MICROSCOPE ASSESSMENTGUIDELINE FOR
StereoMap SOFTWARE

SoftwareVersion: StereoMap 4.0
Manual Version: C

STUM-PE001

STOmics Microscope Assessment Guideline

REVISION HISTORY
Manual Version:
Software Version:
Date:

A1
V1.0 & V2.0
May. 2023

Manual Version:
Software Version:
Date:

B1
V1.0
Oct. 2023

Manual Version:
Software Version:
Date:

C
V4.0
Jun. 2024

Description：• Initial release

Description：• Added microscope evaluation
procedures for Stereoseq Transcriptomics Multiimmunofluorescence (mIF) solution
and Stereo-seq Transcriptomics
H&E solution.
• Added instructions for microscope
hardware evaluation.
• Eliminated the evaluation
requirements for blank Stereo-seq
Microscope Assessment Chip T.
• - Modified compatible image
formats (only compatible with TIFF/
TIF formats).

Description：• Added microscope evaluation
process that is compatible with
Stereo-seq Transcriptomics FFPE
product solution workflow.
• Added validated microscopemodels and their corresponding
input file types.

STOmics Microscope Assessment Guideline

Note: Please download the latest version of the manual and use it with thesoftware specific to this manual.
©2024 STOmics Tech Co., Ltd. at Shenzhen.
All rights reserved.
1. The products shall be for research use only, not for use in diagnostic procedures.
2. The Content on this manual may be protected in whole or in part by applicable
intellectual property laws. STOmics Tech Co., Ltd. and/or corresponding right subjects
own their intellectual property rights according to law, including but not limited to
trademark rights, copyrights, etc.
3. STOmics Tech Co., Ltd. do not grant or imply the right or license to use any copyrighted
content or trademark (registered or unregistered) of us or any third party. Without ourwritten consent, no one shall use, modify, copy, publicly disseminate, change, distribute,or publish the program or Content of this manual without authorization, and shall not
use the design or use the design skills to use or take possession of the trademarks, thelogo or other proprietary information (including images, text, web design or form) of us
or our affiliates.
4. Nothing contained herein is intended to or shall be construed as any warranty,
expression or implication of the performance of any products listed or described herein.
Any and all warranties applicable to any products listed herein are set forth in theapplicable terms and conditions of sale accompanying the purchase of such product.
STOmics Tech Co., Ltd., Shenzhen makes no warranty and hereby disclaims any and all
warranties as to the use of any third-party products or protocols described herein.

STOmics Microscope Assessment Guideline

TABLE OF CONTENTS
CHAPTER 1: INTRODUCTION
1.1. Microscope Assessment Channels				11.2. Assessment Software and Criteria				1
1.3. Assessment Duration					1CHAPTER 2: MICROSCOPE HARDWARE EVALUATION
2.1. Imaging System Requirements				2.2. Imaging Configurations					

56CHAPTER 3. MICROSCOPE IMAGING GUIDELINES
3.1. Pre-assessment Preparation				3.2. Microscope Assessment Procedure			

88

CHAPTER 4. MICROSCOPE IMAGE EVALUATION
4.1. Imaging Quality requirements				4.2. StereoMap Image Assessment		
4.3. Image Examples				

12
13
17

CRITICAL STEPS: Pay extra attention for these steps to avoid experimental setbacks or problematic results.
NOTE: Additional operation tips and guidance.
CAUTION: Proceed with extra care; improper handling or carelessness may cause experimental failure or accidents.

CHAPTER 1
INTRODUCTIONSTOmics Microscope Assessment Guideline

INTRODUCTION

STOmics Stereo-seq Solution quantifies RNA of tissue sections followed by mapping
and visualizing the transcriptomic data on an anatomical image. Typically, a highmagnification microscope is employed to capture high-resolution cellular images oftissues. Hence, the tissue image quality determines the success of downstream analysis
and spatial clustering of gene expression data. This guidebook provides hardware
recommendations, image acquisition and evaluation guidelines, as well as microscopecompatibility test procedures. Additionally, an STOmics-developed image evaluationand processing software – StereoMap is also introduced in this guidebook.

