STED microscopy with pulsed excitation, continuous stimulation, and gated registration of spontaneously emitted fluorescence light

摘要

In a STED fluorescence light microscope pulses of excitation light (3) are applied to a sample, which excite fluorescent entities contained in the sample for fluorescence, and which are focused on at least one focal area. Further, de-excitation light (12) is applied to the sample, which de-excites the excited fluorescent entities and which comprises an intensity zero point in the at least one focal area, as a continuous wave. Fluorescence light emitted by the excited fluorescent entities in the sample is registered after each pulse of the excitation light (3) and overlapping with applying the de-excitation light (13) with high temporal resolution between consecutive pulses of the excitation light (3).

主权项

1. A STED fluorescence light microscopic method of imaging a structure which is marked with fluorescent entities in a sample, the method comprising the steps of:
applying pulses of excitation light to the sample, which excite the fluorescent entities for fluorescence, and which are focused on at least one focal area; applying de-excitation light to the sample, which de-excites the excited fluorescent entities and which comprises an intensity zero point in the at least one focal area, as a continuous wave; registering fluorescence light spontaneously emitted by the excited fluorescent entities in the sample after each pulse of excitation light and overlapping with applying the de-excitation light; and repeating the steps of applying and registering at different positions of the focal area of the excitation light and the intensity zero point of the de-excitation light; wherein, in the step of registering, the fluorescence light spontaneously emitted by the excited fluorescent entities is registered with temporal resolution between consecutive pulses of the excitation light.