Neural circuits in the brain fire on the millisecond time scale. Although two-photon calcium imaging can image this in vivo, its temporal resolution isn’t as high as electrical recordings. Now, researchers from the Brain Research Institute at the University of Zurich have designed a two-photon microscope that makes use of an acousto-optic deflector (AOD) to image at rates up to 500 Hz.
Standard two-photon setups use galvanometric mirrors for scanning and can typically sample at up to 10 to 15 Hz. Using an AOD instead allows movement of the laser focus between any two positions in a field of view in milliseconds and also lets the imaging be concentrated on the structures of interest. However, AODs require correction of spatial and temporal laser beam distortions, which the researchers accomplished by modifying a single-prism compensation method to improve transmission and dispersion compensation over a large field of view.
Using this setup they were able to measure fluorescence from neurons in the mouse neocortex at a 180 to 490 Hz sampling rate, detect single action potential evoked calcium transients with signal-to-noise ratios of 2 to 5, and calculate spike times with near-millisecond precision.
Showing posts with label two-photon microscopy. Show all posts
Showing posts with label two-photon microscopy. Show all posts
Multicolor two-photon microscopy with a new red fluorescent protein
Two-photon microscopy can be used with multiple fluorescent markers to simultaneously observe multiple processes in vivo. Red fluorescent proteins (RFPs) are a popular choice because cellular autofluorescence is reduced at its emission region and it allows greater imaging depth thanks to less light scattering. In addition, longer wavelengths are less damaging to proteins and DNA.
However, using red fluorescent proteins with other colors such as GFP or cyan fluorescent protein (CFP) comes with some challenges. The Ti-Sapphire lasers used for two-photon microscopy are expensive, so most systems have only one laser. This makes it hard to efficiently excite RFPs at the same time as GFPs or CFPs. Also, Ti:Sapphire lasers have low power output in the excitation wavelengths for RFPs.
One way to overcome these problems is to shift the excitation spectrum of RFPs by making RFPs with a large Stokes shifts. One such RFP exists already. Called, mKeima, it can be used for dual-color two-photon microscopy, but its biochemical and photochemical properties leave room for improvement.
Researchers at the Albert Einstein College of Medicine in New York applied random and rational mutagenesis to the monomeric far-red mKate fluorescent protein and produced two RFPs with large Stokes shifts. The RFP variants have six amino acid mutations compared to mKate. Compare to mKeima, the new variants have higher pH stability, better photostability and faster chromophore maturation and monomeric behavior.
They used the new RFPs, named LSS-mKate1 and LSS-mKate2, for two-photon microscopy simultaneously with ECFP and EGFP. Their multicolor intravital imaging study of the motility and Golgi-nucleus alignment of tumor cells as a function of distance from blood vessels in a live mouse xenograft model of breast cancer revealed that breast cancer cells showed polarization towards vessels at a distance of 40 microns.
However, using red fluorescent proteins with other colors such as GFP or cyan fluorescent protein (CFP) comes with some challenges. The Ti-Sapphire lasers used for two-photon microscopy are expensive, so most systems have only one laser. This makes it hard to efficiently excite RFPs at the same time as GFPs or CFPs. Also, Ti:Sapphire lasers have low power output in the excitation wavelengths for RFPs.
One way to overcome these problems is to shift the excitation spectrum of RFPs by making RFPs with a large Stokes shifts. One such RFP exists already. Called, mKeima, it can be used for dual-color two-photon microscopy, but its biochemical and photochemical properties leave room for improvement.
Researchers at the Albert Einstein College of Medicine in New York applied random and rational mutagenesis to the monomeric far-red mKate fluorescent protein and produced two RFPs with large Stokes shifts. The RFP variants have six amino acid mutations compared to mKate. Compare to mKeima, the new variants have higher pH stability, better photostability and faster chromophore maturation and monomeric behavior.