1.1. Microscope Assessment Channelsa.

Stereo-seq Transcriptomics application(ssDNA-stained nuclei image) -> FITC
Channelb. Stereo-seq Transcriptomics with mIF co-detection application (DAPI-stained nuclei
image & mIF images) -> DAPI Channelc. Stereo-seq Transcriptomics H&E application (H&E-stained image) -> Brightfield-Epi
illumination

1.2. Assessment Software and Criteria
All microscope images are evaluated for image quality via the "Tools > ImageQC"
module in StereoMap software, including the scoring evaluation of Trackline clarity
and image clarity. All evaluations are based on the standard of Trackline QC score.
For detailed scoring rules, please refer to Chapter 4.2, Section ImageQC Scoring
Instructions. The image clarity score is for reference only and is not used as a pass/failstandard for image QC.

1.3. Assessment Duration
About half a day.

1

CHAPTER 2
MICROSCOPE HARDWAREEVALUATIONSTOmics Microscope Assessment Guideline

MICROSCOPE HARDWARE EVALUATION

The microscope hardware evaluation mainly evaluates whether the microscope hardware
parameters meet the Stereo-seq experiment requirements. The microscope hardware evaluation
contents are as follows:

It is recommended to make an appointment in advance with your manufacturer's
microscope engineer to accompany the evaluation on site.
Table 1 Overview of fluorescent imaging system requirements

Index/ Parameter

Description

XY stage travel distance of microscope

At least 25*75 mm

Imaging/Evaluation subject

Stereo-seq Microscope Assessment Chip
Slide (Cat. No.: 201CT113)

Focusing approachPre-focus map and/or real-time autofocus

Objective lens

10X (NA ≥ 0.3)

• DAPI (Excitation 358nm, Emission

Fluorescent channel

Camera resolution

461nm)• FITC filter cube (Excitation 480/40nm,
Emission 525/50nm)• TRITC filter cube (Excitation 545/25 nm,
Emission 605/70 nm)• CY5 filter cube (Excitation 620/50 nm,
Emission 690/50 nm)
Camera resolution selection depends on
the objective lens. Please consult yourmicroscope supplier for specific details• Grayscale image: 8bit/16bit (fluorescent
Image bit depth

image)• Color image: 3*8bit/3*16bit (H&E orother brightfield image)

Background balance

Adjustable background balancing function

Distortion correction

Adjustable distortion correction function

Overlap ratio

Adjustable, set it to 10%

Image format

Capable of viewing or exporting stitched
images or Field-of-View (FOV) original images
in TIFF format, 8/16 bit

PC requirement

Windows 10 x 64 system, 16G memory or
beyond3

STOmics Microscope Assessment Guideline

MICROSCOPE HARDWARE EVALUATION

Table 2 Overview of reflected light (epi-illumination) imaging system requirements

Index/ Parameter

Description

XY stage travel distance of microscope

At least 25*75mm

Imaging/Evaluation subject

Stereo-seq Microscope Assessment Chip
Slide (Cat. No.: 201CT113)

Focusing approachPre-focus map and/or real-time autofocus

Fluorescent channel

DAPI, FITC, TRITC, CY5

Objective lens

10X (NA ≥ 0.3)

Camera resolution

Camera resolution selection depends on
the objective lens. Please consult yourmicroscope supplier for specific details

Image bit depth

Grayscale image: 8bit/16bit (fluorescentimage)

Background balance

Adjustable background balancing function

Distortion correction

Adjustable distortion correction function

White balance

Adjustable distortion correction function

Overlap ratio

Adjustable, no less than 5%File format

Capable of viewing or exporting stitched
images or Field-of-View (FOV) original images
in TIFF format, 8/16 bit

Pixel size

Maximum pixel size ≤ 5 µm/pixel

PC requirement

Windows 10 x 64 system, 16G memory or
beyondWhen outputting TIFF images from the microscope's software, please ensurethat the image size and bit depth are consistent with the original format (such as
CZI), as manual conversion of bit depth may lead to differences in QC results.
If the microscope hardware does not meet the above conditions, please switch toa different microscope and re-evaluate. The validated microscopes for Stereo-seq
experiments are listed in Table 2 (Chapter 2.1).

