Two-photon excitation fluorescence microscopy (2PEM) has transformed biological imaging, offering deeper tissue penetration, reduced photodamage, and improved 3D resolution. Here, we explore a cutting-edge 15 fs ultrafast laser for enhanced 2PEM. Our setup includes a single-photon avalanche diode (SPAD) array for image scanning microscopy (ISM) and a 775 nm pulsed laser for STED microscopy.
The ultrashort excitation pulse enables higher peak powers and enhanced image clarity due to wider bandwidth. ISM with a 7x7 SPAD array (Genoa Instruments s.r.l., Genoa, Italy) provides crucial insights into imaging conditions, ensuring high-resolution and high-SNR imaging under sub-optimal and deep imaging scenarios. Additionally, our 775 nm pulsed laser implements STED for super-resolution 2PEF imaging beyond the diffraction limit.
Integrating these techniques in a single microscope platform unlocks powerful nanoscale investigations in tissues, uncovering complex cellular interactions and paving the way for breakthroughs in neurobiology, developmental biology, and cancer research.
KEYWORDS: Image restoration, Reconstruction algorithms, Detection and tracking algorithms, Image resolution, Image processing, Image enhancement, Confocal microscopy, Super resolution, Single photon detectors, Signal to noise ratio
Image Scanning Microscopy (ISM) enables super-resolution at an excellent signal-to-noise ratio thanks to a detector array. The microscope collects a confocal-like image for each detector element, generating a large dataset that requires tailored processing tools to be converted into a single super-resolved image. We propose a novel algorithm to fuse the dataset into an image with enhanced optical sectioning and resolution. Our method exploits the information inherently contained in the dataset to reject out-of-focus contributions and reconstruct an image with a smaller pixel size and a better resolution. The proposed method requires minimal user inputs and outperforms existing reconstruction methods.
Fluorescence laser-scanning microscopy (FLSM) is a widely utilized tool in life-science research. In recent years, this technique has undergone a profound transformation, thanks to the introduction of novel single-photon avalanche diode (SPAD) array detectors.
This study reveals the exciting possibilities of combining the SPAD array detector with single-molecule techniques.
We propose a real-time single-molecule tracking architecture, where the SPAD array effortlessly localizes the molecule of interest, and the beam scanning architecture effectively maintains the molecule at the center of the microscope's detection volume. This approach enables comprehensive three-dimensional tracking throughout the entire cell, offering valuable insights into molecular nano-environments, interactions, and structural changes through fluorescence lifetime information.
Furthermore, utilizing the same FLSM system, we present a novel sequential structure illumination single-molecule localization microscope (similar to MINFLUX). This advanced technique achieves localization precision in the few-nanometer range while simultaneously providing the molecule's fluorescence lifetime.
Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of sub-cellular structures with spatial resolution well below the diffraction limit. The higher the STED beam intensity, the higher the resolution, but at the cost of increased photo-damage, which significantly limits the application of STED microscopy in live specimens. The separation by lifetime tuning (SPLIT) technique uses a time-resolved acquisition and a phasor-based analysis to efficiently distinguish photons emitted from the center and from the periphery of the effective fluorescent region, thus improves the resolution of STED microscopy without increasing the STED beam intensity. Furthermore, the SPLIT method is combined with a deep learning-based phasor analysis algorithm termed flimGANE (fluorescence lifetime imaging based on a generative adversarial network), to improve the robustness of SPLIT-STED allowing improving the resolution up to 1.45 folds at the half of the depletion laser beam intensity.
In the recent years, numerous adaptive optics techniques have emerged to address optical aberrations in fluorescence microscopy imaging. However, many existing methods involve complex hardware implementations or lengthy iterative algorithms that may induce photo-damage to the sample. Our study proposes an innovative approach centered around a novel detector array capable of potentially capturing the probed sample in a single acquisition. Our solution is gentle on the sample and applicable to any laser scanning microscope equipped with a detector array. We demonstrate that the multi-dimensional dataset obtained using the detector array inherently encodes information about optical aberrations. Finally, we propose a convolutional neural network approach to decode these optical aberrations in real-time and with high accuracy, establishing the foundation for a new class of adaptive optics laser-scanning microscopy methods.
