- experts on non-destructive long-term studiesof biological events in cell cultures -

For well-designed research

- non-destructive cell culture analysis -

The HoloMonitor™ Imagine it - Visualize it - Analyze it - PHInalyze it

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concept

The HoloMonitor™ M3 can replace and/or complement many other instruments and techniques of today e.g. microscopes, cell counters and flow cytometry.

Live cell analysisThe HoloMonitor™ M3 is a tool for understanding and visualising bio-logical events in cell culture. Work-ing exclusively with non-destructive methods, Holomonitor is the perfect instrument for long term analysis, us-ing consecutive holographic imag-es of cell cultures that are captured in the normal growth environment of the cells. The HoloMonitor™ M3 is not just another microscope for imag-ing; it is also a powerful cell analyz-er. We therefore like to describe our technique as “Live cell analysing” as we do both analysis and live cell

imaging at once. The HoloMonitor™ concept is based on the fact that it is possible to make detailed cell stud-ies without affecting or manipulating the cells. The HoloMonitor™ M3 has a strong connection between imaging and analysis. It measures cell num-bers and cell proliferation together with morphological parameters. The HoloMonitor™ M3, together with

the software HoloStudio™ 2.0, cre-ates a powerful system for cell biol-ogy studies. This system has minimal influence on the material you are working with and provides data in ways other assays of today cannot do. The procedure is non-destruc-tive, the cultures are unaffected by the analysis and can also be used afterwards for other experiments and analysing methods.

Long term studies on cell cultures - siRNA studies - dose response treatments - toxicology - gene transfections- cell migration - cell differentiation

Short term studies on cell cultures- non-destructive measurement of cell density - viability

Visualisation - imaging- 3D topography- time-lapse imaging

IndexThe Holomonitor ™ M3 concept pages 2-3Holographic Imaging & phase-contrast pages 4-5The Holostudio™ 2.0 software pages 6-73D Imaging pages 8-9Always focused & Cell viability page 10Time-lapse imaging page 11Construction & design page 12Technical specifications page 13The technology platform page 14Microscope options page 15

Cell cultures Cell culturing is one of the most fun-damental tools in biological science for the study of various mechanisms. However, cells are transparent and usually have to be stained in order to be analysed. All handling of the cells affect the parameters your want to study. We therefore introduce the HoloMonitor™ M3, the first totally non-invasive live cell imaging microscope/cell analyzer. Without any damaging cell preparations, analysis can be per-formed directly in the usual growth en-vironment of the cells, thus giving data

with minimal interference.

Cell proliferationWhen you want to produce a pro-liferation curve using the HoloMoni-tor™ M3, the same culture flask can be used every time and no sister cul-tures nor trypsination of the cells are needed. The HoloMonitor™ M3 can be used to measure cell proliferation in many ways, such as cell number, confluence, and total cell volume or as an estimation of dry mass.

Due to the fact that our technology

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does not affect the cells, analysis can be performed with unlimited numbers of exposures. The right temporal reso-lution is important for understand-ing biological events and can only be achieved by multiple time point analysis.

The HoloMonitor™ M3 is primarily de-signed to follow and analyse cell cul-tures, however it can also be used to pinpoint a single cell within a popula-tion. The HoloMonitor™ M3 can help users to increase their scientific quality, and quantity as well as improve the everyday handling of cells in a modern laboratory.

Primary human cheek epitelial cells.

Neuroblastoma cell line, SH-SY5Y . The different colours shows differences in optical thickness in 3D.

Holographic Imaging &Non-destructive cell analysis

More than just a microscope The HoloMonitor™ M3 is actually two microscopes in one, one with bright field phase-contrast optics and one holographic microscope. (If you go for the dual laser option you’ll actually get three microscopes in one, one phase-contrast and two holographic ones!) Bright field phase contrast has been used for decades and is standard in all cell laboratories. Even though the HoloMonitor™ M3 is foremost a holo-graphic instrument, it produces high quality phase-contrast images giving you the best of both worlds.

Until recently, phase contrast micros-copy has been the major tool for non-invasive live cell imaging. The long his-tory and refinement of this technique have made it a standard tool in many

applications. Phase contrast microsco-py can be used to produce very de-tailed images of living cells. However, images produced by this illumination technique cannot be quantified as the parameters studied are focusing dependent.

Quantitative phase imaging Holography is a quantitative phase imaging technique. Quantifying the optical phase shifts associated with cells gives access to information about morphology and dynamics without any staining or other markers. This tech-nique is therefore most suitable for quantitative analysis of morphological parameters of non-stained living cells in proliferation studies.

