A spatial filter was used to improve the beam profile before directing into the microscope.
The two excitation beams were independently modulated and were converged using a beam combiner and then directed into the microscope scan heads.
The beam in the sample arm was coupled into the epifluorescence port of an upright microscope (Axioskop2 MAT, Carl Zeiss, Thornwood, NY) and directed to the microscope objective (40× Achroplan, 0.8 NA or 63× Achroplan, 0.95 NA, Carl Zeiss) by using a short-pass dichroic mirror (635dcspxruv3p, Chroma, Rockingham, VT).
Illumination for confocal imaging was provided by solid state lasers (40 mW 442 nm for CFP; 100 mW 488 nm for EGFP; 100 mW 523 nm for YFP and 500 mW 561 nm for FusionRed) that were directed to the microscope by a custom-designed optical fiber-coupled laser combiner (Spectral Applied Research, Canada; Shin et al., 2010).
As conventional image processing technique based on a single threshold of gray intensity cannot accurately determine the width of both fine and wide cracks, a new double-threshold algorithm is developed and its accuracy is verified by comparing with direct measurement under the microscope.
The first method consisted of direct counting under the microscope of the number of beads or cells inside droplets printed from a solution of known concentration.
A second approach to evaluate thymocyte/TEC adhesion was by direct counting, under the microscope, the numbers of thymocytes which had adhered to TEC cultures.
The set was discarded for research purposes only and does not show any objective wear either using direct observation or the microscope.
The use of Sudan Black B for staining and direct observation under the microscope enables the rapid observation of the qualitative status of lipid production in the cells (Thakur et al. 1988).
We used a method described in (Ackermann et al., 2003) to determine the reproductive output of individual stalked cells of the bacterium C. crescentus by means of direct observation under the microscope.
