David Kleinfeld, the Dr. George Feher Endowed Chair in Experimental Biophysics at UC San Diego, shares his views on training and best practices in neurophotonics, offering words of wisdom for building and strengthening our scientific community.
The compromise between lateral resolution and usable imaging depth range is a bottleneck for optical coherence tomography (OCT). Existing solutions for optical coherence microscopy (OCM) suffer from either large data size and long acquisition time or a nonideal point spread function. We present volumetric OCM of mouse brain ex vivo with a large depth coverage by leveraging computational adaptive optics (CAO) to significantly reduce the number of OCM volumes that need to be acquired with a Gaussian beam focused at different depths. We demonstrate volumetric reconstruction of ex-vivo mouse brain with lateral resolution of 2.2 μm, axial resolution of 4.7 μm, and depth range of ∼1.2 mm optical path length, using only 11 OCT data volumes acquired on a spectral-domain OCM system. Compared to focus scanning with step size equal to the Rayleigh length of the beam, this is a factor of 4 fewer datasets required for volumetric imaging. Coregistered two-photon microscopy confirmed that CAO-OCM reconstructions can visualize various tissue microstructures in the brain. Our results also highlight the limitations of CAO in highly scattering media, particularly when attempting to reconstruct far from the focal plane or when imaging deep within the sample.
KEYWORDS: Neurons, Multiphoton microscopy, Microscopy, Calcium, Signal to noise ratio, In vivo imaging, Neuroimaging, Brain, Brain imaging, Deep tissue imaging, Signal attenuation, Functional imaging, Luminescence, Surface plasmons
We demonstrate that three-photon microscopy (3PM) with 1300-nm excitation enables functional imaging of GCaMP6s labeled neurons beyond the depth limit of two-photon microscopy (2PM) with 920-nm excitation. We quantitatively compared 2PM and 3PM imaging of calcium indicator GCaMP6s by measuring correlation between activity traces, absolute signal level, excitation attenuation with depth, and signal-to-background ratio (SBR). Compared to 2PM imaging of GCaMP6s-labeled neurons, 3PM imaging has increasingly larger advantages in signal strength and SBR as the imaging depth increases in densely labeled mouse brain, given the same pulse energy, pulse width, and repetition rate at the sample surface. For example, 3PM has comparable signal strength as 2PM and up to two orders of magnitude higher SBR as 2PM in mouse cortex around 700-800um. We also demonstrate 3PM activity recording of 150 neurons in the hippocampal stratum pyramidale (SP) at 1mm depth, which is inaccessible to non-invasive 2PM imaging. Our work establishes 3PM as a powerful tool for calcium imaging at the depth beyond 2PM limits.
KEYWORDS: In vivo imaging, Luminescence, Brain, Microscopy, Neuroimaging, Multiphoton microscopy, Deep tissue imaging, Scattering, Signal attenuation, Blood
As a result of the large difference between scattering mean free paths and absorption lengths in brain tissue, scattering
dominates over absorption by water and intrinsic molecules in determining the attenuation factor for wavelengths
between 350 nm and 1300 nm. We propose using longer wavelengths for two-photon excitation, specifically the 1300-nm region, in order to reduce the effect of scattering and thereby increase imaging depth. We present two photon
fluorescence microscopy images of cortical vasculature in in vivo mouse brain beyond 1 mm. We also explore the
capabilities of the 1300-nm excitation for third harmonic generation microscopy of red blood cells in in vivo mouse
brain.
Femtosecond laser pulses have the unique ability to deposit energy into a microscopic volume in the bulk of a material that is transparent to the laser wavelength without affecting the surface of the material. Here we review the use of this capability to disrupt specifically targeted structures in live cells and animals with the goal of elucidating function and modeling disease states. Particular attention will be paid to recent work that uses femtosecond laser disruption to injure cerebral blood vessels that lie below the brain surface in a live, anesthetized rat. By varying the degree of injury, the vessel can be made to leak blood plasma, to rupture, or to clot. This technique thus provides a versatile model of cerebrovascular disorders such as small-scale stroke.
