-
Nature Methods Jul 2023Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution...
Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.
Topics: Animals; Mice; Calcium; Brain; Lighting; Microscopy; Photons
PubMed: 37429962
DOI: 10.1038/s41592-023-01913-z -
Optics Express Jan 2016Brillouin spectroscopy has been used to characterize shear acoustic phonons in materials. However, conventional instruments had slow acquisition times over 10 min per 1...
Brillouin spectroscopy has been used to characterize shear acoustic phonons in materials. However, conventional instruments had slow acquisition times over 10 min per 1 mW of input optical power, and they required two objective lenses to form a 90° scattering geometry necessary for polarization coupling by shear phonons. Here, we demonstrate a confocal Brillouin microscope capable of detecting both shear and longitudinal phonons with improved speeds and with a single objective lens. Brillouin scattering spectra were measured from polycarbonate, fused quartz, and borosilicate in 1-10 s at an optical power level of 10 mW. The elastic constants, phonon mean free path and the ratio of the Pockels coefficients were determined at microscopic resolution.
Topics: Equipment Design; Equipment Failure Analysis; Image Enhancement; Lenses; Lighting; Microscopy, Confocal; Refractometry; Reproducibility of Results; Sensitivity and Specificity
PubMed: 26832263
DOI: 10.1364/OE.24.000319 -
The Review of Scientific Instruments May 2020Light-sheet microscopy offers faster imaging and reduced phototoxicity in comparison to conventional point-scanning microscopy, making it a preferred technique for...
Light-sheet microscopy offers faster imaging and reduced phototoxicity in comparison to conventional point-scanning microscopy, making it a preferred technique for imaging biological dynamics for durations of hours or days. Such extended imaging sessions pose a challenge, as it reduces the number of specimens that can be imaged in a given day. Here, we present a versatile light-sheet imaging instrument that combines two independently controlled microscope-twins, built so that they can share an ultrafast near-infrared laser and a bank of continuous-wave visible lasers, increasing the throughput and decreasing the cost. To permit a wide variety of specimens to be imaged, each microscope-twin provides flexible imaging parameters, including (i) operation in one-photon and/or two-photon excitation modes, (ii) delivery of one to three light-sheets via a trio of orthogonal excitation arms, (iii) sub-micron to micron imaging resolution, (iv) multicolor compatibility, and (v) upright (with provision for inverted) detection geometry. We offer a detailed description of the twin-microscope design to aid instrument builders who wish to construct and use similar systems. We demonstrate the instrument's versatility for biological investigation by performing fast imaging of the beating heart in an intact zebrafish embryo, deep imaging of thick patient-derived tumor organoids, and gentle whole-brain imaging of neural activity in behaving larval zebrafish.
Topics: Equipment Design; Lasers; Light; Microscopy
PubMed: 32486724
DOI: 10.1063/1.5144487 -
The Journal of Physical Chemistry. B Jun 2023Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging nonlinear vibrational imaging technique that delivers label-free chemical maps of cells and...
Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging nonlinear vibrational imaging technique that delivers label-free chemical maps of cells and tissues. In narrowband CARS, two spatiotemporally superimposed picosecond pulses, pump and Stokes, illuminate the sample to interrogate a single vibrational mode. Broadband CARS (BCARS) combines narrowband pump pulses with broadband Stokes pulses to record broad vibrational spectra. Despite recent technological advancements, BCARS microscopes still struggle to image biological samples over the entire Raman-active region (400-3100 cm). Here, we demonstrate a robust BCARS platform that answers this need. Our system is based on a femtosecond ytterbium laser at a 1035 nm wavelength and a 2 MHz repetition rate, which delivers high-energy pulses used to produce broadband Stokes pulses by white-light continuum generation in a bulk YAG crystal. Combining such pulses, pre-compressed to sub-20 fs duration, with narrowband pump pulses, we generate a CARS signal with a high (<9 cm) spectral resolution in the whole Raman-active window, exploiting both the two-color and three-color excitation mechanisms. Aided by an innovative post-processing pipeline, our microscope allows us to perform high-speed (≈1 ms pixel dwell time) imaging over a large field of view, identifying the main chemical compounds in cancer cells and discriminating tumorous from healthy regions in liver slices of mouse models, paving the way for applications in histopathological settings.
Topics: Animals; Mice; Microscopy; Light; Spectrum Analysis, Raman; Nonlinear Optical Microscopy; Lasers
PubMed: 37195090
DOI: 10.1021/acs.jpcb.3c01443 -
Nature Communications 2013Among the applications of optical phase measurement, the differential interference contrast microscope is widely used for the evaluation of opaque materials or...
Among the applications of optical phase measurement, the differential interference contrast microscope is widely used for the evaluation of opaque materials or biological tissues. However, the signal-to-noise ratio for a given light intensity is limited by the standard quantum limit, which is critical for measurements where the probe light intensity is limited to avoid damaging the sample. The standard quantum limit can only be beaten by using N quantum correlated particles, with an improvement factor of √N. Here we report the demonstration of an entanglement-enhanced microscope, which is a confocal-type differential interference contrast microscope where an entangled photon pair (N=2) source is used for illumination. An image of a Q shape carved in relief on the glass surface is obtained with better visibility than with a classical light source. The signal-to-noise ratio is 1.35±0.12 times better than that limited by the standard quantum limit.
Topics: Microscopy; Microscopy, Atomic Force; Microscopy, Confocal; Microscopy, Interference; Photons; Signal-To-Noise Ratio
PubMed: 24026165
DOI: 10.1038/ncomms3426 -
Scientific Reports Nov 2022Super-resolution fluorescence microscopy can be achieved by image reconstruction after spatially patterned illumination or sequential photo-switching and read-out....
