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

Vendor	Base Model	Software	Status
Keyence	BZ-700	BZX Multistack Module	Validated
Keyence	BZ-800	BZX Multistack Module	Validated
Zeiss	AxioObserver	Zen Blue 2.3 or 2.6	Validated
Leica	DMi8	LASX 3.6 or 3.7.1	Validated

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.

The imageable area of three recommended objectives.

Proportional dimensions between outer coverslip and imageable area