They used the new RFPs, named LSS-mKate1 and LSS-mKate2, for two-photon microscopy simultaneously with ECFP and EGFP. Their multicolor intravital imaging study of the motility and Golgi-nucleus alignment of tumor cells as a function of distance from blood vessels in a live mouse xenograft model of breast cancer revealed that breast cancer cells showed polarization towards vessels at a distance of 40 microns.
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Multiphoton GFP photoconversion reveals cilia diffusion dynamics
Researchers measured protein diffusion through a sensory cilium. Courtesy of Peter D. Calvert.Cilia are present on most cells and play important functions in vision, hearing, and smell. Scientist want to know more about the unknown functions of many cilia because mutations in genes that encode cilia proteins can cause serious diseases such as Bardet-Biedl syndrome, Usher syndrome, poly-cystic kidney disease, and retinal degeneration.
Cilia don’t synthesize proteins or membrane, and research is focused on how proteins are delivered to and removed from these organelles. Researchers believe that cilia impose selective barriers to the movement of proteins but haven’t been able to show this experimentally because some of the cilia’s structures have diameters near or below the resolution of light microscopy.
But now, a team of researchers led by Peter Calvert at SUNY Upstate Medical University along with researchers from Lehigh University and the University of California, Davis have measured the mobility of photoactivatable GFP in the connecting cilium of live Xenopus retinal rod photoreceptors and in subcellular compartments bridged by the connecting cilium. In addition, the team measured the overall time for the protein concentration to equilibrate within and between compartments.
The researchers photoconverted GFP at specified coordinates using multiphoton excitation from a Ti:sapphire laser tuned to 820 nm and focused to the diffraction limit. The multiphoton excitation produced spatially well-defined fields of photoconverted molecules and minimized the tissue’s exposure to visible light. They then used confocal excitation at 488 nm to monitor the equilibration of the photoconverted GFP with serial x–y scans that intersected the area of photoconversion.
The results showed that in the connecting cilium diffusion was somewhat slower than expected in an aqueous cell compartment, but that this structure was not a major barrier to protein diffusion. However, the diffusion in the inner and outer segments bridged by the connection cilium was found to be substantially slower than in other cells studied. The authors offer various explanations of what might cause this slower diffusion.
Research Paper:
Use one laser for two multiphoton microscopes
At the Society for Neuroscience conference last week, Olympus introduced new accessories for two-photon confocal microscopy. The new Laser Sharing System, Dual-Port SIM Scanner, and Multi-Point SIM scanner software enhance the capabilities of the company’s FluoView FV1000-MPE multiphoton system.
The Laser Sharing System lets researchers use a single Spectra-Physics Mai Tai DeepSee laser system to perform imaging experiments on two separate Olympus multiphoton systems. It optically redirects laser light to the second multiphoton system, providing flexibility and saving money for those who want to install multiple two-photon instruments. The microscopes can be used simultaneously with the laser if using the same wavelength of light.
The Laser Sharing System lets researchers use a single Spectra-Physics Mai Tai DeepSee laser system to perform imaging experiments on two separate Olympus multiphoton systems. It optically redirects laser light to the second multiphoton system, providing flexibility and saving money for those who want to install multiple two-photon instruments. The microscopes can be used simultaneously with the laser if using the same wavelength of light.
The Dual-Port SIM (Simultaneous) Scanner is for experimental protocols requiring photobleaching with both visible and infrared (IR) light. With the scanner, the user can use both wavelengths without changing any optics or fibers, eliminating laser alignment issues, saving time, and cutting down on hassles that can interfere with imaging.
The SIM scanner software is useful for neuronal spine research in that it lets the user rapidly stimulate multi-point areas. The software has a dedicated neuroscience application and enables control of the scanner, laser, and dataset for stimulation applications used in uncaging or channel rhodopsin experiments. The software visualizes the signal not only from the confocal photomultiplier tube (PMT) but also from electrophysiology patch-clamp equipment through a new interface unit.