The study of neurovascular diseases such as vascular dementia and stroke require novel models of targeted vascular disruption in the brain. We describe a model of microvascular disruption in rat neocortex that uses ultrashort laser pulses to induce localized injury to specific targeted microvessels and uses two-photon microscopy to monitor and guide the photodisruption process. In our method, a train of high-intensity, 100-fs laser pulses is tightly focused into the lumen of a blood vessel within the upper 500 μm of cortex. Photodisruption induced by these laser pulses creates injury to a single vessel located at the focus of the laser, leaving the surrounding tissue intact. This photodisruption results in three modalities of localized vascular injury. At low power, blood plasma extravasation can be induced. The vessel itself remains intact, while serum is extravasated into the intercellular space. Localized ischemia caused by an intravascular clot results when the photodisruption leads to a brief disturbance of the vascular walls that initiates an endogenous clotting cascade. The formation of a localized thrombus stops the blood flow at the location of the photodisruption. A hemorrhage, defined as a large extravasation of blood including plasma and red blood cells, results when higher laser power is used. The targeted vessel does not remain intact.
Photothrombotic microstrokes are produced in rat cortex by 532-nm single-photon optical excitation of an intravenously injected photosensitizer, rose bengal. The dynamics of blood flow and clot formation in the cortical vasculature are observed using two-photon laser scanning microscopy of an intravenously injected fluorescent dye. Flowing and clotted vessels are clearly distinguishable in both large and small vessels, down to individual capillaries, using this technique. We find that by tightly focusing the laser light used to excite the photosensitizer, clots can be formed in individual blood vessels without affecting neighboring vessels tens of micrometers away. We observe many changes in blood flow as a result of localized clot formation, including upstream vascular dilation, clot clearing, i.e. recanalization, and complete reversal of blood flow direction downstream.
We tightly focus femtosecond laser pulses in the bulk of a transparent material. The high intensity at the focus causes nonlinear absorption of the laser energy, producing a microscopic plasma and damaging the material. The tight external focusing allows high intensity to be achieved with low energy, minimizing the effects of self-focusing. We report the thresholds for breakdown and critical self- focusing in fused silica using 110-fs pulses at both 400-nm and 800-nm wavelength. We find that permanent damage can be produced with only 10 nJ (25 nJ) for 400-m (800-nm) pulses, and that the threshold for critical self-focusing is 140 nJ for the 400-nm pulses and 580 nJ for the 800-nm pulses. The critical self-focusing thresholds are more than an order of magnitude above the breakdown thresholds, confirming that self-focusing does not play a dominant role in the damage formation. This lack of self-focusing allows a straightforward interpretation of the wavelength and bandgap dependence of bulk breakdown thresholds. The energies necessary for material damage are well within the range of a cavity-dumped oscillator, allowing for precision microstructuring of dielectrics with a high repetition-rate laser that is roughly one-third the cost of an amplified system.
We present thresholds for optical breakdown in bulk transparent solids and water with 10-fs laser pulses. In solids, we used microscopy and scattering techniques to determine thresholds for plasma formation and permanent damage in a wide variety of materials. Transmission measurements show that damage occurs at energies where there is little absorption of the laser pulse. In water, we used scattering and acoustic techniques to measure the breakdown threshold for 100-fs pulses. In contrast to solids, transmission measurements in water indicate that there is no plasma or bubble formation unless there is significant absorption. For comparison, we also measured breakdown thresholds for 200-ps pulses.
Tightly focused femtosecond laser pulses can be nonlinearly absorbed inside transparent materials, creating a highly excited electron-ion plasma. These conditions exist only in a small volume at the laser focus. This tight confinement and extreme conditions lead to an explosive expansion -- a microexplosion. In solid materials, a microexplosion can result in permanent structural changes. We find that the damage produced by femtosecond pulses in this way is surprisingly small, with only a 200-nm diameter. Material left at the center of the microexplosion is either amorphous and less dense or entirely absent. The threshold for breakdown and structural change is nearly independent of material. Time- resolved measurements of microexplosions in water allow us to observe the dynamics of the explosive expansion. The structural changes in solids resulting from microexplosions allow for three-dimensional data storage and internal microstructuring of transparent solids.
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