Best of both worldsThe HoloMonitor™ M3 contains both holographic and phase-contrast mi-croscopy options. Holographic imag-ing in combination with conventional phase-contrast microscopy will get you both the latest in visualisation and the traditional view of your cells. To-gether with powerful software for cell culture analysis the HoloMonitor™ M3 is the new standard equipment for every cell laboratory. The HoloMonitor™ M3 is suitable for both everyday routine work and frontline experiments.

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Phase contrast

Topographic 3D view of murine cell line

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Same field of view in both phase-contrast mode and holographic mode of Wilm’s tumour cells, WiT 49.

Human prostate cancer cells, PC3, illustrated in a combined phase-contrast and holographic image.

HoloStudio™ 2.0One can call the HoloMonitor™ M3 the heart of this system and new advanced HoloStudio™ 2.0 acquisition and analysis software its brains. The HoloStudio™ 2.0 contains a top of the line image processing module where you can perform analysis of every individual cell imaged. All data are stored in an advanced database where all parameters from every cell are stored. The data can be directly linked into Excel* to produce graphs and data sheets.

The base of the HoloStudio™ 2.0 lays in the capacity of analysing proliferation, morphology and cell migration. HoloStudio™ 2.0 can produce not only statistical information of different morphological parameters in the whole cell culture like cell number, area and phase shift, but it also of morphological parameters for each individual cell in every image captured.

Real time segmentationIn HoloStudio™ 2.0 the user interacts with the program simply by setting adjustable threshold values using adjustable “draw-bars”. Thres-hold values for morphological parameters are illustrated in real time on the screen. This feature makes the program very easy to use

Morphological parametersIn cell cultures, the effect of drugs, genes or proteins that modulate cell proliferation are frequently reflected in morphological parameters prior to actual changes in cell numbers. Changes in cell morphology can therefore often be used as an early indicator of effects of treatment prior to changes in e.g. cell number. The HoloStudio™ 2.0 measures morphological parameters such as elongation, irregularity and thickness. It can be used to select cells with certain characteristics, such as size, width, flatness and roundness. Cells with

many protrusions or enlarged cell organelles can be identified. All data can easily be exported to programs such as Excel for further analysis.

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The HoloStudio™ 2.0Are you curious?

software

(*Phase Holographic Imaging is not affiliated with Microsoft.)

The analytic power of the HoloStudio™ allows the user to perform multiple morphological analysis of every captured image. Parameters like cell area, thickness and other morphological factors are measured for each and every cell. Similar to flow cytometry, each cell can be represented by a dot as in the plots above. However, in contrast to flow cytometry each cell can also be visualised and viewed in its growth environment.

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Easy to use real time segmentation with drawbars that illustrates threshold values makes the HoloStudio™ 2.0 a very user-friendly analytic software.

Every parameter measured in a sequence of captures images can be il-lustrated in clear graphs or plots.

Multiple parameters can be analysed in 3D graphs in the expanded version of HoloStudio™ 2.0.

3D topography The strength of digital holography lays in the possibility to detect the phase shift of the object under ex-amination. This technique is espe-cially suited for transparent objects such as living cells. In addition to traditional microscopy, where an image is projected on a camera, the camera on the HoloMonitor™ M3 registers an interference pattern (For more details see the technology platform section.) Within this interfer-ence pattern all information about

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3D without scanning

3D Imaging

the cells are stored and from this a three dimensional topographic im-age of the cells can be created. It is also possible to virtually select any focus plane from this single interfer-ence pattern. The construction of three dimesional images allows the operator to view the cells from all angles and to detect changes in cell shape in three dimensions and not only two dimesnions.

Another advantage is the possibility to extract shape, density and rela-tive volume of the cells in your sam-ple with only a single exposure.

Dual lasers systemUsing the option of dual lasers on the HoloMonitor™ M3 the 3D rendering can be further enhanced, creat-ing very realistic 3D imaging without scanning.

Wilm’s tumour cell line, WiT 49.

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Human white blood cells.

Human primary cortical neuron.

Human prostate cancer cell line, PC3.

Human primary dendritc cell.

Stay focused An extra benefit of the digital holographic technique is that the focus plane is calculated afterwards in a computer, making the system insensitive to focus disturbances in for instance long time-lapse periods. The computer reconstruction algorithm performs the focusing, giving a total focal depth of up to 4 mm. This is possible since the whole 3D volume of an object is recorded in the hologram. Because of this, no mechanical movement of the imaging system is necessary when focusing, which makes the instrument robust, compact and insensitive to vibrations.

Cell viabilityThe Holomonitor™ can be used to visualise living and dead cells as it measures the refractive index. This can be done without using stains such as trypan blue. Digital holography indirectly measures the refractive index of the cells. We can therefore detect dying cells as the integrity of the plasma membrane deteriorates. The gradient

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Benefits of Always focused & cell viability

digital holography

Dead cell(trypan blue)

Dead cell(Holographic, non stainied)

Living cell

Living cell

As the holographic technology meassures variations in refractive index, dead and dying cells can be detected in their growth environment without the need of any staining. L929 cell line.

over the membrane weakens and is eventually lost. Thereby the difference between the refrective index within the cell and the surrounding media decreases, making the dead or dying cells appear as fried eggs.

