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]

P H A S E

HOLOGRA P HIC

I M A G I N G

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

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

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

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.

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.

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.

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.

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.

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.

Enter your information. Click Next to

continue.

Click Install to start the installaon process.

Click Finish to complete the installation.

HoloMonitor installaon

HoloMonitor is controlled by a computer running the proprietary Hstudio cell analysis and

tracking software, further described in  (page 23).

10 13

HoloMonitor M4 with a motorized stage contains the following parts:

1HoloMonitor with motorized stage 8Main unit power supply

2Objective protective cap 9Laser unit power supply

3Laser unit with optical fiber 10 Laser unit power adapters

4Vessel holders for motorized stage 11 HoloLid Petri

5HoloLid 6-well 12 Cotton swabs

6Transportation plate 13 USB cables

7USB memory with Hstudio 14 Spacer plates for fixed stage (not shown)

1 REMOVAL OF TRANSPORTATION PLATE

• Turn HoloMonitor upside down. Let it

rest on the protecve foam it came in.

• Remove the six screws holding the

plate.

• Turn the instrument back (right side up)

and remove the tape and protecve

foam.

2 REMOVING OBJECTIVE PROTECTIVE CAP

• Remove the cap protecng the objecve.

3 CLEANING OPTICAL SURFACES

• The top of the objecve (A) and the object

beam window (B) are cleaned with a fresh

coon swab dipped in 70% ethanol.

• Use a dry coon swab to remove ethanol

residues.

4 CONNECTING THE LASER UNIT

The optical fiber must be handled gently. Do not forcefully stretch or bend the fiber.

Do not touch the fiber tip.

The laser needs approximately 15 seconds to warm up before the laser light is visible. An

additional 15 minutes is required before it is fully stabilized.

The HoloMonitor laser unit is a Class 2M laser. A Class 2M laser is safe because of the aversion

response, if viewed by a naked eye. However, to avoid any unnecessary discomfort when

handling the disconnected optical fiber, make sure that the laser beam emerging from

the fiber does not radiate directly into somebody’s eye.

• Carefully remove the protecve cap from the Opcal ber p. Save the protecve cap

for future use.

• Gently insert the opcal ber p into the ber

connector. Make sure that the metal protru-

sion (ridge, yellow arrow) is aligned with the

guiding notch in the connector socket (red

arrow) before ghtening the lock ring.

• Connect the Laser unit power supply to the

Laser unit.

• Connect the laser unit power supply to a

power outlet, using one of the Laser unit

power adapters.

5 CONNECTING THE STAGE

• Make sure the motorized stage is connected.

6 CONNECTING THE USB CABLE

• Connect a USB cable in the micro USB con-

nector.

• Connect the USB cable to your computer.

7 CONNECTING POWER

• Connect the Main unit power cable to the

power connector. Make sure the connector is

in the correct posion before ghtening the

lock ring.

• Connect the power cable to a power outlet.

8 ATTACHING THE VESSEL HOLDER

HoloMonitor with motorized stage is deliv-

ered with three different vessel holders, for

microscope slides, standard multi-well plates

and for standard Ø 35 mm Petri dishes.

To ensure that the vessel is correctly fitted on HoloMonitor; first attach the cell culture

vessel to the vessel holder, then attach the vessel holder to the stage by clicking it on from

the side, as seen in the image blow.

Starng the instrument for the rst me

1 CONNECT THE INSTRUMENT

Connect the fully assembled instrument

to the computer using one of the included

USB cables. Drivers will be installed auto-

macally when the USB cable is aached

and power is turned on.

2 START HSTUDIO CALIBRATION

Whenever the Live capture tab is selected

for the rst me aer starng Hstudio, it

will automatically suggest a recalibration.

Select the Live capture tab, remove any

sample from the stage and click Continue

to enter the Calibration wizard. Follow

the   (page 19).

The calibration is performed to optimize

the image quality and is nalized in approx-

imavely one minute.

3 VIEW LIVE IMAGES

If the calibration is in the green area, the instrument is ready to use. Follow the instructions

in section  (page 26)

If the calibration is in the red area, go to  (page 98).

Placing HoloMonitor inside a cell incubator

• Clean HoloMonitor according to the

 (page 18).

• Disconnect the Laser unit power supply

from the power outlet.

• Disconnect the Opcal ber, the USB

cable and the Main unit power supply

cable from the instrument.

