Basic Microscopy Concepts

Introduction to Microscopy Image Stitching

Microscopy image stitching is the process of combining multiple overlapping images (tiles) into a single larger image. This is necessary because:

  1. The field of view of a microscope is limited

  2. High-resolution imaging often requires capturing multiple tiles

  3. Stitching allows visualization and analysis of larger areas

EZStitcher addresses these challenges by:

  • Automatically detecting tile positions

  • Aligning tiles with subpixel precision

  • Blending overlapping regions smoothly

  • Handling multi-channel fluorescence and Z-stacks

Key Microscopy Concepts

Plate-Based Experiments

In high-content screening, samples are typically organized in plates:

Plate Diagram

Key concepts:

  • Plate: A container with multiple wells (e.g., 96-well plate)

  • Well: A single compartment in a plate, identified by a row letter and column number (e.g., A01, B02)

  • Site: A specific location within a well where an image is captured

  • Grid: The arrangement of sites within a well (e.g., 3×3 grid)

EZStitcher processes images on a per-well basis, stitching together all sites within each well.

Multi-Channel Fluorescence

Fluorescence microscopy captures images at different wavelengths to visualize different structures:

Multi-Channel Diagram

Key concepts:

  • Channel: A specific wavelength or color used for imaging

  • Composite: A combined image created from multiple channels

EZStitcher can:

  • Process each channel independently

  • Create composite images from multiple channels

  • Use one channel as a reference for stitching all channels

Z-Stacks

Z-stacks are 3D image stacks captured at different focal planes:

Z-Stack Diagram

Key concepts:

  • Z-Stack: A series of images captured at different focal planes

  • Z-Plane: A single image at a specific focal depth

  • Projection: A 2D representation of a 3D stack (e.g., maximum intensity projection)

  • Best Focus: The plane with the highest focus quality

EZStitcher provides several options for handling Z-stacks:

  • Maximum intensity projection

  • Mean projection

  • Best focus selection

  • Per-plane stitching

For detailed information about Z-stack processing, see the Intermediate Usage guide.

Tiled Images

Tiled images are multiple overlapping images that cover a larger area:

Tiling Diagram

Key concepts:

  • Tile: A single image captured at a specific position

  • Overlap: The region where adjacent tiles overlap

  • Grid Size: The number of tiles in X and Y directions

  • Position: The coordinates of a tile in the final stitched image

EZStitcher handles tiled images by:

  1. Determining the relative positions of tiles

  2. Aligning tiles with subpixel precision

  3. Blending overlapping regions

  4. Assembling the final stitched image