Improving fluorescence confocal microscopy with cryogenically-cooled diode lasers

Recent advances in fluorescence confocal microscopy have focused on exciting multiple dyes, leading to the use of diode laser sources. We show that by varying a diode's operating parameters, diode lasers can address some of the inherent problems associated with multiple dye excitation. Cooling the laser diode caused its emission wavelengths to decrease linearly, its output power to increase five times, and the noise due to reflected laser light for equal fluorescence signals to be reduced five times. We utilized these improvements to produce fluorescence confocal images that minimized the reflected laser intensity, while still efficiently exciting the fluorochrome.