ABSTRACT
This protocol describes the application of laser pulses to image and ablate neuronal tissue for the purpose of automated histology. The histology is accomplished in situ using serial two-photon imaging of labeled tissue and removal of the imaged tissue with amplified, femtosecond pulses. Together with the use of endogenous fluorescent indicators and/or deep penetration of antibody labels and organic dyes, this method may be used to automatically image, reconstruct, and vectorize structures of interest across millimeter to centimeter regions of brain with micrometer resolution.
Subject(s)
Brain/cytology , Brain/physiology , Histological Techniques/methods , Image Processing, Computer-Assisted/methods , Lasers , Optical Devices , Animals , Automation, Laboratory/methods , Fluorescence , MiceABSTRACT
We present a high-speed photon counter for use with two-photon microscopy. Counting pulses of photocurrent, as opposed to analog integration, maximizes the signal-to-noise ratio so long as the uncertainty in the count does not exceed the gain-noise of the photodetector. Our system extends this improvement through an estimate of the count that corrects for the censored period after detection of an emission event. The same system can be rapidly reconfigured in software for fluorescence lifetime imaging, which we illustrate by distinguishing between two spectrally similar fluorophores in an in vivo model of microstroke.
Subject(s)
Brain/cytology , Diagnostic Imaging/methods , Interneurons/physiology , Luminescent Measurements/methods , Photons , Signal Processing, Computer-Assisted , Analog-Digital Conversion , Animals , Cell Death , Green Fluorescent Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microtubule-Associated Proteins/metabolismABSTRACT
We present a prism-based spectrometer integrated into a multifocal, multiphoton microscope. The multifocal configuration facilitates interrogation of samples under different excitation conditions. Notably, the image plane of the microscope and the image plane of the spectrometer are coincident eliminating the need for an intermediate image plane containing an entrance slit. An EM-CCD detector provides sufficient gain for spectral interrogation of single-emitters. We employ this spectrometer to observe spectral shifts in the two-photon excitation fluorescence emission of single CdSe nanodots as a function of excitation polarization.
Subject(s)
Microscopy, Fluorescence, Multiphoton/instrumentation , Spectrophotometry/instrumentation , Cadmium Compounds/chemistry , Cadmium Compounds/radiation effects , Optical Devices , Quantum Dots , Selenium Compounds/chemistry , Selenium Compounds/radiation effectsABSTRACT
Simultaneous spatial and temporal focusing (SSTF) provides precise control of the pulse front tilt (PFT) necessary to achieve nonreciprocal writing in glass wherein the material modification depends on the sample scanning direction with respect to the PFT. The PFT may be adjusted over several orders of magnitude. Using SSTF nonreciprocal writing is observed for a large range of axial focal positions within the sample, and nonreciprocal ablation patterns on the surface of the sample are revealed. Further, the lower numerical aperture (0.03 NA) utilized with SSTF increases the rate of writing.
ABSTRACT
Temporal focusing of spatially chirped femtosecond laser pulses overcomes previous limitations for ablating high aspect ratio features with low numerical aperture (NA) beams. Simultaneous spatial and temporal focusing reduces nonlinear interactions, such as self-focusing, prior to the focal plane so that deep (approximately 1 mm) features with parallel sidewalls are ablated at high material removal rates (25 microm(3) per 80 microJ pulse) at 0.04-0.05 NA. This technique is applied to the fabrication of microfluidic devices by ablation through the back surface of thick (6 mm) fused silica substrates. It is also used to ablate bone under aqueous immersion to produce craniotomies.
Subject(s)
Craniotomy/instrumentation , Laser Therapy/instrumentation , Lasers , Microtechnology/instrumentation , Microtechnology/methods , Animals , Computer Simulation , Equipment Design , Glass , Mice , Nonlinear Dynamics , Silicon Dioxide , Skull/surgery , Ultrasonics/instrumentationABSTRACT
We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences.
Subject(s)
Microscopy, Fluorescence, Multiphoton/methods , Absorption , Algorithms , Animals , Fluorescence , Humans , Microscopy, Fluorescence, Multiphoton/instrumentation , Models, Theoretical , Nonlinear Dynamics , Optics and Photonics/instrumentation , Optics and Photonics/methods , Photobleaching , Photons , Time FactorsABSTRACT
Plasma-mediated ablation makes use of high energy laser pulses to ionize molecules within the first few femtoseconds of the pulse. This process leads to a submicrometer-sized bubble of plasma that can ablate tissue with negligible heat transfer and collateral damage to neighboring tissue. We review the physics of plasma-mediated ablation and its use as a tool to generate targeted insults at the subcellular level to neurons and blood vessels deep within nervous tissue. Illustrative examples from axon regeneration and microvascular research highlight the utility of this tool. We further discuss the use of ablation as an integral part of automated histology.
Subject(s)
Catheter Ablation/methods , Microvessels/surgery , Nerve Tissue/surgery , Animals , Corneal Stroma/surgery , HumansABSTRACT
As a means to automate the three-dimensional histological analysis of brain tissue, we demonstrate the use of femtosecond laser pulses to iteratively cut and image fixed as well as fresh tissue. Cuts are accomplished with 1 to 10 microJ pulses to ablate tissue with micron precision. We show that the permeability, immunoreactivity, and optical clarity of the tissue is retained after pulsed laser cutting. Further, samples from transgenic mice that express fluorescent proteins retained their fluorescence to within microns of the cut surface. Imaging of exogenous or endogenous fluorescent labels down to 100 microm or more below the cut surface is accomplished with 0.1 to 1 nJ pulses and conventional two-photon laser scanning microscopy. In one example, labeled projection neurons within the full extent of a neocortical column were visualized with micron resolution. In a second example, the microvasculature within a block of neocortex was measured and reconstructed with micron resolution.
