A method of determining a focal position for an objective positioned at a measurement location of a sample holder (704, 718) in a microscopy imaging system, the method comprising: moving the objective to a position corresponding to a distance between the objective and the sample holder (704, 718), wherein the position of the objective is a z-position along a Z-axis relative to the sample holder; projecting an off-axis focusing light beam onto the sample holder (704, 718) when the objective is located at the position, wherein the objective focuses the off-axis focusing light beam on the sample holder; observing a reflected light beam resulting from reflection of the off-axis focusing light beam off a conditioned upper surface (706, 720) of the sample holder (704, 718), wherein the conditioned upper surface includes a reflective coating applied to the upper surface; determining the focal position for the objective based on the reflected light beam such that the objective produces an in focus image of a microscopy sample residing at the measurement location when the objective is located at the focal position; directing the reflected light beam (710, 712, 714; 724, 726, 728) onto a linear detector such that the reflected light beam strikes a location on the linear detector; comparing the location on the linear detector to a recorded linear detector location, the recorded linear detector location corresponding to a focused z-position that results in an in focus image when the objective is located at the focused z-position; determining whether the location on the linear detector has shifted away from the recorded linear detector location; and in response to a determination that the location on the linear detector has shifted away from the recorded linear detector location, determining a new z-position for the objective based on the comparison of the location on the linear detector to the recorded linear detector location and identifying the new z-position as the focal position for the objective; characterized in that the method further comprises: configuring the linear detector to monitor a strongest reflected light beam in the plurality of reflected light beams (710, 712, 714; 724, 726, 728) directed onto the linear detector where the strongest reflected light beam results from reflection of the off-axis focusing light beam off the reflective coating applied to the upper surface (706, 720) of the sample holder (704, 718). moving the objective to a position corresponding to a distance between the objective and the sample holder (704, 718), wherein the position of the objective is a z-position along a Z-axis relative to the sample holder; projecting an off-axis focusing light beam onto the sample holder (704, 718) when the objective is located at the position, wherein the objective focuses the off-axis focusing light beam on the sample holder; observing a reflected light beam resulting from reflection of the off-axis focusing light beam off a conditioned upper surface (706, 720) of the sample holder (704, 718), wherein the conditioned upper surface includes a reflective coating applied to the upper surface; determining the focal position for the objective based on the reflected light beam such that the objective produces an in focus image of a microscopy sample residing at the measurement location when the objective is located at the focal position; directing the reflected light beam (710, 712, 714; 724, 726, 728) onto a linear detector such that the reflected light beam strikes a location on the linear detector; comparing the location on the linear detector to a recorded linear detector location, the recorded linear detector location corresponding to a focused z-position that results in an in focus image when the objective is located at the focused z-position; determining whether the location on the linear detector has shifted away from the recorded linear detector location; and in response to a determination that the location on the linear detector has shifted away from the recorded linear detector location, determining a new z-position for the objective based on the comparison of the location on the linear detector to the recorded linear detector location and identifying the new z-position as the focal position for the objective; characterized in that the method further comprises: configuring the linear detector to monitor a strongest reflected light beam in the plurality of reflected light beams (710, 712, 714; 724, 726, 728) directed onto the linear detector where the strongest reflected light beam results from reflection of the off-axis focusing light beam off the reflective coating applied to the upper surface (706, 720) of the sample holder (704, 718).