4

MICROSCOPE HARDWARE EVALUATION

STOmics Microscope Assessment Guideline

2.1. Imaging System Requirements
In general, an imaging system being used should satisfy these requirements:
1. Be able to perform tile scanning to image a minimal area of 10mm x 10mm (standard
Stereo-seq Chip size).
This is required for the standard Stereo-seq Chip (1cm * 1cm). If large-sized
Stereo-seq Chips are used for later transcriptomics experiments, please confirm
the microscope scanning range in advance.
2. Have image stitching function either via the microscope’s software (automatic
stitching) or STOmics image processing software, StereoMap(manually assisted
stitching, for FOV images only).
3. Have an image resolution ≥ 1800 pixels (height) and ≥ 2000 pixels (width).
4. Be able to view and save stitched images in raw file format (e.g., czi in Zeiss) andexport it into TIFF.
5. The computer connected to the microscope is capable of processing large images (>5GB) and supports the installation of third-party image processing software such as
StereoMap.
6. The microscope should also have ALL the properties and functions listed in Table 3
or Table 4.
Table 3 shows microscope models used in the development of STOmics protocols.
Table 3 Validated imaging systems

Brand

Model/ Specification

Output image formats

Leica

Leica DM6-M

Original files in TIFF format

Zeiss
MGI

Zeiss Axio Scan Z1
Zeiss Axio Scan 7
STOmics Microscope
Go Optical

Original Files in CZI format
Original files in TIFF format

The input files required for image registration differ based on the microscopeused. At present, interface adapters for raw image file formats have been
specifically designed for the validated microscope models mentioned in Table 3.
For other microscopes, only stitched large images in TIFF format are supported
as input files.
If none of the above microscopes is available,
a. Ensure that the microscope used fulfills hardware requirements stated in Table 1 orTable 2 of Chapter 2.
b. Perform a microscope compatibility assessment following instructions provided in
Chapter 2.2.

5

STOmics Microscope Assessment Guideline

MICROSCOPE HARDWARE EVALUATION

2.2. Imaging Configurations
Recommended Imaging Configuration for ssDNA - stained nuclei images:
Recommended Fluorescence Configuration:• Light source with a wavelength range of 380 - 680 nm• Monochrome camera (≥ 8 bit)
• FITC filter cube (Excitation 480/30, Emission 525/50)• TRITC filter cube (Excitation 545/25, Emission 605/70)• Maximum pixel size of 5 µm
• Exposure time 1 milli sec - 2 sec
Recommended Imaging Configuration for DAPI - stained nuclei images (mIFapplications):
Recommended Fluorescence Configuration:• Light source with a wavelength range of 380 - 680 nm• Monochrome camera (≥ 8 bit)
• DAPI filter cube (Excitation 375/28nm, Emission 460/50nm)• FITC filter cube (Excitation 480/30nm, Emission 525/50nm)• TRITC filter cube (Excitation 545/25nm, Emission 605/70nm)• CY5 filter cube (Excitation 620/50nm, Emission 690/50nm)• Maximum pixel size of 5 µm
• Exposure time 1 milli sec - 2 sec
Recommended Imaging Configuration for H&E-stained images:• Reflected light (Epi-illumination)• Color camera (3*8 bit)
• White balance• Maximum pixel size of 5 µm
• Exposure time 0.1 milli sec - 100 milli sec

