HoloMonitor M4 Imaging System

Non-Invasive Holographic Time-Lapse Imaging & Cytometry

Analyze live cells without staining or fixing

The HoloMonitor M4 Imaging System is a completely non-invasive live cell microscope and cell analyzer for adherent cells. The system operates with cells in normal lab growth environment, i.e. a common cell culture vessel in an CO2 incubator. The system provides results with minimal sample perturbation and without the need for staining or fixing. This is done via a technique known as digital Phase Holography Imaging (PHI), recently developed and patented by a research group in Sweden. The technology is exclusively licensed to our partner Phase Holographic Imaging AB, and we are granted the opportunity to offer this unique system to researchers in the USA and Canada.

 



LITERATURE
Brochure
User Manual
Publications
Software Demo
 
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What is Phase Holographic Imaging?

 

 

Apoptosis Time-Lapse Video

An etoposide-treated DU145 prostate cancer cell exploding into a cascade of apoptotic bodies.

Cell Division Time-Lapse Video

In this rare video clip, a cell curls up to divide but aborts the cell division process. The cell attempts and fails to divide several times. It is not until after the cell has interacted with its neighbors that it finally manages to divide successfully.

 

To perform digital holography, the M4 applies a coherent 633nm laser to the sample at levels far below any level known to damage cells. The red laser is split into a reference beam and an object beam. After the object beam is passed through the sample, it is merged with the reference beam. This merge creates an interference pattern which is then captured with a digital sensor. Computer algorithms can then convert the interference pattern into a holographic image based on the light phase shifting properties of the sample. Phase shifts are not only visualized but also quantified. This new microscopy technique is called "quantitative" phase (contrast) microscopy, to distinguish it from non-quantitative phase contrast microscopy. Contrary to conventional phase contrast microscopy, this improved technology has the ability to give both quantitative data and beautiful images, transforming phase contrast microscopy into a tool for objective measurements. This can include the following analyses of actively growing cells: accurate cell counting, confluence studies, wound healing analysis, migration measurements and a vast array of morphological analyses.

 

Digital Holography
Live Cell Movie

 

Highly advanced analysis software is integral with every M4 unit. The raw holographic images contain cellular-based information such as area, volume, perimeter length, roughness, irregularity, eccentricity, and position. With this software, images can be quickly viewed and manipulated in 2D or 3D, and with different colors applied to bring out different details (such as refractive “height” pictured above). The HoloMonitor software allows for a host of manipulations of the data to quantify changes in cell shape in three dimensions, or perform cell counts and spatial tracking assays (see some of these below) to name just a few applications. In addition, the user always has complete access to the raw data (tabbed output) which can mined for different phenomena months or years in the future.

 

M4 2D Image M4 3D Image M4 3D Image closeup

 

Auto focus without moving parts? It’s not magic

When creating a focused image from a hologram, the holographic software creates images at different focal distances and selects the focal distance in which the cells are in focus. This allows a holographic microscope to compensate for focus drift, without any mechanical movement which can lead to premature failure of other “incubator”-based microscopes. When a drift occurs, a slightly different focal distance will be selected when creating the viewable image. Holographic images can in fact be refocused at any time after the actual recording, as the entire field of view is captured in each “snap shot”.

 

Image processing with reliable quantification (assisted segmentation)

Segmentation of Adherent CellsConventional phase contrast images of unstained living cells are notoriously difficult for computers to process. This is based upon the refractive index differences between different areas of your sample. However, the HoloMonitor software accounts for these differences, and proven image processing algorithms readily identifies such cells in phase shift images (see the segmentation to the left). This enables cell culture analysis to be automated, which is the foundation of the HoloMonitor products.

 

 

 

 

 

Your cells stay pristine during time-lapse imaging

Time Lapse Imaging of Dividing CellsThe HoloMonitor’s low-light and label-free analysis ensures that artifacts caused by phytotoxicity are kept at an absolute minimum to provide non-invasive, long-term, live cell experiments. During the few milliseconds it takes to capture an image frame, the observed cells are illuminated with an unfocused, low power laser beam. Contrary to conventional illumination, visual lasers generate no heat radiation. And because cells are imaged unstained, cells under observation absorb extremely low levels of energy which avoids phytotoxicity, or chemically-induced effects from the stains themselves. A typical time-lapse video, recorded and processed by the M4 to extract cytometric data is shown to the right (multiple L929 cells divisions). An example of a wound healing time-lapse assay using L929 cells can be dowloaded (126MB) here.

 

 

 

Cell morphology analsysi and visualizaion - quantify in 3D

Measuring Cells in 3D

The sophisticated image processing capabilities of the M4 enables extensive morphological measurement and analysis of unstained adherent cells. A multitude of label-free morphological parameters, such as cell volume, area, height (thickness), contour length, shape, texture and density, are measured for each imaged cell. This data is easily exported as an Excel file.

 

With quantitative phase microscopy images, the pixel intensity corresponds to the variations in optical thickness of unstained cells. This allows for cell images to be digitally enhanced, colored and displayed in three dimensions to create vivid static images or time-lapse videos. Dying cells are identified by their rough texture and loss of contrast. When a cell is dying, cell culture media enters the cell through the punctured cell membrane. This causes a gradual redu­tion in the cells optical density. When the cells optical density reaches the optical density of the surrounding cell culture media it will no longer be visible.

