Fluorescence imaging has emerged as a valuable tool for clinical angiographic and cardiovascular imaging, allowing for visualization and quantification of biological processes. Among the range of fluorescence imaging windows, near-infrared (NIR) imaging has shown great promise as a non-invasive modality for angiographic and cardiovascular imaging. To overcome limitations associated with indocyanine green dye (ICG), we developed a biocompatible DNA-based platform for conjugation with ICG dyes and targeting moieties. The primary objective of this pilot study is to evaluate the efficacy of the DNA-ICG platform for contrast-enhanced NIR-II (>1250 nm) fluorescence imaging in a mouse model. Throughout the experiment, various organs were observed, including the heart, liver, spleen, caecum, and intestines. Notably, vascular structures in the tail, spinal column, and head remained visible for hours after the administration of the contrast agent. The DNA-ICG platform holds promise as an effective imaging tool for angiographic and cardiovascular studies.
Functional nanoscale materials are being extensively investigated for applications in biology and medicine and are ready to make significant contributions in the realization of exciting advancements in diverse areas of diagnostics and therapeutics. Aiming for more accurate, efficient, non-invasive and fast diagnostic tools, the use of near-infrared (NIR) light in the range of the 1st and 2nd biological window (NIR-I: 0.70-0.95 µm; NIR-II: 1.00-1.35 µm) provides deeper penetration depth into biological tissue, better image contrast, reduced phototoxicity and photobleaching. Consequently, NIR-based bioimaging became a quickly emerging field and manifold new NIR-emitting bioprobes have been reported. Since commercially available microscopes are not optimized for this kind of NPs, a new microscopy hyperspectral confocal imager has been developed to cover a broad spectral range (400 to 1700 nm) with high spectral resolution. The smallest spectral variation can be easily monitored thanks to the high spectral resolution (as low as 0.2 nm). This is possible thanks to a combination of an EMCCD and an InGaAs camera with a high resolution spectrometer. An extended number of NPs can be excited with a Ti:Sapphire laser, which provides tunable illumination within 690-1040 nm. Cells and tissues can be mapped in less than 100 ms, allowing in-vivo imaging. As a proof of concept, here we present the preliminary results of the spatial distribution of the fluorescence signal intensity from lanthanide doped nanoparticles incorporated into a system of biological interest. The temperature sub-mm gradient – analyzing the spectral features so gathered through an all-optical route is also thoroughly discussed.
Daniel Heller, Daniel Roxbury, Prakrit Jena, Ryan Williams, Balázs Enyedi, Philipp Niethammer, Stéphane Marcet, Francesca Mangiarini, Marc Verhaegen, Sébastien Blais-Ouellette
The intrinsic near-infrared photoluminescence (fluorescence) of single-walled carbon nanotubes exhibits unique photostability, narrow bandwidth, penetration through biological media, environmental sensitivity, and both chromatic variety and range. Biomedical applications exploiting this large family of fluorophores will require the spectral and spatial resolution of individual (n,m) nanotube species’ fluorescence and its modulation within live cells and tissues, which is not possible with current microscopy methods. We present a wide-field hyperspectral approach to spatially delineate and spectroscopically measure single nanotube fluorescence in living systems. This approach resolved up to 17 distinct (n,m) species (chiralities) with single nanotube spatial resolution in live mammalian cells, murine tissues ex vivo, and zebrafish endothelium in vivo. We anticipate that this approach will facilitate multiplexed nanotube imaging in biomedical applications while enabling deep-tissue optical penetration, exceptional photostability, and single-molecule resolution in vivo.
The efficacy of existing therapies and the discovery of innovative treatments for central nervous system (CNS) diseases have been limited by the lack of appropriate methods to investigate complex molecular processes at the synaptic level. To improve our capability to investigate complex mechanisms of synaptic signaling and remodeling, we designed a fluorescence hyperspectral imaging platform to simultaneously track different subtypes of individual neurotransmitter receptors trafficking in and out of synapses. This imaging platform allows simultaneous image acquisition of at least five fluorescent markers in living neurons with a high-spatial resolution. We used quantum dots emitting at different wavelengths and functionalized to specifically bind to single receptors on the membrane of living neurons. The hyperspectral imaging platform enabled the simultaneous optical tracking of five different synaptic proteins, including subtypes of glutamate receptors (mGluR and AMPAR) and postsynaptic signaling proteins. It also permitted the quantification of their mobility after treatments with various pharmacological agents. This technique provides an efficient method to monitor several synaptic proteins at the same time, which could accelerate the screening of effective compounds for treatment of CNS disorders.
