ABSTRACTUnraveling how neural networks process and represent sensory information and how this cellular dynamics instructs behavioral output is a main goal in current neuroscience. Two-photon activation of optogenetic actuators and fluorescence calcium (Ca2+) imaging with genetically encoded Ca2+ indicators allow, respectively, the all-optical stimulation and readout of activity from genetically identified cell populations. However, these techniques expose the brain to high near-infrared light doses raising the concern of light-induced adverse effects on the biological phenomena being studied. Combing Ca2+ imaging of GCaMP6f-expressing cortical astrocytes as a sensitive readout for photodamage and an unbiased machine-based event detection, we demonstrate the subtle build-up of aberrant microdomain Ca2+ signals in fine astroglial processes. Illumination conditions routinely being used in biological two-photon microscopy (920-nm excitation, 100-fs regime, ten mW average power) increased the frequency of microdomain Ca2+ events, but left their amplitude, area and duration rather unchanged. This increase in local Ca2+ activity was followed by Ca2+ transients in the otherwise silent soma. Ca2+ hyperactivity occurred without overt morphological damage. Surprisingly, at the same average power, continuous-wave 920-nm illumination was as damaging as fs pulses, indicating a linear, heating-mediated (rather than a highly non-linear) damage mechanism. In an astrocyte-specific IP3-receptor knock-out mouse (IP3R2-KO), Near-infrared light-induced Ca2+ microdomains signals persisted in the small processes, underpinning their resemblance to physiological IP3R2-independent Ca2+ signals, while somatic activity was abolished. Contrary to what has generally been believed in the field, shorter pulses and lower average power are advantageous to alleviate photodamage and allow for longer useful recording windows.SIGNIFICANCE STATEMENTImaging the fine structure and function of the brain has become possible with two-photon microscopy that uses ultrashort-pulsed infrared laser light for better tissue penetration. The high peak energy of these light pulses has raised concerns about photodamage resulting from multi-photon processes. Here, we show that the time-averaged rather than the peak laser power matters. At wavelengths and with laser powers now commonly used in neuroscience brain damage occurs as a consequence of direct infrared light absorption, i.e., heating. To counteract brain heating we explore a strategy that uses even shorter, more energetic pulses but a lower time-averaged laser power to produce the same image quality while making two-photon microscopy less invasive.
Dual Near Infrared Two-Photon Microscopy for Deep-Tissue Dopamine Nanosensor Imaging