The VCM100 can be also be configured for simultaneous multi-channel fluorescence detection. Figure 1 represents the imaging system setup.
Figure 1: VCM100 configured as two channel confocal fluorescence microscope
In this setup, the emission signal is still collected into the single mode fiber for spatial filtering, but then the fluorescent signal is split using a dichroic and sent to two different PMT detectors. The spectral range is determined by the optical system (for example, .the choice of dichroic filters or emissions filters). Alternatively, the fluorescence signal could also be split before the fiber collimator using a dichroic filter if one prefers to separate the two channels into two different single mode fibers. The VCM100 scan head is customizable, and users are encouraged to create their own design, (e.g. free space coupling is useful for multiphoton setups).
Figure 2 shows a longitudinal cross-section of a Pisum seed imaged with VCM100 using the two channel fluorescence setup shown above. The green channel was assigned to the light collected between 569 and 600 nm, while the red channel collected the light from 505 to 555 nm. For the image shown below, a line average was applied during data collection to increase the signal-to-noise ratio.
Figure 2. Pseudo-Color confocal fluorescence imaging of human skin (a) and fern leaf (b) mounted on a standard microscope slide. These images show the psuedo-color projection and cross-sectional images created from a series of individual Z-slices taken by the VCM100 with an infinity corrected 60X water immersion objective. The samples were excited with fiber coupled 405 nm light from a laser diode. The emission signal was separated by a dichroic filter and directed through a single mode fiber to a PMT for detection.
Figure 3: Fluorescent image of a pine pollen grain taken with the modified multi-channel VCM100. (a) A single optical slice of the pollen grain, and (b) 3D rendering of the sample image data.
Figure 4: 3D rendering of the multi-channel confocal fluorescence image data acquired from a pine root sample fixed on a microscope slide.