• Put on the protecve cap on the Opcal

ber p. Do not touch the ber p.

• Place HoloMonitor inside the cell incubator. Make sure there is space for the Motorized

stage to operate.

• Pull the Opcal ber, the USB cable and the Main unit power supply cable through the

access port of the incubator and reconnect them to the instrument.

• Finally, reconnect the Laser unit power supply to the power outlet.

• After at least three hours of acclimatization, calibrate HoloMonitor according to the

 (page 19) to ensure that the instrument operates opmally before

proceeding with any experiment.

Be careful when handling the optical fiber so it is not stretched, pulled, bent or harmed

in any way.

To avoid build-up of internal condensation, the power must be switched on at all times

while HoloMonitor is inside the incubator.

Do not lift HoloMonitor by holding the motorized stage. Only lift holding the blue and

white metal of the instrument.

Cleaning instrucons

1 CLEANING OPTICAL SURFACES

• The top of the objecve (A) and the object beam

window (B) are cleaned with a fresh coon swab

dipped in 70% ethanol.

• Use a dry coon swab to remove ethanol residues.

• To validate that the opcal surfaces are clean,

calibrate HoloMonitor according to the 

procedure (page 19), to ensure that the Diag-

nosc Values are within the green ranges.

• Repeat cleaning, if the Diagnosc Values are not

in the green ranges.

An instrucon video of how to clean the instrument is available at www.phiab.se/support.

2 CLEANING INSTRUMENT EXTERIOR

• The instrument can be wiped down with a damp cloth (ethanol or water). Cleaning must be

performed so that no liquid enters the interior of the instrument.

Calibraon procedure

HoloMonitor should be re-calibrated before use. Image calibration gives a background image

that is used by Hstudio to lower background disturbances and increase the image quality.

The calibration procedure takes approximately one minute.

1 PERIODIC IMAGE CALIBRATION

Start the calibraon procedure by clicking

the Calibration button in the Calibra-

tion side panel in the Live capture tab

in Hstudio.

2 CALIBRATION WIZARD

The three Diagnostic values measure

the image quality. All values should be

in the green range. If this is not the case,

please follow the 

(page 18). After cleaning, click Recali-

brate to refresh the Diagnostics values.

3 CALIBRATION WARNING

If Hstudio fails to perform a calibration, a

warning will appear. After verifying that

HoloMonitor is properly installed and

cleaned, click OK.

4 FURTHER PROBLEMS

If any of the Diagnostic values remain outside the green range after several cleaning

attempts and overnight drying (see ), contact the technical support at

suppor[email protected] for further assistance.

Long-term storage

• Long-term storage of HoloMonitor should be at room temperature outside the incubator.

Use a dust cover, if the instrument is not in use for a longer period of me.

• All cords and the laser ber can be kept inside the incubator at all mes if it is inconvenient

to remove them. When detaching the laser ber, use the protecve cap of the laser ber.

• Before re-installing HoloMonitor into the incubator, run the  (page 19)

to assure that the opcal surfaces are clean.

If the cables are left inside the incubator for a longer period of time, remember to wipe

them off regularly with 70% ethanol to reduce the risk of incubator contamination.

Table of contents

Popular Medical Equipment manuals by other brands

NanoVibronix

NanoVibronix Wound Shield user manual

DHG

DHG Raizer M user manual

Boston Scientific

Boston Scientific Vercise 92290361-01 quick start guide

Otto Bock

Otto Bock 1E57 Lo Rider Instructions for use

College Park

College Park Capital hydraulic knee user guide

Organon

Organon Follistim Pen Instructions for use

Stryker

Stryker Hygia Reprocessed Currie ALP Sleeve IFU Instructions for use

Fukuda Denshi

Fukuda Denshi HBX-800 Service manual

TheraPlate

TheraPlate Revolution owner's manual

Oxypharm

Oxypharm NOCOSPRAY 2 user manual

Optos

Optos P200TxE user guide

Roche

Roche Accu-Chek Mobile user manual

bort medical

bort medical DorsoFX Instructions for use

LiNA

LiNA Xcise manual

ConMed

ConMed 1000 SES Service manual

Dentsply Sirona

Dentsply Sirona Propex IQ Getting started guide

Nasco Healthcare

Nasco Healthcare Basic Buddy LF03693 instruction manual

mk-dent

mk-dent AM1016 instruction manual

PHI HoloMonitor M4 Manual instruction

Popular Medical Equipment manuals by other brands