6CHAPTER 3
MICROSCOPE IMAGINGGUIDELINES

STOmics Microscope Assessment Guideline

MICROSCOPE IMAGING GUIDELINE

3.1. Pre-assessment Preparation
Materials• Stereo-seq Microscope Assessment Chip Slide (STOmics, Cat. No. 201CT113)• One OCT/FFPE tissue block• Microscope photograph image - Table 2 for input file formats.
• Experimental Procedure Manual - Table 4 for more details• Software Operation Manual: StereoMap User Manual
Materials required for the experimental procedures differ based on the specificexperimental workflows. For more details, please refer to the STOmics Document List.
Table 4 Overview of user manuals corresponding to different evaluation channels

Evaluation Channels

OCT embedded fresh frozen Formalin-fixed and paraffin(FF) tissue blockembedded (FFPE) tissue block

FITC Channel

Stereo-seq Transcriptomics Stereo-seq Transcriptomics Set
Set for Chip-on-a-slide User
for FFPE User Manual
ManualDAPI Channel

1. Stereo-seq
Transcriptomics Set for mIF
User Manual
2. Stereo-CITE ProteoTranscriptomics Set User
ManualStereo-seq Transcriptomics Set
for FFPE User Manual

Epi-illumination

Stereo-seq Transcriptomics
Set H&E User Manual

Stereo-seq Transcriptomics Set
for FFPE User Manual

3.2. Microscope Assessment Procedure
This section applies to the microscope evaluation process of various product solutions
based on STOmics Stereo-seq technology. Please select the appropriate evaluation
procedures according to the project needs.
Please contact and make an appointment with a microscope engineer of the
microscope brand you are using before the evaluation. Some microscope image
parameters (such as FOV height, FOV width, and image scale) will be usedduring the evaluation process, and obtaining such information may require theassistance of the microscope engineer. After imaging the Stereo-seq Microscope
Assessment Chip Slide and passing the QC, please confirm the image parametersand do not change them before the official Stereo-seq experiment.

8

STOmics Microscope Assessment Guideline

MICROSCOPE IMAGING GUIDELINE

3.2.1. Introduction to Experimental Procedure
The tissue preparation process for microscope evaluation is aligned with the various
Stereo-seq experimental workflows, concluding with fluorescence or bright-fieldimaging. The specific workflow chosen depends on the embedding conditions of thetissue and channel requirements. Select the corresponding Stereo-seq User Manuals
by referring to Table 4 in Chapter 3, Section 1 to access the latest version on the official
STOmics Website.

Figure 1. Workflow for Microscopic Evaluation for Stereo-seq Experiments

3.2.2. Precautions for Experimental Operations
Stereo-seq Slide Cassette Assemble

Please refer to the Stereo-seq Chip Slide Assemble User Manual. For a demonstration video of
Stereo-seq Slide Cassette assembling and removal, please refer to the link or by scanning the QR
code: https://en.stomics.tech/resources/videos/list.html

Tissue Sectioning Instructions
Select the tissue sectioning method based on the tissue block type. Stereo-seq provides
solutions for both frozen tissue blocks and paraffin blocks. Frozen sections are 10 μm
thick, while paraffin sections are 5 μm thick for regular tissues and 4 μm thick for highfat content tissues (e.g., breast tissue).

