Single and multiphoton excitation fluorescence in-line cytometry for real-time bioprocess metabolic monitoring

An in-line analytical system for monitoring cell status, the system comprising:

• a probe for insertion into a cell culture, the probe including:
• a focusing optic for focusing excitation energy to an excitation cytometry volume in the cell culture, and
• a collection channel for fluorescence signals;
• an excitation source for directing excitation energy to the probe focusing optic;
• a first detection subsystem optically coupled to the collection channel of the probe and configured to detect a first peak in fluorescence intensity in a first fluorescent signal from a first fluorescing cell fluorophore;
• a second detection subsystem optically coupled to the collection channel of the probe and configured to detect a second peak in fluorescence intensity in a second fluorescent signal from a second fluorescing cell fluorophore;
• and
• a signal processor, responsive to the first and second detector subsystems and configured to identify when the first and second peaks occur substantially simultaneously indicating the presence of a cell at the excitation cytometry volume instead of background fluorescence in order to measure cellular status using the intensity levels of the first and second peaks.

An in-line cytometry system for monitoring cell status in agitated bioreactors, the method comprising:

• focusing excitation energy to an excitation cytometry volume that is smaller than a cell to fluorescence cells when present at a focal region of the excitation energy;
• randomly sampling cells that are moving across the excitation volume;
• and
• generating fluorescence time traces that contain cells peaks as the cells flow in the agitated bioreactor.