Microscope Requirements - Overview
This section presents an overview of the microscope requirements. Specific microscope requirements are listed within each microscope section.
Introduction
Akoya’s CODEX® system integrates with inverted epi-fluorescent microscopes across a number of makes and models with the following:
Minimal hardware requirements
Vendor-specific microscope software versions
The CODEX® Instrument Management (CIM) software is co-installed on the microscope control PC and integrates with the microscope control software’s user interface for the synchronization of the CODEX® Assay cycles and image capture. The CIM was designed to be rapidly adaptable to optional hardware components and software modules beyond the minimal microscope requirements. By establishing common vendor-specific configurations, Akoya is able to rapidly adopt the scripting for the wide range custom install configurations. Additional configurations and models will be updated as validated.
Integrated Microscope Makes and Models
Dye Set and Filter Cube Configuration
CODEX uses one of two dye groupings; one recommended for standard operation (Grouping 1) and one for tissues with autofluorescence (Grouping 2). Antibodies come with two options for reporters with Alexa 488 or Alexa 750 for panel design.
Filter Position
Excitation
Emission
Reporter
Dye Set Group 1
Dye Set Group 2
DAPI Filter
358 nm
461 nm
Nucler Stain
✓
✓
FITC Filter
492 nm
517 nm
Alexa Fluor® 488
✓
X
TRITC Filter
560 nm
575 nm
ATTO 550
✓
✓
Cy5 Filter
648 nm
668 nm
Cy5
✓
✓
Cy7 Filter
753 nm
775 nm
Alexa Fluor® 750
X
✓
Microscope Minimum Hardware Requirements
The minimal hardware requirements for compatibility with the CODEX fluidics and Assay : Vendor-specific makes, models and part numbers are listed below. Zeiss and Leica microscopes have a wide variety of custom configurations, so minimal requirements are listed.
Component
Description
Objectives
Medium: Air
Objective: 20x
Working distance: > 1mm (Will affect imageable area)
NA: >0.75
Light Source
LED or MH White light (380-750 nm) with 3-4W output power
The use of an LED or Halogen lightsource will determine the use of either Filter cubes or an LED filter.
Camera
Detector Type: EMCCD or sCMOS
Bit Depth: Supports 16 bit output
Resolution: >1 megapixel
Color Channels: Monochrome, not color
Pixel Size: ~6.5 um pixel (5.5-7.5)
Special Requirements: Detect AF750 if applicable
Stage
Automated, motorized stage with X, Y, Z capabilities
Field Diaphragm
Recommended square diaphragm to vary light
Spill Guard
Helps protect microscope from potential liquid spills. Spill guard connects to spill sensor provided by Akoya to stop instrument run in case of liquid detection
Dark Room
Not Required for Keyence
Objective Considerations
Note : Check the individual camera specification sheets to determine the optimal quantum efficiency, um per pixel ratio, and specifications for your experimental use.
Magnification - 20x
Akoya recommends a 20x Objective. For additional information about higher magnification objectives, please contact your local sales representative.
Higher magnification objectives such as 40x or 63x, can be used but the workflow is to be optimized by the user and will result in increased CODEX run times for the same tissue area, and subsequently increased data processing time and storage requirements.
Lower magnifications such as 2x, 5x, or 10x can be used to initially scan the tissue area on the coverslip to set the imaging area. Segmentation of data captured at these magnifications is not supported.
Numerical Aperture
The numerical aperture of the objective needs to be ≥0.75. Numerical apertures of less than 0.75 will have a lower image resolution that adversely affects data processing.
Objective Type
Only air objectives have been validated by Akoya Biosciences.
Immersion objectives (oil or water) may be used at users own risk. Imaging area size will be adversely affected.
The immersion objective will need to maintain a seal with the coverslip during the duration of the entire CODEX® experiment. To ensure the immersion liquid seal does not break, image size and run time will need to be optimized by the user.
Imageable Area on Coverslip
The imageable area on the coverslip will be different based on the geometrical shape of the objective. Note : Tissues should always be placed in the center of the coverslip.
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