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 higher

Validated

Leica

DMi8

LASX 3.6

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, Leica and Nikon microscope 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

Required for Nikon, Zeiss, and Leica

Not Required for Keyence

Camera Recommendations

For Zeiss and Leica microscopes, we recommend the following cameras:

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