Super-resolution fluorescence microscopy can be achieved by image reconstruction after spatially patterned illumination or sequential photo-switching and read-out. Reconstruction algorithms and microscope performance are typically tested using simulated image data, due to a lack of strategies to pattern complex fluorescent patterns with nanoscale dimension control. Here, we report direct electron-beam patterning of fluorescence nanopatterns as calibration standards for super-resolution fluorescence. Patterned regions are identified with both electron microscopy and fluorescence labelling of choice, allowing precise correlation of predefined pattern dimensions, a posteriori obtained electron images, and reconstructed super-resolution images.
Topics: Calibration; Lighting; Electrons; Microscopy, Fluorescence; Algorithms
PubMed: 36418420
DOI: 10.1038/s41598-022-24502-0 -
Journal of Assisted Reproduction and... Jul 2014To assess irradiance and total energy dose from different microscopes during the in-vitro embryonic developmental cycle in mouse and pig and to evaluate its effect on...
PURPOSE
To assess irradiance and total energy dose from different microscopes during the in-vitro embryonic developmental cycle in mouse and pig and to evaluate its effect on embryonic development and quality in pig.
METHOD
Spectral scalar irradiance (380-1050 nm) was measured by a fiber-optic microsensor in the focal plane of a dissection microscope, an inverted microscope and a time-lapse incubation system. Furthermore, the effect of three different red light levels was tested in the time-lapse system on mouse zygotes for 5 days, and on porcine zona-intact and zona-free parthenogenetically activated (PA) embryos for 6 days.
RESULTS
The time-lapse system used red light centered at 625 nm and with a lower irradiance level as compared to the white light irradiance levels on the dissection and inverted microscopes, which included more energetic radiation <550 nm. Even after 1000 times higher total energy dose of red light exposure in the time-lapse system, no significant difference was found neither in blastocyst development of mouse zygotes nor in blastocyst rates and total cell number of blastocysts of porcine PA embryos.
CONCLUSIONS
Our results indicate that red light (625 nm, 0.34 W/m(2)) used in the time-lapse incubation system does not decrease the development and quality of blastocysts in both mouse zygotes and porcine PA embryos (both zona-intact and zona-free).
Topics: Animals; Blastocyst; Embryo, Mammalian; Embryonic Development; Female; Fiber Optic Technology; Humans; Light; Mice; Pregnancy; Swine; Zona Pellucida; Zygote
PubMed: 24854483
DOI: 10.1007/s10815-014-0247-7 -
PloS One 2016Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also...
LIGHT SHEET MICROSCOPY IN THE MUSEUM
Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology.
DESIGN PRINCIPLES OF AN EDUCATIONAL LIGHT SHEET MICROSCOPE
To maximize educational impact, we radically reduced a standard light sheet microscope to its essential components without compromising functionality and incorporated stringent safety concepts beyond those needed in the lab. Our eduSPIM system features one illumination and one detection path and a sealed sample chamber. We image fixed zebrafish embryos with fluorescent vasculature, because the structure is meaningful to laymen and visualises the optical principles of light sheet microscopy. Via a simplified interface, visitors acquire fluorescence and transmission data simultaneously.
THE EDUSPIM DESIGN IS TAILORED EASILY TO FIT NUMEROUS APPLICATIONS
The universal concepts presented here may also apply to other scientific approaches that are communicated to laymen in interactive settings. The specific eduSPIM design is adapted easily for various outreach and teaching activities. eduSPIM may even prove useful for labs needing a simple SPIM. A detailed parts list and schematics to rebuild eduSPIM are provided.
Topics: Animals; Biology; Embryo, Nonmammalian; Germany; Green Fluorescent Proteins; Imaging, Three-Dimensional; Light; Lighting; Microscopy; Microscopy, Fluorescence; Museums; Optics and Photonics; Zebrafish
PubMed: 27560188
DOI: 10.1371/journal.pone.0161402 -
International Journal of Molecular... Jan 2022Optical microscopy has vastly expanded the frontiers of structural and functional biology, due to the non-invasive probing of dynamic volumes in vivo. However,... (Review)
Review
Optical microscopy has vastly expanded the frontiers of structural and functional biology, due to the non-invasive probing of dynamic volumes in vivo. However, traditional widefield microscopy illuminating the entire field of view (FOV) is adversely affected by out-of-focus light scatter. Consequently, standard upright or inverted microscopes are inept in sampling diffraction-limited volumes smaller than the optical system's point spread function (PSF). Over the last few decades, several planar and structured (sinusoidal) illumination modalities have offered unprecedented access to sub-cellular organelles and 4D (3D + time) image acquisition. Furthermore, these optical sectioning systems remain unaffected by the size of biological samples, providing high signal-to-noise (SNR) ratios for objective lenses (OLs) with long working distances (WDs). This review aims to guide biologists regarding planar illumination strategies, capable of harnessing sub-micron spatial resolution with a millimeter depth of penetration.
Topics: Imaging, Three-Dimensional; Lighting; Microscopy, Fluorescence; Signal-To-Noise Ratio; Single Molecule Imaging; Time-Lapse Imaging
PubMed: 35163562
DOI: 10.3390/ijms23031643 -
Nature Methods Nov 2022Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores....
Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photobleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle three-dimensional (3D) imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, an LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150 nm, combined with lower phototoxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1 Hz.
Topics: Imaging, Three-Dimensional; Lighting; Microscopy, Fluorescence; Photobleaching
PubMed: 36280718
DOI: 10.1038/s41592-022-01635-8