Scanner

The very initial iSPIM and diSPIM in the Shroff lab used a homebuilt scanner system with galvo. ASI developed a “scanner” that uses a MEMS mirror to create a light sheet. Several variants exist including versions with a cylinder lens for fixed sheet, a version based on ASI's C60-CUBE-III which is ideal for phototargeting or TIRF or other applications where it's important to fill the BFP (in contrast with light sheet with low-NA beams), and with an anti-striping option.

The same scanner body can accommodate a cylindrical lens to make a stationary light sheet.

The advantages of the cylindrical lens version include:

• faster imaging speed is possible (e.g. by ~2x) because you don't have to blank the beam during camera readout and beam flyback
• cost $5k less each b/c not paying patent royalty fee
• for many samples bleaching will be less by spreading out photon dose over longer time, ditto for avoiding fluorophore saturation

The disadvantages of the cylindrical lens version include:

• can't use the virtual slit trick for partial confocality
• no stroboscopic effect to minimize motion blur
• Gaussian intensity profile of illumination intensity across field ⇒ may need flat field correction

The resonance of a typical (1.2mm dia.) MEMS mirror is on the order of 2.5kHz, you have to stay far away from that or else the mirror can self-destruct. So 1kHz is the approximate upper bandwidth. How fast you can realistically make a light sheet is several times smaller and depends on the size of the sheet and whether you “fly back” and make all sheets in the same direction (default) or not. There is a lowpass filter in the electronics that can be set (by default to 400Hz).

A good rule of thumb with standard objectives is that both the scan and max flyback can keep up with the camera readout speed but not go too much faster. E.g. with 512 rows the readout time is 2.5ms and we can do a 2.5ms scan and flyback in 2.5ms for 200fps.

If you are really interested in FCS and/or pushing the diSPIM speed I suggest a cylindrical lens scanner which generates a static sheet, then no scanning is involved. Besides not worrying about scan/flyback, you can also leave the laser on during camera readout so in that earlier example you can actually do 400fps (the camera limit) instead of 200fps.