In the past decade, the efficacy of existing therapies and the discovery of innovative treatments for Central Nervous System (CNS) diseases have been limited by the lack of appropriate methods to investigate complex molecular processes at the synaptic level. In order to better understand the fundamental mechanisms that regulate diseases of the CNS, a fast fluorescence hyperspectral imaging platform was designed to track simultaneously various neurotransmitter receptors trafficking in and out of synapses. With this hyperspectral imaging platform, it was possible to image simultaneously five different synaptic proteins, including subtypes of glutamate receptors (mGluR, NMDAR, AMPAR), postsynaptic density proteins, and signaling proteins. This new imaging platform allows fast simultaneous acquisitions of at least five fluorescent markers in living neurons with a high spatial resolution. This technique provides an effective method to observe several synaptic proteins at the same time, thus study how drugs for CNS impact the spatial dynamics of these proteins.
A new type of Raman spectroscopy hyperspectral imager based on Bragg tunable filter has been developed by University of Montreal and Photon etc. The technology of Bragg tunable filter significantly reduces the acquisition time by selecting a single wavelength in a full camera field and scanning the wavelength with a high efficiency. The transmission is continuously tunable over 400 nm range with a spectral resolution of 0.2 nm. We here present the principle of this novel Raman imaging system as well as hyperspectral images of a Si/Ti structured wafer and carbon nanotubes taken with a spectral resolution of 0.2 nm on the whole field of view of the microscope.
Volume Bragg grating technology has enabled the development of a new type of staring
hyperspectral camera. Based on Bragg Tunable filters, these hyperspectral cameras have both high
spectral and spatial resolution, and significantly higher sensitivity than competing technologies like
push broom spectrometer, liquid crystal tunable filters, or acousto-optic tunable filters. They are
minimally sensitive to polarization and their spectral isolation can reach 106. Here we thus present
an innovative tool to collect SWIR hyperspectral data with high spectral and spatial resolution. This
new instrument is based on a 3nm bandwidth Bragg Tunable Filter, continuously tunable from
1.0um and 2.5um. Because high spectral resolution also means less light per channel, a low noise
custom HgCdTe (MCT) camera was also developed to meet the requirement of the filter. The high
speed capability of more than 300 fps and the low operating temperature of 200K (deep cooled
option to 77K) allow full frame 500 spectral channel datacube acquisitions in minimal time. Basic
principle of this imaging filter will be reviewed as well as the custom MCT camera performances.
High resolution hyperspectral measurements will be demonstrated between 1.0um and 2.5um on
different objects.
A new type of Raman spectroscopy hyperspectral imager based on Bragg tunable filter has been developed by
University of Montreal and Photon etc. The technology of Bragg tunable filter significantly reduces the acquisition time
by selecting a single wavelength in a full camera field and scanning the wavelength with a high efficiency. The
transmission is continuously tunable over 400 nm range with a spectral resolution of 0.2 nm. We here present the
principle of this novel Raman imaging system as well as hyperspectral images of Si taken with a spectral resolution of
0.2 nm on the whole field of view of the microscope.
EMCCDs are devices capable of sub-electron read-out noise at high pixel rate, together with a high quantum efficiency
(QE). However, they are plagued by an excess noise factor (ENF) which has the same effect on photometric measurement
as if the QE would be halved. In order to get rid of the ENF, the photon counting (PC) operation is mandatory, with the
drawback of counting only one photon per pixel per frame. The high frame rate capability of the EMCCDs comes to the
rescue, at the price of increased clock induced charges (CIC), which dominates the noise budget of the EMCCD. The CIC
can be greatly reduced with an appropriate clocking, which renders the PC operation of the EMCCD very efficient for faint
flux photometry or spectroscopy, adaptive optics, ultrafast imaging and Lucky Imaging. This clocking is achievable with
a new EMCCD controller: CCCP, the CCD Controller for Counting Photons. This new controller, which is now commercialized
by Nüvü cameras inc., was integrated into an EMCCD camera and tested at the observatoire du mont-M'egantic.
The results are presented in this paper.
