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.
We demonstrate a combination of polarization-resolved with fluorescence optical scanning microscopy that offers polarizing based information along with molecular view of sample as a versatile tool for imaging the chromatin organization.
Light-sheet microscopes with an extended depth of field (EDOF) offer a simple but powerful route toward fast volumetric imaging. However, methods for EDOF typically result in a loss of signal-to-noise ratio. Here, we propose a parallelization strategy as a simple solution. By illuminating multiple acoustically generated light sheets at different axial positions within the EDOF, and following an encoding sequence, information from several in-focus planes can be simultaneously retrieved. After applying a decoding algorithm, volumetric images are reconstructed with enhanced signal and level of detail. Our strategy paves the way for exploiting the full speed capabilities of EDOF light-sheet systems.
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.
This review presents a selection of advanced microscope designs employing acousto-optical deflectors (AODs). In the designs covered, AODs are used as tunable diffraction gratings to control complex illumination patterns at the Fourier plane of an objective lens. This approach allows us to generate desired illumination patterns at the focal plane of a light microscope. In simple terms, I will describe two established designs, the 3D Random-Access Multi-Photon Microscope and the Standing-Wave Super-Resolution Microscope, as well as two new schemes including the Random-Access STED Microscope and the Frequency-Encoded Multi-Beam Microscope. All instruments mentioned here were designed to overcome the throughput limitations of previously used light microscopes in experimental Neuroscience.
Here, we report sub-diffraction resolution in two-photon excitation (TPE) fluorescence microscopy achieved by merging
this technique with continuous-wave (CW) stimulated-emission depletion (STED). We show an easy-to- implement and
promising laser combination based on Ti:Sapphire ultrafast laser source for two-photon excitation and a commercial
Leica TCS STED-CW microscope for resolution enhancement. Images of fluorescent nanoparticles produce comparative
similar resolution to the one photon excitation. Two-photon excitation STED microscopy achieves approximately 3-4
fold improvement in resolution in the radial direction over conventional 2PELSM. Further improvements in resolution
are theoretically achievable, suggesting that 2PE STED microscopy will permit nanoscale imaging, for instance, of
neuronal structures located in relatively intact brain tissue.
We show that after two-photon excitation of the photo initiator we are able to inhibit the polymerization process with a
second beam of different wavelength. For achieving sub-diffraction resolution this is one of the key elements.
Remarkably is that these experiments can be performed with a commercially available STED microscope slightly
modified for two-photon-excitation (TPE). First experiments featuring an enhanced resolution will be presented.
Extracellularly distributed collagen and chondrocytes seeded in gelatine and poly--caprolactone scaffolds are visualized by two-photon excitation microscopy (TPEM) and second-harmonic generation (SHG) imaging in both forward and backward nondescanned modes. Joint application of TPEM and SHG imaging in combination with stereological measurements of collagen enables us not only to take high-resolution 3-D images, but also to quantitatively analyze the collagen volume and a spatial arrangement of cell-collagen-scaffold systems, which was previously impossible. This novel approach represents a powerful tool for the analysis of collagen-containing scaffolds with applications in cartilage tissue engineering.
Several endogenous protein structures give rise to second harmonic generation (SHG) - second order nonabsorbative
energy doubling of an excitation laser line. The orientation of collagen fibers within tissues such as tendons or ligaments
is of primary importance. In this study, we propose a method to map the orientation of collagen fibers of a tendon. The
method uses only few images acquired at specific polarizations of the input laser beam by rotating the sample on the
stage. This procedure is implemented both in backward and forward scattering pathways. The improved details should
clarify the backscattered signal nature.
The vertebrate retina is an array of “narrow-capture” photoreceptive elements of diverse cellular types that allow the fine spatial resolution characteristic of vision. Imaging of photoreceptors and of the whole retina has been previously reported; however, both were achieved exclusively after fixation. We report our development of a new technique for imaging live bovine retinas ex vivo. Using this technique, we conducted fluorescence confocal laser scanning microscopic imaging of bovine retinas. Eyecups were incubated with conventional fluorescent mitochondrial probes (MitoTracker and JC-1). Unexpectedly, we found that, besides the retinal mitochondria, the rod outer segments that are devoid of mitochondria were also stained. No other neuron was stained. Both protonophores, which decrease mitochondrial membrane potential, or inhibit electron transport strongly inhibited the selective association of dyes with both retinal rod outer segments and mitochondria. This is the first time that living rod outer segments were visualized by this technique. This finding may shed light on previous reports of the existence of a proton potential across the disk membranes and on the mechanism of the adenosine tri-phosphate (ATP) supply for phototransduction, which still requires investigation.
Vertebrate retinal rod outer segment disks house the proteins involved in the phototransduction cascade that converts light into neuronal signal. We develop a technique for the immunofluorescent labeling of osmotically intact isolated rod outer segment disks for confocal laser scanning microscopy imaging. Osmotically intact Ficoll-flotation isolated bovine disks are directly labeled with antibodies in solution. For the first time, osmotically intact single disks can be visualized. Thus, imaging of purified disks, based on advanced optical techniques, may serve as a powerful complement to other methods in studies on phototransduction. In fact, even though much is known about the rod outer segment photoresponse, some unanswered questions remain, particularly about ATP supply, light adaptation, and morphogenesis.
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.
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