumetric samples because the image is constructed sequentially point by point while the laser is raster scanning the sample, imposing a strong limit on the acquisition speed. 3-D imaging using CLSM, MPM, or SDCS, is not suited for large biological specimens due to the objective high numerical aperture and short working distance commonly utilized in those techniques
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