Technologies available

Single-molecule imaging and tracking (SMIT) in live cells to quantify protein dynamics and target-search mechanisms in live cells

Single-molecule imaging and tracking (SMIT) is a powerful tool for examining the biological structure and function of individual molecules. It offers a level of detail not achievable in ensemble studies, as it uncovers molecular behaviors that are otherwise hidden by the averaging of data. It further proposes—
•    Diffusion dynamics of proteins in live cells
•    Target-search mechanism of proteins
•    Residence time of protein binding
•    Fraction of bound and unbound molecules

Single-molecule Localization Microscopy (SMLM), STORM, PALM, for high-resolution structure imaging (x,y resolution: 20 nm; z resolution: 50 nm)

Single-molecule localization microscopy (SMLM) encompasses a family of advanced imaging techniques that significantly enhance spatial resolution compared to standard diffraction-limited microscopy, allowing for visualization of biological structures at the molecular level.

Single-molecule localization microscopy (SMLM) involves selectively activating the fluorescence of a small subset of molecules at any given moment, allowing each molecule to be individually localized.

Stochastic Optical Reconstruction Microscopy (STORM) creates a super-resolution image by accurately localizing individual fluorophores in three dimensions and across multiple colors It also can generate images of molecular structures resolved to under 20 nm.

In PALM microscopy, photoactivatable fluorophores are activated by lasers to resolve fine spatial details of tightly packed molecules. These fluorophores emit briefly and bleach, with the laser selectively activating random fluorophores in small groups to map their positions accurately.

All these advanced microscopic techniques offer—

• High-resolution localization and structure analysis

• Molecular clustering

• Stoichiometry

6D imaging (x, y, z, time, multichannel, multiposition) in live and fixed cells.

6D super-resolution imaging (by combining 6 dimensions) provides a comprehensive view of complex biological systems, enabling researchers to study spatial structures, dynamic interactions, multi-channel (color) information, and molecular orientations. It proposes —

• x, y, z, time, channels imaging altogether

• Live cell imaging

• Studying dynamics of cellular processes

Specification of Microscope Available at Facility

Inverted completely motorized (latest model) microscope with Infinity optical corrected optical system, equipped with

• Magnification changer1.0x to 1.5x

• Objectives: High-performance, Objectives suitable for Bright field/Fluorescence/DIC, Observation
  1) Plan Apochromat 20x with N.A 0.80
  2) Plan Apochromat 40x with N.A 0.95
  3) Plan Apochromat oil immersion 60x or 63x with N.A 1.42 or higher
  4) Plan Apochromat/Apo 100x oil TIRF with N.A. 1.47 or higher 200         ml immersion oil for objective lenses

• Tokai Hit Onstage Incubation System With Live Cell Imaging and Perfect Focus System (PFS): STXG-WSKMX-E Stage Top Incubator for XY manual/motorized stage Controller: With built-in digital gas mixer for 100%CO2 gas. STX-CSG Dish Attachment and Fixing Lid for slide glass, chamber slide, and chambered coverglass use cylinder use Multi-well plate and 35/60mm dish use.

• Epi-fluorescence attachment

• Epi-Fluorescence Filter cubes: highest quality bandpass fluorescent filter cubes for DAPI, CFP, YFP, GFP, TRITC, mCherry, CY5, and FRET (CFP-YFP).

• Camera 1: ORCA-Fusion BT For STORM-ORCA-Fusion BT degital SCMOS camera set CoaXPress (Made in Japan) Ultra Low readout Noise, Faster frame rate with back-thin boosted high QE.Exceptional photon- detection. and collection. 95% QE, 89.1 Frames/S @2304 X 2304 Pixels

• Camera 2: Prime 95B, High Resolution Prime 95B Scientific CMOS, 95% Quantum Efficiency 6.5μm x 6.5μm Pixel Area 1.0e-Read Noise with Correlated Multi Sampling (CMS) 43 fps @ 16-bit/ 12-bit (CMS) 63 fps @ 11-bit PrimeEnhance increases SNR 3-5x

• Motorized TIRF & Localization-based Super Resolution System​

• System Control and Application Software: NIS-ELEMENTS AR / CN, 3D Volume View 1, NIS-D Z DRIVE 1, NIS-A 3D Measurement 1, NIS-A 6D 1, NIS-A N-STORM Analysis 1, NIS-A RT Acquisition 1, NIS-A, Illumination Sequence 1, NIS-D Splitter DV 1, NIS-A CA FRET

Single-molecule FRET (smFRET) for studying protein conformational dynamics

Single-molecule FRET (smFRET) has become a valuable technique for measuring changes in the distance between two molecules within the 10-100 Å range, which is highly relevant to molecular biological processes. It offers —

• Protein conformation dynamics

• Biomolecular interaction dynamics

Single-molecule Fluorescence In Situ Hybridization (smFISH) to quantify transcription heterogeneity in the cell population

Single-molecule fluorescence in situ hybridization (smFISH) — also known as smRNA FISH or RNA FISH — is a cutting-edge technique for studying gene expression in single cells. It enables visualization and quantification of specifically labeled RNA molecules at single-molecule resolution; and offers—

• Quantification of transcription dynamics and regulation

State-of-the-art image processing such as 2D/3D image analysis, 3D rendering, virtual reality

Image processing in microscopy is crucial because it enhances the ability to quantitively analyze, interpret, and extract meaningful information from microscopic images of biological samples. Advanced image processing software such as 2D/3D deconvolution offers several image processing techniques such as —

• 3D deconvolution

• 3D rendering

• Virtual reality

• Quantitative microscopy (intensity and volume measurements)

• Image visualization and presentation