9

STOmics Microscope Assessment Guideline

MICROSCOPE IMAGING GUIDELINE

Tissue Mounted Chip Imaging Guidelines
1. Make sure to keep the chip aligned with the edge of the imaging stage and do not tilt
the chip.
2. Adjust the imaging parameters to ensure that the exposure of the tissue area is wellbalanced and the image, as well as the tracklines, is clear. If there is any problem,
please contact the microscope engineer to troubleshoot within time.
3. It is recommended to use 'manual focus' for imaging. Select the modeling points firstat the four corners of the chip and then select a few points within and outside of thetissue by focusing on them respectively, to obtain clear tracklines and tissue staining
images at the same time. Please use 'autofocus' mode with caution. The autofocus
might be tricky to adjust and fail to focus on both tracklines and the tissue, whichmight result in Image QC failure.
4. During manual focus imaging, first select the appropriate focal plane, exposure,and lighting parameters to identify the tissue contour clearly. Then, adjust theseparameters in the blank areas to identify the tracklines clearly. Avoid setting lighting
parameters too low or too high, as this can impact the image quality.
5. If there are regions (more than 4 FOVs) that have not been adequately infiltrated withmounting medium or glycerol, additional modeling points should be added to theseregions.
6. Do not touch the surrounding surface during microscope scanning. Avoid introducing
any vibration sources near the microscope, such as moving and walking.
7. Ensure the duration is within 30 minutes between tissue mounting to imaging.
Do not leave your chip in the same position for a long time to avoid fluorescencesignal quenching. Turn off the laser when the chip is not being imaged to prevent
prolonged exposure, which may lead to local fluorescence quenching.
8. Compressed or scaled images will affect the QC and stitching results. Please ensurethat the TIFF images exported by the microscope are not compressed or scaled to
avoid image distortion affecting the downstream analysis.
9. After imaging, please use StereoMap software to QC the image immediately. If QC
does not pass, please try to image it again.

10CHAPTER 4
MICROSCOPE IMAGE
EVALUATIONSTOmics Microscope Assessment Guideline

MICROSCOPE IMAGE EVALUATIONIn this step, the obtained tissue images are input into the StereoMap software, wherethe "Tools" module is utilized for evaluation. The final assessment criterion relies onachieving a passing score for image trackline clarity evaluation. If Cellbin analysis isrequired, the image clarity score also needs to be taken into consideration.

4.1. Imaging Quality requirements
In general, a qualified image should fulfil the following requirements:
a. High image resolution: ≥ 1800 pixels (height) and ≥2000 pixels (width)
When exported an image file into TIFF format, ensure the file is not compressed to
avoid resolution loss.
b. The imaged tissue contour and nuclei should be clear and within focus
c. Tracklines on the chip should also be clear and within focus
1. To have both the imaged tissue and tracklines within focus, select multiple focal
points when performing imaging.
2. Set 3 focal points within the tissue and 3 other focal points in the background.
3. Adjustment of contrast and brightness might be required after imaging, to havethe tracklines clearly visible.
d. No stitching errors
e. Avoid photobleachingf.

Tissue area should not exceed 80% of the chip area

Figure shown here as an example of a high-quality mouse brain fluorescent image thathas passed QC:

Figure 2

All the factors c. to f. will affect the image trackline QC score in Image Quality Control
evaluation, which has a scale from 0 to 100. A minimal score of 60 is required to pass
the evaluation, the higher the score, the more accurate the image registration willbe, which has a direct impact on data clustering.
12

STOmics Microscope Assessment Guideline

MICROSCOPE IMAGE EVALUATION

Please ensure that both the tissue and tracklines are in focus during imaging. If
the microscope does not have automatic focusing capability or is not suitable,
it is recommended to use manual focusing mode. It is typically necessary to set
three focal points inside and outside the tissue to ensure comprehensive coverage.
After imaging, adjust the contrast and brightness appropriately to clearly view thetracklines. During imaging, adjust the exposure parameters based on the clarityof the tissue/cells. Refer to the imaging guidelines for specific operations, andavoid overexposing tissues just to see track lines clearly. After imaging, rely on
passing QC results or manually adjust brightness and contrast to view track lines
as needed.
Imaging factors that will influence cell segmentation and cellbin results:
Capture images which cell boundaries can be clearly identified with naked eyes.
Image clarity score should be above 80.
Image (nuclei-stained image) MUST pass QC.
Make sure that the stitching error is ≤ 5 pixel (Contact and make an appointment
with the manufacturer's microscope engineer for assistance if your image showsa stitching error > 5 pixel)
5. Tissue type and cell density will affect final cell segmentation in different aspects.
1.
2.
3.
4.