Viability can be studied continuously in the same growth vessel without side effects on the cell cultures. The results can be given as dot plots, where each dot can be tracked backed to the corresponding cell.

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Infinite number of exposures without cell influence

Two properties make digital holography superior for time-lapse studies and cell motility. One is the fact that any number of exposures from the HoloMonitor™ M3 does not affect the cells and the second is the fact that the focus is calculated after the capture of a hologram. The red lines indicate how the cells have migrated over time. Individual cells can also be monitored for changes in morphology as they move.

Time-lapse Imaging

of thousands of exposures without damaging the cells. Since no stains or damaging focused light beams are used there are absolutely minimal effects on the cell cultures.

Cell migration For those who are interested in cell migration and cell tracking the HoloStudio™ 2.0 can be had with the extra cell migration software module. The fact that you can take infinite numbers of exposures on the HoloMonitor™ M3 without affecting the cells makes this system perfect for

Time-lapse studiesA picture tells a thousand words, a movie more than a million! The fact that the HoloMonitor™ M3 does not interfere in any way with the observed cells on the instrument and causes no bleaching or light toxicity, makes it perfect for long term time lapse studies with multiple exposures.

When using the HoloMonitor™ M3 together with a heating stage and/or a micro-incubator mounted on the instrument the cell culture can be monitored for weeks with tens

cell migration and motility studies.

The focusing of the image takes place in the software after storing of the digital interference patterns.This makes the technique especially well suited for time lapse studies as the object does not go out of focus.

Phase Holographic Imaging recommend IBIDI* heating stages and OKO-labs* micro incubators.

(*Phase Holographic Imaging AB is not affiliated with IBIDI GmbH nor OKO-labs.)

Images taken from a time-lapse sequence showing human dentritic cells changing morphology over time interval of 90 seconds.

Construction &Imagine it - Visualize it - Analyze it - PHInalyze it

design

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The designThe chassis of the boldly designed HoloMonitor™ M3 is made out of cast aluminium to ensure stability and endurance. It is also ergonomically designed and made to work in a modern laboratory environment. The interior is as sturdy as the outside as all optical and mechanical parts are mounted into a solid frame of aluminium.

The absence of ocular on this instrument also sets it apart from traditional microscopes. The oculars are not needed as the instrument is connected to a computer and a 24” widescreen LCD monitor.

Colour optionsThe HoloMonitor™ M3 comes in red and silver metallic. However, the instrument can be had in any colour combination the official PHI-AB colours which are PHI-red, PHI-silver, PHI-grey and PHI-white.

This is the new exciting design of the HoloMonitor™ M3 which is designed by the well know Swedish designer Johan Behrin. “Working with medical and scientific instruments performance and reliability is crusial. When you are able to combine this with new and exciting design you´ll get something really special. Working in a laboratory environment should never be dull.”

Johan Behrin, designer

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Technical specifications

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SystemMeasurement technique: single wavelength transmission digital holographic microscopyImage types: intensity and quantitative phase contrast image (DH mode)

phase contrast image (PC mode)Light source: monochromatic laser source (DH mode)

Luxeon 1 watt LED (PC mode)Sample stage: manual or automated XY stage up to 150 mm × 120 mm travel

range automated Z stage up to 20mm travel rangeCamera 1280 × 1024 pixels, 8 or 10 bits (hologram)

2048 × 1536 pixels, 8 bit RGB (phase contrast)Available objectives: standard objectives 10-100xObjective mounting: 4-position objective turret with manual or automated functionComputer: PC workstation with latest Intel processor.Software: Holostudio™ imaging and cell analyze softwarePerformanceLateral resolution: objective dependent, down to 500 nm 100x objectiveField of view: objective dependent, up to 4 mmWorking distance: objective dependent, from 0.3 to 18 mmDigital focusing range: up to 50× depth of field (objective dependent)Grabbing time: down to 4 μs in a single image grab (no scanning mechanism,

insensitive to vibrations)Spatial sampling: 1024 × 1024 pixels (hologram)Acquisition rate: 27.5 fps (1024 × 1024 pixels)Reconstruction rate: 4 fps (512 × 512 pixels), 1 fps (1024 × 1024 pixels)Sample illumination: ~0.1 mW/cm2Maximum container height: 60mmPower requirementsInput voltage: 230 VAC - 50/60 HzPower requirements (w/o computer):

max. 83 W

Dimensions & weightDimensions (L × W × H): 650 × 360 × 560 mmWeight: 35 kgSamplesCells Eukaryotic adherent monolayer cells in any standard cell culture

vessel or suspension cells in IBIDI µ-slides.Culture vessels Any standard culture flask or petri dish, multi well plates down to

24-wells, any IBIDI µ-slide

Micro incubator PHI-AB recommends a microscope stage incubator from OKO-labs

platform

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Open platform system

Schematic view of the technique behind the Holomonitor™ M3.

