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no way to truly avoid it.’ According to Aswani,


minimising sample damage while imaging live cells ultimately comes down to limiting the sample’s exposure to light, with shutters in the case of lamps, and turning LEDs off when the sample is not being imaged. The use of pulsed lasers or pulsed LEDs may also reduce sample damage, she added. It is thought that this could be due to the ability of the cell to recover from being exposed constantly to light, thereby generating fewer reactive oxygen species. ‘So, you can give the cells time to recover before you blast them again with light, which leads to reduced phototoxicity,’ said Aswani.


For advanced microscopy applications, Excelitas has demonstrated that it’s X-Cite XLED1 high-power light source, when operated in live cell mode or pulsed mode, results in lower phototoxicity in live cells when compared to continuous light exposure. In a separate study, the company also found that cell


The goal is to


image in a similar way to fluorescence, but without having to add any dyes or stains


proliferation is higher when a sample is imaged with LED than when imaged with a mercury lamp for the same exposure time, although these have only been initial findings.


But although pulsed light is typically associated with less photodamage, the extremely high speeds of multiphoton lasers can also create issues concerning sample damage.


Since two-photon excitation is a non-linear process, scientists quickly realised that shorter laser pulses at the sample resulted in higher fluorescence signal because


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of the higher peak power. ‘For this reason, lasers designed for multiphoton microscopy typically have pulse durations between about 50 and 150 femtoseconds,’ said Marco Arrigoni, director of marketing for Coherent’s scientific market segment. ‘However, with some biological specimens, if not properly managed, the shorter pulses also produce the undesirable effect of a higher rate of photobleaching.’ Very short pulses (i.e. 50-100fs)


are more difficult to manage since their larger bandwidth results in a higher material dispersion; by the time the pulses travel through all the microscope optics and reach the sample, their duration may be longer than at the laser output. ‘Because of this, lasers like Coherent’s Chameleon Discovery and Vision incorporate a so-called pre-chirp feature that conditions the laser pulses to have the shortest duration right at the sample plane,’ Arrigoni said, adding that the laser pre- chirp feature can also be used to detune the pulses and make them purposely longer, so that the user can find the optimum pulse duration for different experiments. Interestingly, the ideal laser parameters for minimising tissue damage in three-photon imaging, a newer technique that enables greater imaging depths in the brain, are quite different than with two-photon microscopy. Professor Chris Xu and co- workers at Cornell University recently reported that higher energies at lower repetition rates, with correspondingly lower average power, are paramount for minimising thermal sample damage, Arrigoni pointed out. ‘This newer imaging technique is leading to the adoption of lasers, like the Coherent Monaco, which produce a more energetic pulse train at 1MHz repetition rate rather than the 80MHz of more conventional mode-locked lasers,’ commented Arrigoni. l


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