KEYWORDS: Clocks, Electron multiplying charge coupled devices, Cameras, Charge-coupled devices, Photon counting, Signal to noise ratio, Video, Human-machine interfaces, Data acquisition, Data communications
In order to make faint flux imaging efficient with an EMCCD, the Clock Induced Charges (CIC) must be reduced to a minimum.
Some techniques were proposed to reduce the CIC but until now, neither commercially available CCD controller
nor commercial cameras were able to implement them and get satisfying results. CCCP, the CCD Controller for Counting
Photons, has been designed with the aim of reducing the CIC generated when an EMCCD is read out. It is optimized for
driving EMCCDs at high speed (≥ 10MHz), but may be used also for driving conventional CCDs (or the conventional
output of an EMCCD) at high, moderate, or low speed. This new controller provides an arbitrary clock generator, yielding
a timing resolution of ~20 ps and a voltage resolution of ~2mV of the overlap of the clocks used to drive the EMCCD.
The frequency components of the clocks can be precisely controlled, and the inter-clock capacitance effect of the CCD can
be nulled to avoid overshoots and undershoots. Using this controller, CIC levels as low as 0.001 - 0.002 ¯e per pixel per
frame were measured on a 512×512 CCD97 operating in inverted mode, at an EM gain of ~2000. This is 5 to 10 times
less than what is usually seen in commercial EMCCD cameras using the same EMCCD chip.
KEYWORDS: Electron multiplying charge coupled devices, Photon counting, Signal to noise ratio, Charge-coupled devices, Photons, Clocks, Quantum efficiency, Temperature metrology, Signal processing, Interference (communication)
CCCP, a CCD Controller for Counting Photons, is presented. This new controller uses a totally new clocking architecture
and allows to drive the CCD in a novel way. Its design is optimized for the driving of EMCCDs at up to 20MHz of pixel
rate and fast vertical transfer. Using this controller, the dominant source of noise of EMCCDs at low flux level and high
frame rate, the Clock Induced Charges, were reduced to 0.001 - 0.0018 electron/pixel/frame (depending of the electron
multiplying gain), making efficient photon counting possible. CCCP will be deployed in 2009 on the ESO NTT through
the 3D-NTT1 project and on the SOAR through the BTFI project.
The Smart Tunable Filter is a new spectrograph providing a continuous field of view of 4'×4' sampled at 56 mas using
the technology of imaging Bragg Tunable Filter (BTF) and a scanning Fabry-Perot tunable filter. 48 holographic gratings
photoinscripted helicoidally in a doped glass cylinder and preceded by 4 tunable BTF are used to study 52 emission lines
selected between the main OH night sky lines. The Smart Tunable Filter offers the advantage to be an "opto-ecological"
system providing a complete separation between spectral and spatial information avoiding problems from image slicing
and slit effects. The design can fully operate at cryogenic temperatures for IR purpose. The Fabry-Perot mode operates at
a spectral resolution of 5000 and can be removed to provide a 100 spectral resolution over the 48 fixed gratings. Main
results obtained in laboratory from the first prototype developed in collaboration with Photon etc. Inc. are presented.
Merit factors to compare this instrument to similar ones are studied versus sciences cases.
We report on the science case high level specifications for a wide field spectrograph instrument for an Extremely Large
Telescope (ELT) and present possible concepts. Preliminary designs are presented which resort to different instrument
concepts: monolithic integral field (IFU), multi-IFU, and a smart tunable filter. This work is part of the activities performed
in the work package 'Instrumentation' of the 'ELT Design Study', a programme supported by the European Community,
Framework Programme 6.
We discuss the possibility of improving the optical efficiency of ELT instruments by reducing the number of optical surfaces with highly aspheric optics generated by deformable mirrors (DMs). Preliminary analysis shows that a 2 aspheric mirror design could in principle replace a traditional optical design with a complex series of ~ 10 lenses, providing a potential gain in efficiency of ~ 20% as well as a significant gain in compactness. New OH suppression systems based on technologies from the photonics world become available that may ultimately allow to reduce the near IR sky brightness by 2 to 4 magnitudes, depending on wavelength. The potential performance of an ELT with OH suppression is similar in imaging and significantly higher in spectroscopy than JWST.
