System and method for fluorescence microscopy with detection of light emission from multiple fluorochromes

A microscopy system for separating fluorescence signals emitted from a sample labeled with a plurality of predetermined fluorochromes, the microscopy system comprising:

• a light source arrangement configured to emit light;
• a light guide for guiding light from the light source arrangement to the sample for excitation of the plurality of predetermined fluorochromes;
• a filter arrangement configured to allow separation of fluorescence emitted by the plurality of predetermined fluorochromes labeling the sample, and
• a detection device configured for receiving light transmitted through the filter arrangement,
• wherein
• the microscope filter sets can separate signals from at least four different fluorochromes that emit light within the visible light spectra,
• a pair of fluorochromes comprising a first and a second fluorochrome are selected from the at least four different fluorochromes,
• the light source arrangement is configured to sequentially emit light within at least four different excitation wavelength intervals,
• the excitation wavelength interval for the second fluorochrome is selected to reduce excitation of the first fluorochrome,
• the filter arrangement comprises at least four matched filters configured to allow light to pass through within at least four different emission wavelength intervals,
• the emission wavelength interval for the first fluorochrome is selected to reduce light emission bleedthrough from the second fluorochrome, and
• the pair of fluorochromes are selected as a fluorochrome having an excitation wavelength interval comprising 546 nm forming the first fluorochrome and a fluorochrome having an excitation wavelength interval comprising 602 nm forming the second fluorochrome, or the pair of fluorochromes are selected as a fluorochrome having an excitation wavelength interval comprising 436 nm forming the first fluorochrome and a fluorochrome having an excitation wavelength interval comprising 500 nm forming the second fluorochrome.