SDDRC - Introduction to microscopy скачать в хорошем качестве

SDDRC - Introduction to microscopy

Join the San Diego Digestive Research Center for this comprehensive overview of microscopy and histology methods in digestive research. This first lecture covers a range of cutting-edge microscopy techniques, including whole-animal imaging, intravital microscopy, 3D imaging, slide scanning, and high-resolution confocal microscopy. Discover how these powerful tools can strengthen your research questions and enhance the impact of your publications. Learning Objectives Master Microscopy Fundamentals: Understand the physics of fluorescence, the Stokes shift, and why numerical aperture is more critical than magnification. Navigate Core Technology: Identify the right tool for your project, from whole slide scans to super-resolution. Optimize Image Quality: Learn to manage different types of noise (thermal, read, and photon) and use histograms to ensure quantitative accuracy. Advanced Imaging Techniques: Explore specialized methods like Intravital Microscopy for live-cell tracking and RareCyte Orion for high-plex spectral imaging. Safety and Best Practices: Review BSL-2 protocols for transporting human cell lines and infectious samples to the core facility. 00:54 – Bright Field vs. Fluorescence Imaging 01:54 – Bit Depth and Digital Image Formats 02:22 – Fluorescence Demos: Quinine and Curcumin 04:22 – The Science of Fluorophores and the Stokes Shift 05:36 – Introduction to Non-Invasive Luminescence Imaging 06:18 – Core Equipment: Zeiss Axio Scan and Keyence BZX 06:57 – Live Cell and Intravital Imaging on the Leica SP8 07:23 – Advanced Spectral Scanning and QuPath Analysis 09:10 – The Importance of Absolute Calibration in Luminescence 09:44 – Understanding Immune Responses to Fluorescent Proteins 11:44 – The Shadowing Effect and Light Scattering in Tissue 13:05 – The Biological Optical Window: Why Red Light Penetrates Better 14:49 – Technical Deep Dive: Camera Detectors and Bit Depth 15:55 – Managing Noise: Thermal, Read, and Photon (Shot) Noise 18:52 – How to Use Histograms and Lookup Tables (LUTs) 22:39 – Objective Lens Secrets: Magnification vs. Numerical Aperture (NA) 24:30 – Point Spread Function (PSF) and Resolution Limits 25:53 – Super-Resolution Methods (Airyscan, SIM, STORM, STED) 28:00 – Wide Field vs. Confocal: Making the Right Trade-off 29:30 – Meet the Zeiss LSM 780, 880, and 980 Confocals 31:17 – Molecular Dynamics: Fluorescence Recovery After Photobleaching (FRAP) 31:29 – BSL-2 Biosafety Protocols for Imaging Core Users 34:12 – Intravital Case Study: Tracking B-cell Movement 37:34 – Visualizing Immune Cell Migration in the Gut 40:47 – High-Throughput Islet Analysis with Slide Scanning 42:17 – High-Plex Imaging with the RareCyte Orion 44:51 – The Cyclic Process of Experimental Design 47:06 – Best Practices for Publishing Research Images Microscopy Core Expert Tips Always Report Numerical Aperture (NA): When writing your materials and methods, magnification alone is insufficient; the NA drives resolution and photon collection far more than magnification. Avoid Image Saturation: Once you hit the "ceiling" of your detector, you can no longer quantify how much brighter a signal is. Always set your levels using your brightest positive control. Mind the "Biological Optical Window": When imaging through whole tissue, green and blue wavelengths are heavily absorbed by blood; use near-infrared fluorophores for better penetration and lower autofluorescence. Standardize Contrast Adjustments: For publication, any brightness or contrast adjustments must be applied identically across all experimental and control panels to ensure a fair comparison. Secure BSL-2 Samples for Transport: When moving human cell lines to the core, seal dishes with Parafilm and use a tightly closed secondary container to prevent hazardous spills. Notes and links to publications and resources: https://docs.google.com/document/d/1K...