An entirely new type of imaging tunable filter has been developed by Photon etc. and the California Institute of Technology. The Volume Bragg Grating based device is able to select a single wavelength for each pixel in a full camera field. The demonstration tabletop prototype was able to select images with a 2 nm bandwidth from 400 to 750 nm. Data cubes were produced through a wavelength scan from which a spectrum per pixel can be extracted. The prototype showed no image distortion, a very stable instrument profile, and high efficiency. The compact and robust tunable filter can operate from 350 nm to 2.5 mm with bandwidths from 3 Å to 200 nm, showing a great potential for both ground based and space astronomy.
KEYWORDS: Signal to noise ratio, Electron multiplying charge coupled devices, Signal processing, Charge-coupled devices, Photon counting, Quantum efficiency, Digital signal processing, Numerical simulations, Amplifiers, Interference (communication)
Thorough numerical simulations were run to test the performance of three processing methods of the data coming out from an electron multiplying charge coupled device (EMCCD), or low light level charge coupled device (L3CCD), operated at high gain, under real operating conditions. The effect of read-out noise and spurious charges is tested under various low flux conditions (0.001 event/pixel/frame< f < 20 events/pixel/frame). Moreover, a method for finding the value of the gain applied by the EMCCD amplification register is also developed. It allows one to determine the gain value to an accuracy of a fraction of a percent from dark frames alone.
Recent advances in photosensitive glass and holographic recording technologies open new possibilities for astronomical instrumentation. We have developed novel instrument concepts using thick Volume Phase Holographic gratings: a multiband filter for OH line suppression and a single order, widely tunable filter. We present these technologies along with other current and future uses of these promising holographic gratings.
A novel concept for a very efficient tunable filter for the optical and near infrared is proposed. The filter consists of two parallel volume phase holographic Bragg gratings used in a dispersion-recombination configuration. The passband profile is determined by the first order grating efficiency curve which changes in wavelength according to the angle of incidence. The filter can be continuously tuned over ranges of wavelengths larger than 600 nm by adjusting the tilt of the gratings, while bandwidth can be selected between 0.1 and 200 nm. The conceptual design is presented, along with predicted performances. Potentially useful instruments for large volume surveys
and targeted observations are explored, along with application in areas other than astronomy.
A user-friendly and automatic illuminator with adjustable wavelength and optical power has been developed to obtain precision quantum efficiency (QE) curves of astronomical CCD as well as optical transmission measurements for cryogenic holographic gratings and other optical components. Integrating commercial components with custom mechanical parts and control software, this equipment is able to illuminate a target with light of controlled intensity and wavelength. This facility is primarily intended for testing of Volume Phase Holographic (VPH) gratings at low temperature as well as obtaining CCD quantum efficiencies. A Labview control application runs on a desktop computer allowing full automation of the spectrophotometer. The apparatus includes a Quartz-Tungsten light source, neutral density filters, a monochromator, visible and near-infrared power meters, as well as collimating and focusing optics. Rotation mechanisms allow the characterization of gratings for all angles of diffractions. For CCD testing, network commands allow the facility to get raw images, compute and record QE curves for further detector characterization.
Near-infrared emission from atmospheric OH radicals is known to severely affect astronomical observations. Until now, only complex dispersive instruments were partially capable of removing this unwanted background, which is composed of hundreds of narrow emission lines. Recent development in photosensitive glass and holographic recording technologies now allow the elaboration of filters with a large number of narrow reflecting bands well matched to OH lines. This technology shows promise for removing many tens of lines in the J, H, and K bands. That would result in a many fold increase in imaging and low resolution signal-to-noise ratio. Filters with 10 lines have been tested and show the appealing possibilities of these new devices.
Characterization of Volume Phase Holographic gratings at cryogenic temperatures have been conducted using a new test facility at Caltech. The new test bench includes a cryostat that allows large angles for incident and diffracted light. Gratings under tests are shielded from thermal background, and precisely and uniformly temperature controlled. Preliminary results are presented and show little temperature dependence of the efficiency function.
We present details of the design, operation and calibration of an astronomical visible-band imaging Fourier transform spectrometer (IFTS). This type of instrument produces a spectrum for every pixel in the field of view where the spectral resolution is flexible. The instrument is a dual-input/dual-output Michelson interferometer coupled to the 3.5 meter telescope at the Apache Point Observatory. Imaging performance, and interferograms and spectra from calibration sources and standard stars are discussed.
We have acquired spatial-spectral datacubes of astronomical objects using the Livermore visible-band imaging Fourier transform spectrometer at Apache Point Observatory. Each raw datacube contains hundreds of thousands of spectral interferograms. We present in-progress demonstrations of these observations.
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