Biomedical Engineering Core

Our Zeiss LSM 780 is an older model, point scanning fluorescence optical microscope which acquires optical sections in a 3D biological sample labeled with a fluorescent dye or biosensor.  The imaging depth is limited by optical scattering and is typically around 100 microns and a non-cleared biological tissue sample. The microscope operates with one-photon excitation with resolution limited by diffraction. 

A laser is used to excite the sample point-by-point and the fluorescence from every point is spatially filtered by a pinhole and detected on photomultiplier tube.  The full image of the sample is reconstructed from these individual point signals. The expected resolution is about 200 nm laterally (x,y) and 1 micron axially (z). The resolution of the microscope depends on the objective used, the size of the pinhole and the image sampling rate (number of points per (x,y) scan and the axial interval between z sections), with all these parameters set by the user.

The microscope acquires a spectrum of the sample's fluorescence and is typically able to discriminate between 3 different fluorescence bandwidths imaged in 3 separate channels. Additional fluorescence channels may be used with careful adjustments of the emission parameters and channel crosstalk correction. The speed of image acquisition is limited by the scanning rate, which is usually limited by the time needed to acquire the individual image points with sufficient signal-to-noise ratio. An individual (x,y) frame typically takes 200ms and a z-stack may take up to a few seconds depending on the number of sections required.