Fluorescence confocal laser-scanning microscopy (LSM) is one of the most popular tools for life science research. This popularity is expected to grow thanks to single-photon array detectors tailored for LSM. These detectors offer unique single-photon spatiotemporal information, opening new perspectives for gentle and quantitative superresolution imaging. However, a flawless recording of this information poses significant challenges for the microscope data acquisition (DAQ) system. We present a DAQ module based on the digital frequency domain principle, able to record essential spatial and temporal features of photons. We use this module to extend the capabilities of established imaging techniques based on single-photon avalanche diode (SPAD) array detectors, such as fluorescence lifetime image scanning microscopy. Furthermore, we use the module to introduce a robust multispecies approach encoding the fluorophore excitation spectra in the time domain. Finally, we combine time-resolved stimulated emission depletion microscopy with image scanning microscopy, boosting spatial resolution. Our results demonstrate how a conventional fluorescence laser scanning microscope can transform into a simple, information-rich, superresolved imaging system with the simple addition of a SPAD array detector with a tailored data acquisition system. We expected a blooming of advanced single-photon imaging techniques, which effectively harness all the sample information encoded in each photon.
We propose a straightforward implementation of two-photon image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced single-photon avalanche diode (SPAD) array detector and a novel blind image reconstruction algorithm is shown to dramatically improve the optical resolution of two-photon imaging, in various test samples. We show how our computational ISM approach is able to adapt to changing imaging conditions, thus ensuring optimal image quality. We also show how our recently introduced blind deconvolution approaches can be integrated into the image reconstruction workflow to further improve the image quality.
Stimulated emission depletion (STED) microscopy is a powerful bioimaging technique that theoretically provides molecular spatial resolution while preserving the most important assets of fluorescence microscopy. When combined with two-photon excitation (2PE) microscopy (2PE-STED), subdiffraction resolution may be achieved for thick biological samples. The most straightforward implementation of 2PE-STED microscopy entails introduction of an STED beam operating in continuous wave (CW) into a conventional Ti:sapphire-based 2PE microscope (2PE CW-STED). In this implementation, resolution enhancement is typically achieved using time-gated detection schemes, often resulting in drastic signal-to-noise/-background ratio (SNR/SBR) reductions. Herein, we employ a pixel-by-pixel phasor approach to discard fluorescence photons lacking super-resolution information to enhance image SNR/SBR in 2PE CW-STED microscopy. We compare this separation of photons by lifetime tuning approach against other postprocessing algorithms and combine it with image deconvolution to further optimize image quality.
Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of macromolecular complexes and sub-cellular structures with spatial resolution well below the diffraction limit. However, resolution in the double-digit nanometer range can be obtained only using high intensity depletion laser, at the cost of increased photo-damage, which significantly limits STED applications in live specimens. To minimize this, we use the separation by lifetime tuning (SPLIT) technique, in which phasor analysis is used to efficiently distinguish photons emitted from the center and from the periphery of the excitation spot of a STED microscope. Thus, it can be used to improve the resolution without increasing the STED beam intensity. Our approach utilizes a combination of pulsed excitation and pulsed depletion lasers to record the time-resolved photons by FastFLIM. The photons stream are successively analyzed using the SPLIT technique, demonstrating that the resolution improves without increasing the depletion laser intensity.
If a scanning illumination spot is combined with a detector array, we acquire a 4 dimensional signal. Unlike confocal microscopy with a small pinhole, we detect all the light from the object, which is particularly important for fluorescence microscopy, when the signal is weak. The image signal is basically a cross-correlation, and is highly redundant. It has more than sufficient information to reconstruct an improved resolution image. A 2D image can be generated from the measured signal by pixel reassignment. The result is improved resolution and signal strength, the system being called image scanning microscopy. A variety of different signal processing techniques can be used to predict the reassignment and deconvolve the partial images. We use an innovative single-photon avalanche diode (SPAD) array detector of 25 detectors (arranged into a 5× 5 matrix). We can simultaneously acquire 25 partial images and process to calculate the final reconstruction online.
Stimulated emission depletion (STED) microscopy is a powerful bio-imaging technique since it provides molecular spatial resolution whilst preserving the most important assets of fluorescence microscopy. When combined with twophoton excitation (2PE) microscopy (2PE-STED), the sub-diffraction imaging ability of STED microscopy can be achieved also on thick biological samples. The most straightforward implementation of 2PE-STED microscopy is obtained by introducing a STED beam operating in continuous wave (CW) into a conventional Ti:Sapphire based 2PE microscope (2PE-CW-STED). In this implementation, an effective resolution enhancement is mainly obtained implementing a time-gated detection scheme, which however can drastically reduce the signal-to-noise/background ratio of the final image. Herein, we combine the lifetime tuning (SPLIT) approach with 2PE-CW-STED to overcome this limitation. The SPLIT approach is employed to discard fluorescence photons lacking super-resolution information, by means of a pixel-by-pixel phasor approach. Combining the SPLIT approach with image deconvolution further optimizes the signal-to-noise/background ratio.
Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of macromolecular complexes and sub-cellular structures with spatial resolution below the diffraction limit. The spatial resolution of STED is limited by power of the depletion laser at the specimen plane. Higher depletion laser power will improve resolution, but at the cost of increased photo-bleaching, photo-toxicity, and anti-stoke emission background. This degrades the signal-to-noise ratio, and can significantly limit STED applications in living specimens. Here, we present an efficient multi-color STED microscopy method based on the digital frequency domain fluorescence lifetime imaging (FastFLIM) and the phasor plots. Our approach utilizes a combination of pulsed excitation and pulsed depletion lasers to record the time-resolved photons by FastFLIM. We demonstrate that the resolution is improved without increasing the depletion laser power by digital separation of the depleted species from the partially depleted species based on their different decay kinetics. We show the utility of this novel STED method applied in both fixed and live cellular samples, and also show its application to fluorescence lifetime correlation spectroscopy (FLCS) measurements. By combining fluorophores with different fluorescence lifetimes, we simultaneously record two-color STED images of cells labeled with Atto655 and Alexa647 in a single scan by using a single pair of excitation and depletion lasers. This novel approach shortens the data acquisition time while minimizing the photo-toxicity caused when using two separate depletion lasers.
There are basically two types of microscope, which we call conventional and scanning. The former type is a full-field imaging system. In the latter type, the object is illuminated with a probe beam, and a signal detected. We can generalize the probe to a patterned illumination. Similarly we can generalize the detection to a patterned detection. Combining these we get a range of different modalities: confocal microscopy, structured illumination (with full-field imaging), spinning disk (with multiple illumination points), and so on. The combination allows the spatial frequency bandwidth of the system to be doubled. In general we can record a four dimensional (4D) image of a 2D object (or a 6D image from a 3D object, using an acoustic tuneable lens). The optimum way to directly reconstruct the resulting image is by image scanning microscopy (ISM). But the 4D image is highly redundant, so deconvolution-based approaches are also relevant.
ISM can be performed in fluorescence, bright field or interference microscopy. Several different implementations have been described, with associated advantages and disadvantages. In two-photon microscopy, the illumination and detection point spread functions are very different. This is also the case when using pupil filters or when there is a large Stokes shift.
One of the key frontiers in optical imaging is to maximize the spatial information retrieved from a sample while minimizing acquisition time. Confocal laser scanning microscopy is a powerful imaging modality that allows real-time and high-resolution acquisition of two-dimensional (2D) sections. However, in order to obtain information from threedimensional (3D) volumes it is currently limited by a stepwise process that consists of acquiring multiple 2D sections from different focal planes by slow z-focus translation. Here, we present a novel method that enables the capture of an entire 3D sample in a single step. Our approach is based on an acoustically-driven varifocal lens integrated in a commercial confocal system that enables axial focus scanning at speeds of 140 kHz or above. Such high-speed allows for one or multiple focus sweeps on a pixel by pixel basis. By using a fast acquisition card, we can assign the photons detected at each pixel to their corresponding focal plane allowing simultaneous multiplane imaging. We exemplify this novel 3D confocal microscopy technique by imaging different biological fluorescent samples and comparing them with those obtained using traditional z-scanners. Based on these results, we find that image quality in this novel approach is similar to that obtained with traditional confocal methods, while speed is only limited by signal-to-noise-ratio. As the sensitivity of photodetectors increases and more efficient fluorescent labeling is developed, this novel 3D method can result in significant reduction in acquisition time allowing the study of new fundamental processes in science.
In a stimulated emission depletion (STED) microscope the region from which a fluorophore can spontaneously emit shrinks with the continued STED beam action after the excitation event. This fact has been recently used to implement a versatile, simple and cheap STED microscope that uses a pulsed excitation beam, a STED beam running in continuous-wave (CW) and a time-gated detection: By collecting only the delayed (with respect to the excitation events) fluorescence, the STED beam intensity needed for obtaining a certain spatial resolution strongly reduces, which is fundamental to increase live cell imaging compatibility. This new STED microscopy implementation, namely gated CW-STED, is in essence limited (only) by the reduction of the signal associated with the time-gated detection. Here we show the recent advances in gated CW-STED microscopy and related methods. We show that the time-gated detection can be substituted by more efficient computational methods when the arrival-times of all fluorescence photons are provided.