Cell segmentation will be greatly compromised if captured images fail to meet no.
1- 4, thus do not recommend to proceed with cellbin analysis.

4.2. StereoMap Image Assessment
StereoMap is an offline image processing software for Stereo-seq applications, primarily
involving image quality assessment, manual adjustment modules (image calibration,
tissue segmentation, and cell segmentation), and interactive visualization. Image
quality evaluation mainly evaluates the clarity of the tracklines and overall image clarityof the microscopic image, ensuring suitability of the image for further downstream
bioinformatics analysis.

4.2.1. Software Evaluation Workflow
1. Download and install the latest version of StereoMap software from the STOmics
website and refer to the StereoMap user manual for installation instructions.
2. Execute Image quality control (ImageQC) module: StereoMap->Tools-> ImageQC.
Different image files and formats are required to input into the software dependingon the microscope models. Refer to the StereoMap User Manual for details.

13

STOmics Microscope Assessment Guideline

MICROSCOPE IMAGE EVALUATION

4.2.2. ImageQC Scoring Instructions
Trackline QC Score (applicable for ssDNA, DAPI, H&E, DAPI&mIF)
The ratio of derivable template points or area within 5 pixels to the template points orarea possessed by the corresponding image of the whole chip is calculated respectively.
The maximum of the two ratios is taken, and the maximum value is linearly mapped
to a percentile score. If the ImageQC result of the trackline is 'Pass', meaning that thealgorithm can successfully derive the global template.

• TemplateValidArea: The proportion of the surrounding area formed by the points ofthe derivation template with error less than 5 pixels when compared to the overall
image area. As shown in this figure, the ratio of the pixel area surrounded by theyellow area to the pixel area of the entire chip = 460718882 pixels/604856088 pixels =0.76• TemplateRecall: The proportion of derived template points with an error of less than
5 pixels to the theoretical template points possessed by the corresponding image ofthe chip. As shown in this figure, the ratio of track points in the yellow doted matrix
to the template points of the entire chip = 2636/4760 = 0.5538

Description

Trackline Score

ImageQC Results Staining TypeTemplateValidArea,
TemplateRecall) < 0.1

< 60

Fail

ssDNA/DAPI/DAPI&mIF(TemplateValidArea,
TemplateRecall) ≥ 0.1

≥ 60

Pass

ssDNA/DAPI/DAPI&mIF(TemplateValidArea,
TemplateRecall) = 1

= 100

Pass

ssDNA/DAPI/DAPI&mIF(TemplateValidArea,
TemplateRecall) < 0.01

< 60

Fail

H&E

(TemplateValidArea,
TemplateRecall) ≥ 0.01

≥ 60

Pass

H&E

(TemplateValidArea,
TemplateRecall) = 1

= 100

Pass

H&E

Demonstration of Image trackline QC calculation process

14

STOmics Microscope Assessment Guideline

MICROSCOPE IMAGE EVALUATION

Image clarity score (applicable for ssDNA & DAPI)Image clarity score is calculated using a trained deep learning model which slices the
input image, and divides the segmented image into five types: 1. good, 2. moderately
blurry, 3. severely blurry, 4. slightly overexposed and 5. heavily overexposed,
assigns different weighing points to each category, and finally calculates the clarity scoreof the entire image. The calculation formula is as follows:n represents the categories, 1 to 5 represents the 5 types of 'clarity', w represents
the weighing points corresponding to the categories, and c represents the counts
corresponding to the categories.
Clarity Types

Representative
Number

Weighing Points Staining Type

Good

1

1

ssDNA/DAPI/DAPI&mIF

Moderately blurry2

0.2

ssDNA/DAPI/DAPI&mIF

Severely blurry

30

ssDNA/DAPI/DAPI&mIF

Slightly overexposed

4

0.9

ssDNA/DAPI/DAPI&mIF

Heavily overexposed

50

ssDNA/DAPI/DAPI&mIF

This score shows the clarity of the tissue image and is used as a prediction of the final
cell segmentation results. The higher the score, the better the quality of the tissue image
and the higher the success rate of cell segmentation. The table below shows how theclarity score corresponds to the segmentation effect.
Clarity Score

