Multicolor fluorescence microscopy using static light sheets and a single-channel detection

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.

Serial imaging of micro-agents and cancer cell spheroids in a microfluidic channel using multicolor fluorescence microscopy

“…Crosstalk analysis is carried out to visualize the effect of excitation light and emitted fluorescence signals on the acquired individual fluorescence images [ 14 , 39 ]. Fluorophores are excited with single, double, and triple combinations of the center wavelengths at 470 nm, 565 nm, and 775 nm.…”

“…In numerous application domains ranging from single-cell visualization to rapid diagnosis, multicolor fluorescence microscopy is recognized as a key emerging technology [11][12][13][14][15][16][17][18][19][20][21][22][23]. The working principle of this microscopy technique is based on performing imaging using spectrally-different conventional fluorescent dyes [11], endogenous fluorophores [22], fluorescent proteins [24], or quantum dots [25].…”

“…Since the delay associated with the spectrum band change is removed, the acquisition is limited by the frame rate of the imaging sensor. Depending on the spectrum of the fluorophores, the acquired images suffer from crosstalk [14]. To minimize the crosstalk, emitted light from the fluorophores is separated using dichroic mirrors and direct to individual imaging sensors using a common optical path [16,18,[20][21][22].…”

Multicolor light‐sheet microscopy for a large field of view imaging: A comparative study between Bessel and Gaussian light‐sheets configurations

“…It consists of three different CW‐lasers (Coherent, Obis; operating at 445, 488, and 561 nm). The lasers are sequentially turned on/off and synchronized with a single camera's acquisition frame rate for rapid multicolor image acquisition [20]. The system can work either under DSLM configuration, using Bessel or Gaussian beams for the excitation, or under a SPIM configuration.…”

Morphological and Calcium Signaling Alterations of Neuroglial Cells in Cerebellar Cortical Dysplasia Induced by Carmustine

“…In LSFM, a light-sheet excites a single fluorescent plane within the sample, while the excited fluorescent plane is imaged through a microscope objective and a camera placed orthogonal to the excitation light-sheet. LSMF can perform wide-field optical sectioned (tomographic-like) imaging with good optical resolution over volumetric samples at different length scales [ 21 , 22 , 23 , 24 ].…”