PHI HoloMonitor M4 Manual instruction

HoloMonitor®M4
Setup and Operaon Manual
February 2018, Hstudio 2.7.3, rev. 8
This manual is issued by:
Phase Holographic Imaging PHI AB
Scheelevägen 22 | 223 63 Lund | Sweden
www.phiab.se | suppor[email protected]
© 2018 Phase Holographic Imaging PHI AB | All rights reserved
P H A S E
HOLOGRA P HIC
I M A G I N G

2© 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se |[e-mail]
Contents
The manual 5
Instrument safety 6
Warranty 7
Introducon to HoloMonitor® M4 8
Computer requirements 9
Hstudio installaon 9
HoloMonitor installaon 12
Starng the instrument for the rst me 16
Placing HoloMonitor inside a cell incubator 17
Cleaning instrucons 18
Calibraon procedure 19
Long-term storage 20
Sample requirements 21
HoloLids™ 22
Introducon to Hstudio 2.7 23
View live images 26
The theory of focusing 26
Praccal focusing 28
Moving the motorized sample stage 32
Change how live images are displayed 33
Change default cell and medium refracve index 36

3© 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se |[e-mail]
Capture images 37
Capture a single image 37
Capture a me-lapse sequence at one posion 38
Capture me-lapses at several posions in parallel 39
Saving me-lapses in separate groups 42
Automacally generate random capture posions 44
Add capture posions manually 46
Focus vericaon 47
View and adjust captured images 48
View an image 48
View one posion of several in parallel me-lapses 49
Change how captured images are displayed 50
Re-focus a captured image 51
Cell idencaon 53
Change how idened cells are displayed 54
Adjust cell idencaon 55
Make adjustments for individual cells 58
Cell tracking 60
Basic cell tracking 60
Start tracking 60
Cell tracking warnings 63
Displaying cell tracking 64
Adjusng cell tracking 65
Tracking cell morphology 68
Mulple analyses 70
Export tracking data 70
Analyze and export data 71
Measure cells interacvely 71
Analyze results in plot 72
Mulple analyses 74
Display results in scaer plot 75
Display results in histograms 80
Export plots and cell data 81

4© 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se |[e-mail]
Cell count 83
Adjust the histogram proporons 84
Remove data from plot 85
Export results 85
Export images and movies 86
Add and remove image frames 86
Edit the images 87
Create an AVI movie 90
Export images 92
Database handling and backup 93
Switch to exisng database 93
Switch to a new database 94
Move a database 94
Copy and move individual images, groups or projects 95
Backup/restore data 95
Transfer data from one computer to the next 96
Troubleshoong 98
Live capture 98
View images 103
Idenfy cells 104
Track cells 105
Analyze data 105
Export images 105
Morphological parameters 106
Holographic microscopy 106
Cell morphology parameters 107
References 112

5© 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se |[e-mail]
The manual
In the first part of the manual, Instrument, there is information on instrument safety and
handling.
In the second part of the manual, Software, there are detailed guides of how to perform
different procedures using Hstudio, such as how to capture an image and how to analyze
the images.
In the third part of the manual, there are trouble shooting guides for the software and the
hardware.
The PDF version of the manual is interactive, with internal links. Clicking the link will refer
you to the section of the manual the link refers to. The symbol below is found throughout
the manual. Clicking it will bring you back to the previous view.
Interactive button for previous view.

6© 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se |[e-mail]
Instrument safety
HoloMonitor M4 was designed and tested in compliance with low voltage direcve 2006/95/EC
and electromagnec compability 2004/108/EC. References to relevant harmonized standards
used, or references to the specicaon in relaon to which conformity is declared, are EN61010-
1:2001 safety requirements for electrical equipment for measurement and EN60825-1:2007
safety for laser products.
This manual includes information and warnings which must be observed by the user. It
contains information notes as well as information of importance to safety of personnel and
property. If the equipment is used in a manner not specified in the manual, the protection
provided by the equipment may be impaired.
Symbols are categorized as follows:
Important information.
Risk for physical damage on person or property.
Risk for electrical shock that might cause damage to person or property.
Risk for laser light causing damage to person or property.

7© 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se |[e-mail]
Warranty
The manufacturer guarantees that the instrument has no material or producon defects
when delivered, according to EU direcve 99/44/EC sale of consumer goods and associated
guarantees. If the manufacturer is informed within 6 months after delivery, the manufacturer
is obliged to rectify such defects. It is the manufacturers decision whether to repair the
instrument or replace the instrument with an instrument free of defects. No guarantee is
provided for defects caused by natural wear (wearing parts in particular) or improper use.
The instrument manufacturer is not liable for damage caused by faulty operation, negligence
or any other meddling with the instrument, particularly the removal or replacement of
instrument components, or the use of accessories from other manufacturers. This forfeits
all the claims against warranty.
With the excepon of the procedures specied in this manual, no maintenance or repair
of HoloMonitor may be performed by users. Repairs may only be performed by specially
authorized personnel.

8© 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se |[e-mail]
Introducon to HoloMonitor® M4
HoloMonitor M4 is a live cell imaging me-lapse cytometer, providing imaging and quan-
caon of unstained living adherent cells directly in their culture vessels. It can be used for
a wide range of applications, including:
• Cell count and proliferaon
• Cell culture quality assessment
• Cell morphology
• Cell molity and migraon
• Cell division/mitosis
• Toxicology
HoloMonitor is based on digital holography microscopy. This technique is based on measure-
ments of how the cells shi the phase of light that passes through the cells, and does not
require any kind of labeling or staining.
HoloMonitor is incubator compatible, allowing experiments to be performed in a cell culture
incubator or a hypoxia chamber. The cells can be analyzed while growing undisturbed in a
range of different cell culture vessels, see www.phiab.se/support/vessel.
HoloMonitor is equipped with a fixed sample
stage, or with a computer-controlled motorized
XYZ-stage.
Applicaons are described in applicaon notes,
such as cell molity, cell death, cell cycle and
toxicology. Applicaon notes are available at

and associated protocols at www.phiab.se/
support/protocol.

9© 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se |[e-mail]
Computer requirements
HoloMonitor is controlled by the proprietary Hstudio software running on an external
computer. Hstudio needs to be installed prior to installation of the HoloMonitor base unit.
Make sure the computer fulfills the minimum requirements:
 Windows 10, 64-bit
 Intel Core i7
 16 GB RAM (8 GB minimum)
 556 GB SSD (256 GB minimum) and external USB3 hard drive
for backup and transfer
 Full HD (1920x1080) or higher
The files associated with Hstudio are stored in the folder C:\PHI, which must not be used
for anything else. User created data is stored under C:\PHI as well. However, the user data
location can be changed at any time.
Hstudio installaon
1 BACKUP
If Hstudio already is installed, make a backup according to the instructions in Database
 (page 93).
2 UNINSTALL PREVIOUS VERSION
Uninstall previous Hstudio version prior to
installaon. Previously created user data
will not be removed. Go to
Control Panel → Uninstall
3 INSTALLATION
Locate the installation executable file
HstudioM4setup.exe on the PHI USB
memory and double click it to begin in-
stallation.
Accept the user account control question
by clicking Yes.

10 © 2018 Phase Holographic Imaging PHI AB | All rights reserved | www.phiab.se |[e-mail]
4 PREREQUISITES INSTALLATION
The installation will start with prerequi-
sites installation which launches separate
installation programs from third-party
vendors. Depending on the computer
configuration this step may not occur.
Click Install to continue and wait for the
installation to complete.
5 HSTUDIO INSTALLATION
Progression of installation.
Click Next to continue.