Requires: multi-camera splitter with dichroic chain, matched cameras, pentaband or multiband excitation dichroic. All channels share the same exposure timing and stage position. Benefits: no mechanical overhead, true simultaneity for dynamic samples, maximum throughput. Limitations: requires sufficient spectral separation, more complex alignment.
Acquisition Strategy
Simultaneous Acquisition
All channels at once
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Definition
Capturing multiple fluorescence channels in a single exposure using spectral separation and multiple detectors. Eliminates filter wheel switching and enables true co-temporal imaging of dynamic samples.
Technical Details
Simplified
Taking a photo with multiple cameras at exactly the same instant, each camera seeing a different color. Nothing has to move between shots—you get all your colors in the time it takes for one exposure.
Why It Matters
Simultaneous 4-channel acquisition (after sequential DAPI) is the key to achieving sub-minute Z-stacks with the 5-camera system. Without simultaneous detection, the same data would take 10× longer to acquire.
Practical Example
With pentaband dichroic (405/488/561/594/647), channels 2-5 (A488, Atto565, A594, CY5.5) are acquired simultaneously on cameras 2-4. Only DAPI requires separate acquisition to avoid bleedthrough. 5-channel Z-stack overhead: ~1 dichroic switch total.