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Taking into account the axial magnification, which equates to the square of lateral magnification of 60X, introduced by the microscope objective, one can estimate the accuracy of the autofocusing in the object space as 0.42μm, and DOF is calculated to be 0.91µm using Eq. (2).
Common-path Digital Holographic Microscopy (CP-DHM) is a new technique which can physically compensate the phase curvature introduced by the microscope objective.
We followed a nanodisk laser inside a macrophage cell over 8 h and found that its intensity varied over time, presumably because cellular motion induces changes in the position and orientation of the nanodisk, thus affecting the amount of emission that is collected by the microscope objective.
The emitted light from fluorescent tracer particles is then collected by the microscope objective and recorded by a camera.
The fluorescence emitted from the region beyond the aperture was collected by the microscope objective and passed through a bandpass filter (386 nm) and then focused onto a photomultiplier tube (PMT, Hamamatsu, HC125-2) for detection.
The object beam is loosely focused onto the sample by a condenser lens to illuminate the field of view, and the diffracted light is collected by the microscope objective.
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The two-photon images in Figures 1G and H were acquired in the same piece of tissue, simply by moving the microscope objective along the z direction.
For single-molecule recordings, the recording chambers were cooled to approximately 14°C by cooling the microscope objective lens assembly with recirculating chilled water.
Cells were imaged either at 30°C, or 36.8°C by heating the microscope objective with a flexible resistive heater (Omega, Stamford, CT) utilizing an on off controller (Minco, Minneapolis, MN), which maintained the temperature at the objective within ±0.1°C as readout by a 100 Ω platinum thermistor (Minco).
The different spot sizes were produced by changing the magnification of the microscope objective and confirmed by measuring the irradiance profile using a scanned knife-edge technique.
Imaging depth is controlled by raising and lowering the microscope objective.
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