Hasil untuk "Microscopy"

Lihong V. Wang

Philipp J. Keller, Annette D. Schmidt, J. Wittbrodt et al.

A. Gruber, A. Dräbenstedt, C. Tietz et al.

Z. Wang

R. Nixon, J. Wegiel, Asok Kumar et al.

E. Meijering, Mathew Jacob, J. Sarria et al.

B. Cappella, G. Dietler

K. M. Zinn

C. Prater, H. Butt, P. Hansma

R. Henderson, P. Unwin

Junjie Yao, Lidai Wang, Joon-Mo Yang et al.

J. Fuhrmann

Tyler J. Chozinski, Aaron R. Halpern, Haruhisa Okawa et al.

R. Fernández-Leiro, S. Scheres

Fei Chen, A. Wassie, Allison Coté et al.

The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy of RNA. We developed a small-molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, postexpansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity as well as single-molecule precision in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) separates RNAs and supports amplification of single-molecule signals (i.e., via hybridization chain reaction) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine.

Lilian Magermans, Assia Benachir, Nathan P. Spiller et al.

Speckled illumination enhances widefield fluorescence microscopy by enabling optical sectioning and super resolution. In random illumination microscopy, sequences of speckled illumination patterns are used to excite fluorescent samples and images are reconstructed based on a statistical analysis of the intensity fluctuations. Although random illumination microscopy has been shown to give excellent performance, its widespread implementation is hindered by the high cost and complexity of the generation of suitable speckled illumination patterns, which is achieved using digital micro-mirror devices or spatial light modulators. Here, we present a zwitterion-doped liquid crystal (LC) device capable of generating independent, high-contrast speckle patterns with a tunable decorrelation time in the 0.1 s to 0.1 ms range under visible laser illumination. This LC-based dynamic speckle generator is applied to widefield random illumination fluorescence microscopy of tissue and cell samples, where it enables optical sectioning with a 2 micron axial resolution, and a 1.5-fold improvement in lateral spatial resolution. Owing to its low cost and simplicity, this LC speckle generator offers an attractive alternative to digital micro-mirror and spatial light modulator devices for implementing widefield random illumination microscopy.

Assia Benachir, Xiangyi Li, Eric M. Fantuzzi et al.

Biological and biomedical samples are routinely examined using focused two-photon (2P) fluorescence microscopy due to its intrinsic axial sectioning and reduced out-of-focus bleaching. However, 2P imaging often requires excitation intensities that can damage samples through ionization and radical formation. Additionally, the lateral resolution of 2P microscopy is lower compared to linear one-photon (1P) fluorescence microscopy. Widefield 2P microscopy, using cameras, holds promise for reducing photo-toxicity while maintaining high image acquisition rates. Widefield imaging trades the high power and short integration times of sequential single point scanning for the low power and extended integration times of parallel detection across millions of pixels. However, generating effective axial sectioning over arbitrarily large fields of view (FOVs) has remained a challenge. In this work, we introduce 2P Random Illumination Microscopy (2P-RIM), an easy-to-implement 2P widefield technique, that achieves low photo-damage, fast imaging, micrometric axial sectioning, and enhanced lateral resolution for arbitrarily large FOVs. By using widefield speckled illuminations in conjunction with an image standard deviation matching algorithm, 2P-RIM demonstrated multicolor imaging over FOVs greater than 200 um, lateral resolution 220 nm, axial sectioning 2 um, and peak excitation powers about 10 times lower than those used in focused laser scanning microscopy.

Shivasubramanian Gopinath, Joseph Rosen, Vijayakumar Anand

Fresnel incoherent correlation holography (FINCH) is a self-interference-based incoherent digital holography method. In FINCH, light from an object point is split into two beams, modulated differently using two lenses with different focal distances, and creates a self-interference hologram. At least three phase-shifted holograms are recorded and synthesized into a complex hologram, which reconstructs the object image without twin image and bias noises. Compared with conventional imaging, FINCH exhibits a longer depth of focus (DOF) and higher lateral resolution. In this study, we propose and demonstrate a new method termed post-engineering of axial resolution in FINCH (PEAR-FINCH), which enables post-recording DOF engineering for the first time. In PEAR-FINCH, a library of FINCH holograms catalogued with unique axial characteristics, DOF, and focus location is recorded by changing the focal distance of one of the diffractive lenses. Selected holograms from this library are combined to engineer new axial characteristics not achievable in FINCH. A two-step reconstruction, involving numerical back-propagation and deconvolution with a point spread hologram, is implemented. Experiments with multiplane objects having large axial separations confirm that PEAR-FINCH achieves a substantially extended DOF compared with direct imaging and FINCH. PEAR-FINCH will be promising for applications in biomedical imaging, holography, and fluorescence microscopy.

Kohki Horie, Keiichiro Toda, Takuma Nakamura et al.

Quantitative phase microscopy (QPM) and interferometric scattering (iSCAT) microscopy are powerful label-free imaging techniques and are widely used for biomedical applications. Each method, however, possesses distinct limitations: QPM, which measures forward scattering (FS), excels at imaging microscale structures but struggles with rapidly moving nanoscale objects, while iSCAT, based on backward scattering (BS), is highly sensitive to nanoscale dynamics but lacks the ability to image microscale structures comprehensively. Here, we introduce bidirectional quantitative scattering microscopy (BiQSM), an innovative approach that integrates FS and BS detection using off-axis digital holography with bidirectional illumination and spatial-frequency multiplexing. BiQSM achieves spatiotemporal consistency and a dynamic range 14 times wider than QPM, enabling simultaneous imaging of nanoscale and microscale cellular components. We demonstrate BiQSM's ability to reveal spatiotemporal behaviors of intracellular structures, with FS-BS correlation analysis providing insights into proteins, lipids, and membranes. Time-lapse imaging of dying cells further highlights BiQSM's potential as a label-free tool for monitoring cellular vital states through structural and motion-related changes. By bridging the strengths of QPM and iSCAT, BiQSM advances quantitative cellular imaging and opens new avenues for studying dynamic biological processes.

Alexander Holmberg, Nils Mechtel, Wei Ouyang

We present a domain adaptation of video diffusion models to generate highly realistic time-lapse microscopy videos of cell division in HeLa cells. Although state-of-the-art generative video models have advanced significantly for natural videos, they remain underexplored in microscopy domains. To address this gap, we fine-tune a pretrained video diffusion model on microscopy-specific sequences, exploring three conditioning strategies: (1) text prompts derived from numeric phenotypic measurements (e.g., proliferation rates, migration speeds, cell-death frequencies), (2) direct numeric embeddings of phenotype scores, and (3) image-conditioned generation, where an initial microscopy frame is extended into a complete video sequence. Evaluation using biologically meaningful morphological, proliferation, and migration metrics demonstrates that fine-tuning substantially improves realism and accurately captures critical cellular behaviors such as mitosis and migration. Notably, the fine-tuned model also generalizes beyond the training horizon, generating coherent cell dynamics even in extended sequences. However, precisely controlling specific phenotypic characteristics remains challenging, highlighting opportunities for future work to enhance conditioning methods. Our results demonstrate the potential for domain-specific fine-tuning of generative video models to produce biologically plausible synthetic microscopy data, supporting applications such as in-silico hypothesis testing and data augmentation.