Dr. Paul Maddox, Assistant Professor,
The University of North Carolina at Chapel Hill, Biology Department
Conventional light sheet fluorescence microscopy (LSFM) is performed with two objectives oriented orthogonally to each other so that one objective introduces the light sheet and the other detects the fluorescence signal. However, this orientation requires the detection objective to be placed slightly away from the sample to prevent the two objectives colliding in space. Therefore, a long working distance detection objective is necessary which means that high NA, oil-immersion objectives are incompatible with the conventional LSFM design.
This presents a problem for the detection of cellular or subcellular structures which require a high NA detection objective and coverslip-based mounted samples for the superior resolution and light collection efficiency.
The Mizar TILT overcomes this problem by removing the illumination objective and introducing a tilted light sheet through a photomask and cylindrical lens which can be made to converge at the working distance of high NA objectives. In this way, high magnification and high NA (60x, 1.49), oil-immersion objectives can be used to image coverslip-based mounted samples.
Like all light sheet systems, the Mizar TILT is designed to minimize photodamage and photobleaching to live samples by reducing the light source intensity and reducing exposure times. This allows for longer acquisitions to be made to monitor live processes over longer timescales.
One way to reduce exposure times is to use a more sensitive camera. CMOS devices are typically used in LSFM for the combination of a large field of view and fast speed but the sensitivity isn’t that high. EMCCD cameras are more sensitive than CMOS devices but suffer from small fields of view and slow speeds which makes them unappealing for LSFM applications.
The Prime 95B Scientific CMOS [sCMOS] camera is, right now, the best solution we have found for TILT imaging. Coupling the Prime 95B to the TILT generates an extremely powerful imaging system!