The SIM scanner software is useful for neuronal spine research in that it lets the user rapidly stimulate multi-point areas. The software has a dedicated neuroscience application and enables control of the scanner, laser, and dataset for stimulation applications used in uncaging or channel rhodopsin experiments. The software visualizes the signal not only from the confocal photomultiplier tube (PMT) but also from electrophysiology patch-clamp equipment through a new interface unit.
More information here.
New biotech optics catalog includes microscopy products
Optical components provider Edmund Optics has released a catalog featuring product lines for researchers and product developers in the biotech field. Marisa Edmund, the company’s vice president of marketing says that experience in micro optics manufacturing, asphere production, and complex assemblies, coupled with expertise in thin-film coating design makes Edmund Optics uniquely qualified to service all areas of biotech research, development and production.
The company has more than 16,900 off-shelf-products and can also design and fabricate custom components. Some of the microscopy products in the catalog include a variety of objective lenses, broad-wavelength lens systems such as UV-NIR triplets that are useful for two-photon microscopy, and filters for various microscopy applications including filters with high damage thresholds.
Learn more on the company’s new biotech applications webpage. Request a print catalog here or view an interactive catalog here.
The company has more than 16,900 off-shelf-products and can also design and fabricate custom components. Some of the microscopy products in the catalog include a variety of objective lenses, broad-wavelength lens systems such as UV-NIR triplets that are useful for two-photon microscopy, and filters for various microscopy applications including filters with high damage thresholds.
Learn more on the company’s new biotech applications webpage. Request a print catalog here or view an interactive catalog here.
New version of FCS/fluorescence lifetime imaging software
Picoquant has released V 5.1.2 of its SymPhoTime fluorescence lifetime imaging and correlation software. New features include online-TCSPC histogramming during data acquisition and the ability to reconstruct an instrument response function (IRF) from a measured decay curve (especially useful for multi-photon excitation setups). The software fully supports the HydraHarp 400 for data acquisition and analysis, and it speeds up FLIM data analysis by calculating prehistogrammed images. The software can be used in applications such as scanned imaging, single molecule detection, FLIM, FCS, and confocal microscopy. The company will host a SymPhoTime training day on Sept. 14.
Microscopy images reveal how B cells are activated
Researchers at Sydney's Garvan Institute of Medical Research and the University of California San Francisco have captured microscopy images that reveal some important new details of how the immune system works.
The researchers were interested in knowing what drives the immune system's B cell activation and where it occurs. B cells produce antibodies in response to a specific invaders’ antigens. These antibodies help fight the invader at that time as well as in the future. Scientists have not understood how these B cells get exposed to the invader’s antigens if macrophages and dendritic cells are constantly destroying the invaders.
The researchers studied these immune interactions in a living mouse’s lymph nodes--where invaders are brought to be destroyed by macrophages and dendritic cells. Intravital and multiphoton microscopy images revealed highly specialized 'subcapsular sinus (SCS) macrophages' embedded in the lining of the lymph nodes’ sinus. The images showed the heads of these macrophages capturing the antigen on one side of the lining and their tails delivering it to B cells on the other side of the lining. In other words, the intact antigen passes through the subcapsular sinus almost like it is on a conveyer belt. This finding helps to clarify other groups’ observations of unusual macrophages in the subcapsular sinus and B cells residing nearby, and it clarifies how and where the B cells become activated. View videos of findings are here.
Research paper: Tri Giang Phan, Jesse A Green, Elizabeth E Gray, Ying Xu & Jason G Cyster, Nature Immunology, Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation. Published online: June 7, 2009, doi:10.1038/ni.1745.
Image courtesy of the U.S. National Cancer Institute's Surveillance, Epidemiology and End Results (SEER) Program.
Automatic tracking of in vivo dendritic spines
A nueron's dendrites have tiny protrusions called dendritic spines. These spines have the important job of receiving input from a neighboring neuron, and thus are the subject of intense study. Most of these studies are done in vitro, but in vivo imaging of dendritic spines is possible with specialized equipment.In vivo imaging allows spines to be watched over time in a natural environment and allows observations during disease progression. However, counting and comparing dendrites from image to image taken with time-lapse microscopy is quite labor intensive.