Cell Segmentation Results

80 ~ 100

High success rate for automated cell segmentation

50 ~ 80

Moderate success rate for automated cell segmentation

0 ~ 50Low success rate for automated cell segmentation

Automated cell segmentation is being done via Stereo-seq Analysis Workflow (SAW)
pipelines.
The image clarity score does not affect the final QC assessment results and is onlyused as a reference to assess the feasibility of cell segmentation.
15

STOmics Microscope Assessment Guideline

MICROSCOPE IMAGE EVALUATION

Microscope Stability Assessment Scoring Instructions(applicable for DAPI& mIF)
Microscope Stability Stitching Evaluation Score: Using an algorithm based on thesimilarity of overlapping regions, we divided FOVs into two types: FOVs with clear
features in overlapping areas that can be detected by the algorithm and total FOVs.
According to the ratio of FOV with clear features among the entire FOV that can bedetected by the algorithm and the average offset score between each FOV and theadjacent FOV, the stitching score of each FOV is eventually calculated. This score is forevaluation of the stitching stability of the microscope, mainly for DAPI & mIF images, andfor predicting image registration results. The higher the score, the higher the registration
accuracy.
A good standard is that the boundaries of overlapping area features can be discerned by
the naked eye. The definitions and calculation formulas of each index are as follows:· FOV with clear features in detected overlapping areas: The number of FOVs
that the algorithm can detect, represented in "a".
· Total FOV: The total number of FOV of the entire chip, represented in "b".
· FOV offset score with clear features in the overlapping area: Offset score
calculated from the offset of each FOV and its adjacent FOV, represented in "c".
· Average offset score: The ratio of FOV offset score to the detected FOV numbers,
represented in "d".

Example of overlapping area with clear features. The features of the overlapping
area are clear when the ratio is greater than 5%.

16

STOmics Microscope Assessment Guideline

MICROSCOPE IMAGE EVALUATION

Image Calibration Assessment Scoring Instructions(applicable for DAPI& mIF)
Image calibration assessment: It utilized a similarity calibration algorithm to evaluate
the offset between DAPI and its corresponding IF images. The passing criteria are whenthe maximum offset does not exceed 20 pixels and the image similarity is greater than
1%. This score mainly evaluates the coincidence degree of DAPI and IF images, which
predicts the image registration results.
Calibration evaluation is only for single-channel images, and the default DAPI and
IF offsets for multi-channel images are 0.
Please refer to the Help Document section within the software for detailed
instructions on software usage.

4.3. Image Examples
Examples below are images of good and bad qualities for comparison.

Tracklines

Qualified image

Out-of-focus tracklines

Unqualified
image

Tracklines tilted

Stitching errors (Tracklines) 1

17

STOmics Microscope Assessment Guideline

MICROSCOPE IMAGE EVALUATION

Tissue clarity

Qualified image

Overexposure

Unqualified
image

Out-of-focus tissue image

Indistinct tissue boundaries

Image stitching

stitching line

Qualified image

Unqualified
image

Stitching errors (Tissue image) 1

18

STOmics Microscope Assessment Guideline

MICROSCOPE IMAGE EVALUATION

Epi-Illumination for H&E

Qualified image

Epi-Illumination overexposure

Unqualified
image

Epi-Illumination stitchingartifacts 2

Unadjusted white balance

1. To solve stitching errors, please perform a microscope stitching calibration.
2. The appearance of distinct stitching lines is usually due to a strong brightness
contrast among different tile images (FOV). Hardware repair/recalibration might berequired, please contact the manufacturer’s microscope engineer.
19