Patent ID: 6468803
Filing Date: 2002-10-22
Classification: G01N

Abstract:
A method for calibrating the sample height of a sample in a chamber of a sample analyzer held within an optical scanning instrument, said method comprising:a) providing said chamber for receiving a sample, in said sample analyzer; b) depositing a sample into said chamber wherein said sample contains a fluorescent dye; c) illuminating said sample with excitation light; d) scanning the sample with said instrument to detect the fluorescence from said sample; e) measuring fluorescence intensity values in particle-free locations within a calibration area of such height, h, that the measured fluorescence intensity is independent of the height value; f) extrapolating to the integrated fluorescence intensity, Ical, for a total calibration area of size, A; g) measuring fluorescence intensity values in particle-free locations within a single-particle area; h) extrapolating to the integrated fluorescence intensity, Isca, for a total area of size, A; i) determining an effective height, H, in the calibration area by using the equation H=&Integral;-h/2h/2&it;E&it;(z)*D&it;(z)&it;dzâ€ƒwhere E&it;(z)=[sin&it;(uex&it;(z)/4)uex&it;(z)/4]2â€ƒwhere uex&it;(z)=2*Ï€Î»ex*(NA)2*zâ€ƒwhere D&it;(z)=[sin&it;(uem&it;(z)/4)uem&it;(z)/4]2â€ƒwhere uex&it;(z)=2*Ï€Î»ex*(NA)2*zâ€ƒand wherein NA is the numerical aperture of the scanning instrument's objective lens, &lgr;ex is the excitation center-wavelength, &lgr;em is the emission center-wavelength, and z is a position within the sample on an axis parallel to the scanning instrument's imaging optics; j) calculating a quantity K where K=IscaIcal*H;k) determining an integration limit, d, that is a solution of the equation: &Integral;-d/2d/2&it;E&it;(z)*D&it;(z)&it;â€ƒ&it;&dd;z=Kâ€ƒwhereby d is equal to the unknown sample height in the single-cell area that is to be determined; and l) obtaining the calibrated sample height.