Researchers at the Methodist Hospital Research Institute & the Methodist Hospital, Weill Cornell Medical College in Houston, TX recently published a paper detailing a technique that automates spine detection and tracking in a live animal model. They tested the technique on multiphoton microscopy images of an anesthetized mouse model of Alzheimer’s disease. Their method was able to map the dendritic backbone and its associated spines, quantify spine length and area, track the growth or loss of spines, and deal with poor image quality.
As we learn more about neurodegenerative disease such as Alzheimer’s disease, in vivo imaging of animal models will be key for studying how the brain changes during disease progression or reacts to therapies. Automating key parts of image analysis will greatly aid these studies.
Free full text .pdf of research paper: Jing Fan, Xiaobo Zhou, Jennifer G. Dy, Yong Zhang and Stephen T. C. Wong, An Automated Pipeline for Dendrite Spine Detection and Tracking of 3D Optical Microscopy Neuron Images of In Vivo Mouse Models, Neuroinformatics.
Researchers at the Methodist Hospital Research Institute & the Methodist Hospital, Weill Cornell Medical College in Houston, TX recently published a paper detailing a technique that automates spine detection and tracking in a live animal model. They tested the technique on multiphoton microscopy images of an anesthetized mouse model of Alzheimer’s disease. Their method was able to map the dendritic backbone and its associated spines, quantify spine length and area, track the growth or loss of spines, and deal with poor image quality.
As we learn more about neurodegenerative disease such as Alzheimer’s disease, in vivo imaging of animal models will be key for studying how the brain changes during disease progression or reacts to therapies. Automating key parts of image analysis will greatly aid these studies.
Free full text .pdf of research paper: Jing Fan, Xiaobo Zhou, Jennifer G. Dy, Yong Zhang and Stephen T. C. Wong, An Automated Pipeline for Dendrite Spine Detection and Tracking of 3D Optical Microscopy Neuron Images of In Vivo Mouse Models, Neuroinformatics.
Two-photon microscopy with 113.5-nm resolution
Two-photon microscopy is very useful for imaging living tissues, but the method’s restricted excitation volume typically comes with the trade-offs of lower spatial resolution (especially in the axial direction) and slower imaging than one-photon imaging. Multi-focal multiphoton microscopy–which uses multiple focal spots excitation instead of a single spot–looks promising for overcoming these drawbacks.
Researchers at the Chinese Academy of Science in Shanghai and Sun Yat-sen University in Guangzhou, China have reported their work to improve on the multi-focal technique in the latest issue of the Journal of Microscopy. They produced high-speed two-color two-photon microscopy with improved transverse and axial resolutions by using a 3-D optical lattice made by multi-beam interference and two excitation wavelengths. With 400 and 800 nm excitation they were able to produce 113.5 nm resolution in the transverse and axial directions while imaging faster than is possible with traditional two-photon microscopes.
Research paper: Journal of Microscopy, Two-colour two-photon confocal microscopy with isotropic three-dimensional resolution and parallel excitation, Volume 234 Issue 2, Pages 205 – 210.
Researchers at the Chinese Academy of Science in Shanghai and Sun Yat-sen University in Guangzhou, China have reported their work to improve on the multi-focal technique in the latest issue of the Journal of Microscopy. They produced high-speed two-color two-photon microscopy with improved transverse and axial resolutions by using a 3-D optical lattice made by multi-beam interference and two excitation wavelengths. With 400 and 800 nm excitation they were able to produce 113.5 nm resolution in the transverse and axial directions while imaging faster than is possible with traditional two-photon microscopes.
Research paper: Journal of Microscopy, Two-colour two-photon confocal microscopy with isotropic three-dimensional resolution and parallel excitation, Volume 234 Issue 2, Pages 205 – 210.
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