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Label-Free Multiphoton FLIM of Moving Bacteria

Live bacteria are often difficult to image in their natural environment because they are extremely mobile. To obtain reasonable image quality extremely short image acquisition time has to be used which, however, leads to pixel photon numbers insufficient for accurate lifetime analysis. Higher excitation power does not solve the problem because it impairs the viability of the bacteria or even causes severe photodamage. We therefore developed a technique that records a sequence of fast single-frame images, identifies the bacteria in these images by phasor analysis, and combines the photons of the corresponding pixels in a single decay curve of high photon number. This curve is processed by multi-exponential decay analysis in the time-domain, yielding precision multi-exponential decay parameters of the bacteria.

Keywords: FLIM, Label-Free, NADH, Metabolic Imaging, Moving Bacteria, Phasor Plot

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