Microsecond melting and revitrification of cryo samples with a correlative light electron microscopy setup

A method of controlling an apparatus for observing dynamic systems by time-resolved microscopy, the apparatus comprising an optical microscope including a cryo stage for receiving a cryo sample and maintaining the cryo sample at a cryogenic temperature, one or more illumination sources operable to temporarily heat at least a portion of the cryo sample such that the portion of the cryo sample temporarily melts to a liquid state and revitrifies, and an image detector associated with the optical microscope for detecting magnified images of the portion of the cryo sample generated by the optical microscope, the method comprising:

• recording a first image of the portion of the cryo sample;
• after recording the first image, energizing the one or more illumination sources to apply an illumination power level to temporarily heat the portion of the cryo sample such that the portion of the cryo sample melts to a liquid state and revitrifies;
• after the portion of the cryo sample revitrifies, recording a second image of the portion of the cryo sample;
• computing a difference image of the portion of the cryo sample by subtracting the first image from the second image;
• analyzing the difference image;
• and
• maintaining or adjusting the illumination power level depending on the step of analyzing the difference image.

A method of correlative light-electron microscopy comprising:

• transferring a cryo sample to an apparatus comprising an optical microscope including a cryo stage for receiving the cryo sample and maintaining the cryo sample at a cryogenic temperature, a heating system including one or more illumination sources, and an image detector associated with the optical microscope for detecting magnified images of the cryo sample generated by the optical microscope;
• operating the heating system to temporarily heat at least a portion of the cryo sample such that the portion of the cryo sample temporarily melts to a liquid state and revitrifies;
• transferring the cryo sample to an electron microscope;
• and
• investigating the cryo sample using the electron microscope.