KEYWORDS: Point spread functions, Optical filters, Confocal microscopy, Signal to noise ratio, Microscopy, Phase only filters, Image filtering, Optical resolution, Optical transfer functions, Image resolution
The aim of this work is to propose and analyze optical schemes to obtain an improvement of resolution in optical
fluorescence microscopy. This goal can be achieved by implementing interfering illumination beams. We start from the insertion, on the illumination arm of the confocal microscope, of appropriately phase plates inducing laterally interfering beams, and then we propose to exploit two-photon excitation, too. We plan to implement solutions for shaping also the axial component of the point spread function by use of phase-only pupil filters and binary filters. In order to implement such schemes we use a computational simulation mainly based on a vectorial approach coupled to experimental procedures utilizing ultra-thin fluorescent layers and thick gels containing immobile fluorescent molecules as 2D and 3D phantoms, respectively. As well, image processing and successive views can be recombined to get a final isotropic improvement of resolution.
Confocal laser scanning (CLS) and two-photon excitation (TPE) microscopy are powerful techniques for 3D
imaging of biological samples. Although CLS and TPE microscopy images are better than standard epifluorescence
images, they still undergo degradation due to blurring and random noise because of the inherent nature
of the physical phenomenon (diffraction and photon counting noise) involved. The aim is to obtain the real
object from the degraded noisy image. This problem belongs to inverse problems and is found to be very notorious
in nature. Several algorithms such as maximum likelihood (ML) based algorithm, have been proposed to reduce these artifacts. Unfortunately, ML based algorithm tends to generate noise artifacts, so regularization constraints based on some prior knowledge have to be integrated to stabilize the solution. This is termed as maximum
a-posteriori (MAP) technique. We propose a MAP approach in which the image field is suitably modeled
as Markov random field (MRF), forcing the image distribution to be Gibbs distribution. The prior knowledge
is incorporated through the potential function in the Gibbs distribution. We proposed potential functions based
on white-noise prior, smoothest prior and fuzzy logic. MAP approach has the advantage of include the available
prior knowledge in the restoration procedure. In other words, inclusion of prior knowledge makes the notorious
inverse problem well-posed. Various evaluations such as visual inspection and Csiszar I-divergence are performed
on the CLS microscopy restored images to study the characteristics of the proposed approach (in both simulated
and real data). It is observed that the noise artifacts are considerably reduced and the desired images characteristics
(edges and minute features as islets) are retained in the restored images. The algorithm is extended in
the third dimension for 3D-image restoration application. The proposed algorithm is found to perform better
than existing image restoration algorithm in microscopy. The algorithm is stable, robust and tolerant at various
noise (Poisson) intensities. The convergence of the proposed algorithm is empirically observed. We hope that
the proposed algorithm will find wide applications in microscopy and biomedical imaging.
Layer-by-Layer or self-assembly techniques can be used to prepare Fluorescent polymer samples on glass coverslips
serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general
for sectioning microscopy. Layers can be realized as ultra-thin (<< 100 nm) or thin (approx. 100 nm)
characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to
directly measure the z-response of different scanning optical microscopes. In two-photon excitation microscopy z-sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized
in a Sectioned Imaging property chart (SIPchart), we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the z-response of the system over a full field of view area. These samples are also useful for monitoring photobleaching
behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional
technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be
effciently used for real time alignment of the microscope.
We report about a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (paGFP). This special form of the molecule increases its fluorescence intensity when excited by 488 nm after irradiation with high intensity light at 413 nm1. The aim in this work was to evaluate the use of two-photon interactions for activation of the molecules2. Therefore experiments were performed using fixed and living cells which were expressing the paGFP fluorophore and microspheres whose surface was modified by specific adsorption of the chromophores. The latter objects were used to investigate the ability of different wavelengths to activate the paGFP due to the anticipated more homogeneous density distribution. The molecular switches were activated in a range of wavelength from 720 nm to 840 nm. The optimal wavelength for activation was then chosen for cell imaging. A comparison between the conventional activation with a single photon at 413 nm and two-photons demonstrates clearly the advantages using non linear processes: much smaller volume in the cell can be activated unlike to a whole cell activation in single photon excitation regime.
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