Abstract: A method of analyzing a mixed fluorescence response of a plurality of fluorophores in a microscopic sample includes reconstructing individual fluorescence responses from a mixed fluorescence response using spectral un-mixing based on reference emission spectra for fluorophores to be reconstructed, and a procedure for determining and validating reference emission spectra including providing a plurality of image acquisition settings for a sequence of images of the sample equal to, or greater than, the plurality of fluorophores and including an illumination setting for each image, acquiring the sequence of images using the plurality of image acquisition settings and storing each image together with the corresponding illumination setting, determining candidate reference emission spectra for the fluorophores to be reconstructed from the sequence of images of the sample using one or more reference emission spectra determination algorithms, and conditionally using the candidate reference emission spectra as the refe

Abstract: A holographic optical tweezers for manipulating a micro- or nano-size particle, the optical tweezers including a light source configured to emit first and second light beams; a light focusing apparatus configured to focus the first and second light beams to generate focused light beams, which create optical forces; and a trapping assembly configured to receive the first and second focused light beams and form a trap for holding the particle with the optical forces. The trapping assembly includes first and second micromirrors attached to a microscope coverslip.