Simultaneous 2-D analysis of multiple fluorescent probes using multispectral imaging microscopy

This research discusses the use of a new multispectral imaging system in fluorescence microscopy applications. We present the results of experiments designed to demonstrate the utility of this instrument for biological investigations. A phantom object was constructed using fluorescent microspheres and imaged to demonstrate the ability of the system to differentiate microscopic structures based on spectral properties. Excellent spectral isolation of subcellular structures, differentially labeled with fluorescent probes, was also obtained in images of neurons and rat sinusoidal epithelial (RSE-1) cells demonstrating that separation of subcellular structures in biological imaging applications is possible with high spatial resolution (up to 0.25 microns) and excellent signal to noise ratio (SNR>30). A final imaging experiment using this device with a confocal micro-endoscope shows the flexibility of the developed instrument when used with different imaging systems. In addition, the results indicate that multispectral investigations of tissue in-vivo may be possible.