Researchers and industrial scientists have been using confocal laser scanning microscopy for more than two decades due to its ability to image fluorescent samples with superior image quality when compared to standard bright field and epi-fluorescent microscope technologies. The key design feature that makes this possible is the use of a confocal iris, pinhole, or other aperture located within the optical system—used to spatially separate the desired fluorescent signal from the out-of-focus background fluorescence—making this technique ideal for optical sectioning. Combing this with image processing software allows cross-sectional imaging, 2D projections and psuedo-3D rendering of the optically sectioned sample.
Thorlabs has recently developed the VCM100, a video-rate laser scanning microscope system that is both compact and fully user customizable. A few sample fluorescent images obtained by this system are shown below.
Figure 1: Pseudo-Color 3D confocal fluorescence imaging of a collection of pollen grains mounted on a standard microscope slide and optically sectioned using a 60X objective. Sample was excited at 405 nm using a laser diode (DL5146-152), and the emission signal was selected using a dichroic mirror (FD05D) and an emission filter (505 + 15 nm). Total Z-sectioning was 80 µm in depth.
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: Confocal fluorescence images of the head of a peach worm, 440x430 µm. (1-8) images of Z- slices separated by 10 µm in depth. ( 9) pseudo color projection image of 256 slices. Total Z depth range is 80 µm.