Digital holography can register both the amplitude and phase of transmitted light that has passed through an object. By creating an interference pattern of the transmitted light and an undisturbed reference light, a hologram of the object can be recreated. The hologram contains 3D information of the object of both shape and density.

A major advantage of holographic imaging versus traditional imaging is that in holography both the phase and the amplitude of the light are recorded. In traditional optical microscopy only the amplitude is recorded, which means that only objects that absorb or scatter light can be observed. When recording the phase of the transmitted light the “optical thickness” of the object can be measured. This optical thickness is equal to the thickness of the object times the refractive index of the object. Another way to

About digital holography In 1947, Denis Gabor discovered the principles of holography, a now well-established imaging technique. He was awarded the Nobel price in physics in 1971 for his discoveries. Built on traditional holography, digital holography is a method of acquiring and processing holographic measurement data from interfering wave fronts. The scattered light from an object illuminated with a coherent light (usually a laser beam in the visible spectrum) interferes with the light from a reference source and is recorded on a CCD camera. The image can then be numerically reconstructed in a computer. Digital holography typically delivers three-dimensional (3D) surface or optical thickness data.

The technology platform

express it is to say that the optical thickness is proportional to how much the light has been slowed down, while moving through the object. By imaging the phase shift, instead of the amplitude, objects that are invisible in a traditional microscope are made visible. This makes, for instance, staining of cells to increase the contrast unnecessary.

Refractive index The recording of the optical thickness gives the possibility to measure the difference in index of refraction of the objects to the surroundings. This gives the possibility to separate different types of objects in a blend since the index of refraction is material specific. In addition, if the indexes of refraction of the objects are known, a volume distribution of the objects can be calculated.

The technology

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Microscope options

Objectives The Holomonitor™ M3 has an objective turret for four different objectives and can be mounted with objectives from 10-100X. (Dependent on magnification they are either made by Olympus or Nikon.)

Motorized objective turret The objective turret can be fully mo-torized. This is a useful feature that makes switching between holo-graphic and phase contrast mode easier.

Motorized stageEvery Holomonitor™ has a motorised z-axis stage for focusing of the phase contrast as standard feature (in the

(*Phase Holographic Imaging AB is not affiliated with Nikon, Olympus, nor NVIDIA.

holographic mode focus is set in the software after an holographic interference pattern is taken). As an option, a fully x,y and z-axis motorized stage can be had to facilitate for instance multi well plate evaluations.

Dual lasers Using a single laser, the Holomonitor™ M3 produces a 3D topographic image of the cells based on optical density in each pixel measured. By using dual lasers with different wavelengths the instrument can produced 3D images with spatial differences i the z-axis, for realistic 3D.

Computer and softwareThe system is delivered with a PC workstation with the latest Intel processor and 24” widescreen. The system can also be used on a Macintosh computer with Vista.

Calculation time can be even further reduced with the option of an extra acceleration card Tesla from NVIDIA.

Closeup of the motorized stage and objective turret of the Holomonitor™ M3.

Customize your system

Phase Holographic Imaging ABIdeon Bioincubator

BioMedical Center D10221 84 Lund

SWEDEN Phone: +46 (0)462865970

Fax: +46 (0) 2220296E-mail:info@phiab.se

Phase Holographic Imaging is the leading expert on non-destructive long term studies of biological events in cell cultures, for example studying the effects of drug dose responses, toxicology, modulation of gene expression or protein evalu-ation studies.

The technology of the HoloMoni-tor™ M3 is based on digital hologra-phy which has several advantages over traditional light microscopy. Using traditional microscopy, cells which are transparent need to be

stained before visualization and analysis. All staining affect the cells and are also often harmful, toxic or even lethal to the cell cultures. With our technology, no staining is neces-sary, no toxic agents or damag-ing light is used and the analysis is performed directly in the cell culture growth environment. The HoloMoni-tor™ M3 can help users to increase their scientific quality and quantity as well as improve the everyday handling of cells in a modern labo-ratory.

The HoloMonitor™ M3 also provides unique features like 3D-visualization of cells and focus in the software after the hologram is taken, espe-cially useful for time-lapse and cell migration studies.

The Holomonitor™ M3 is not just another microscope for imaging; it is also a powerful cell analyzer work-ing exclusively with non-destructive methods. We therefore like to describe our technique as “Live cell analysing” as we do both analysis and live cell imaging at once.