Population estimation of human embryonic stem cell cultures.

Traditionally, the population of human embryonic stem cell (hESC) culture is estimated through haemacytometer counts, which include harvesting the cells and manually analyzing a fraction of an entire population. Obviously, through this highly invasive method, it is not possible to preserve any spatial information on the cell population. The goal of this study is to identify a fast and consistent method for in situ automated hESC population estimation to quantitatively estimate the cell growth. Therefore, cell cultures were fixed, stained, and their nuclei imaged through high-resolution microscopy, and the images were processed with different image analysis techniques. The proposed method first identifies signal and background by computing an image specific threshold for image segmentation. By applying a morphological operator (watershed), we split most physically overlapping nuclei, leading to a pixel area distribution of isolated signal areas on the image. On the basis of this distribution, we derive a nucleus area model, describing the distribution of the area of cell debris, single nuclei, and small groups of connected nuclei. Through the model, we can give a quantitative estimation of the population. The focus of this study is on low-density human embryonic stem cell populations; hence cultures were measured at days 2-3 after seeding. Compared with manual cell counts, the automatic method achieved higher accuracy with <6% error.

Three dimensional sidewall measurements by laser fluorescent confocal microscopy.

Precise three dimensional (3D) profile measurements of vertical sidewalls of concave micro-structures are impossible by conventional profiling techniques. This paper introduces a simple technique which can obtain 3D sidewall geometry by means of laser fluorescent confocal microscopy and an intensity gradient algorithm. The measurement principle is: when a concave micro-structure is filled up with fluorescent solution, the position where the maximum intensity variation lays represents the profile of the micro-structure in the fluorescent 3D volume image. The physical essence behind this measurement principle is analyzed in this paper in detail. The strengths and limitations of this technique are studied by experiments or by illustrations. The factors that are able to improve the measurement accuracy are discussed. This technique has demonstrated the capability for measuring of 3D geometry of various concave features, such as vertical, buried and other micro channels with sub-mum (RMS) measurement accuracy and repeatability.

Measurement of a fiber-end surface profile by use of phase-shifting laser interferometry.

We describe a laser interferometric system in which two objectives are used to measure surface profile on a connectorized fiber-end surface. By the use of the proposed illumination design a He-Ne laser as a point light source is transformed to an extended light source, which is beneficial to localize interference fringe pattern near the test surface. To obtain an optimal contrast of the interference fringe pattern, the flat mirror with an adjustable reflection ratio is used to suit different test surfaces. A piezoelectric transducer attached on the reference mirror can move precisely along the optical axis of the objective and permits implementation of four-step phase-shifting interferometry without changing the relative position between the CCD sensor and the test surface. Therefore, an absolutely constant optical magnification can be accurately kept to capture the interference fringe patterns resulting from a combination of light reflected from both the reference flat mirror and the test surface. The experimental result shows that surface profile on a fiber-end with surface features such as a small fiber diameter of 125 microm and a low reflection ratio of less than 4% are measurable. Measurements on a standard calibration ball show that the accuracy of the proposed setup is comparable with that of existing white-light interferometers and stylus profilometers.