 

Optical ThicknessIn the image to the right, the optical height, or thickness, of a hypothetical spherical cell (top image ) displays as a smooth peak in 3D quantitative phase contrast images (bottom image). "h" is the physical height of the cell at an image point, "nc" is the optical density of the cell and "nm" is the optical density of the surrounding cell culture media.

 

The displayed shape of an adherent cell — which does not curve inwards as a spherical cell does — is much closer to the actual physical shape. However, it should always be kept in mind that the height of an image point depends on physical height and the optical density of the cell in the image point. The HoloMonitor software allows you to adjust the refractive index for different types of cells and media will enable you to get the most accurate images.

 

The morphology data is easily plotted in histograms and scatter pots such as the examples below

 

Cell Thickness Comparison

Morphology changes of etoposide treated L929 cells

Untreaded L929 cells (blue)consist of a large group of cells in the G1 phase and a smaller group of contracted cells preparing for cell division (red). Treated cells (blue) exhibit a very diferent morphology profile. Nearly all cells have either contracted into a dense, early stage, apoptotic cell or into a less dense, late stage, apoptotic cell.

 

 

 

 

 

 

 

 

 

The HoloMonitor software allows for cell morphology measurements to be plotted as histograms, interactive scatter plots (such as the plot below of cell area (Y-axis) versus cell thickness (X-axis)), or exported to Microsoft Excel or OpenOffice for further processing. When hovering over a data point in the interactive scatter plots (below screenshot), the corresponding cell image will be identified and displayed for visual inspection. Cell subpopulations may be identified and analyzed by interactively defining scatter plot regions as well.

 

M4 HoloMonitor Scatter Plot

 

 

Wound healing and cell migration assays

Cell migration can be defined as the movement of cells from one location to another in response to biological/chemical signals or environmental events. Migration of cells plays an essential role in many normal biological processes. Therefore, it is not surprising that impaired migration can contribute to pathological conditions in many disease areas like immune responses, tumor metastasis and angiogenesis. Wound healing assays mimic in vivo cell migration. They are in vitro assays widely used in all kind of research related to directed cell migration.

 

The HoloMonitor wound healing assay reveals data not only on a cell population level in terms of gap closure, but also on an individual cell level. Cells at the edge of the gap can be tracked and detailed and non-biased data on migration into the wound area are easily achieved. As all HoloMonitor applications, the wound healing assay is cell-friendly and based on label-free live-cell imaging. 

 

Wound Healing

 

Cell counting and confluence

Time Lapse Imaging of Dividing Cells

 

The included HoloStudio software can count cells and measure cell confluence by analyzing a sufficient number of randomly selected images of the cell culture. It takes less than a minute to count a culture with live adherent cells. Best of all, no trypsin treatment or staining is necessary. Simply place the HoloMontior M4 in the incubator as the cells are growing in their normal cell culture flask, and the software will do the rest. It’s that easy!

 

 

 

 

 

 

 

 

 

 

Spatial and Morphological Cell Tracking

The included HoloStudio spatial cell tracker can track cell speed and the direction of individually selected cells, allowing cell motility and migration studies. Cell movements can either be plotted in a spatial plot or overlaid with a high degree of certainty.

 

 

Overlay of chemotaxis cell tracks created by the HoloMonitor from Phase Holographic Imaging Plot of chemotaxis cell tracks created by the HoloMonitor from Phase Holographic Imaging
a
b
c

HT-1080 cells were grown in a gel matrix on a µ-Slide Chemotaxis 3D slide from IBIDI. The cells were imaged once every 5 minutes for 16 hours. Bovine serum was used to stimulate cell motion.

(a) Time-lapse video. Cells are artificially colored to show the optical thickness. Purple cells are optically thicker.

(b) Overlay of cell tracks. Lighter cells are opti­cally thicker.

(c) Spatial plot of cell movement.

SPECIFICATIONS
Type Holographic transmission microscopy
Objective 20x holographic
Stage Fixed
Field of View 0.25 mm x 0.25 mm
Actual Image Size 567 x 567 µm
Working distance 0.5 - 2 mm
Lateral Resolution 1 µm
Digital Focusing Range 1.5 mm
Exposure Time 5 ms, non scanning
Light Source External red laser unit (635nm)
Sample illumination 0.2 mW/cm²
Image Size 1024×1024 pixel
Optional XYZ Automated Stage Travel 100 x 70 x 10 mm
Optional XYZ Automated Stage Repeatability 5 µm
Cells Mono-layer of adherent eukaryotic cells in any of the supported cell culture vessels.
Vessel Compatibility 6-well, 24-well, 96-well, IBIDI µ-slide, microscopy slide and 35mm Petri dish
Computer Minimum Requirements Windows 10 or higher, 64-bit, 8 GB RAM, Intel core i7 and full HD display (1920 × 1080) or higher
Operating Environment 10 – 40°C, Up to 95% humidity
Power Requirements 100 – 249 VAC, 47 – 63 Hz
Weight
3.9 kg (5.5kg w/ motorized stage)
Unit Size
250×160×180 mm (260×200×180 mm w/ motorized stage)
Warranty 1 year parts and labor


Ordering

ITEM #
SIZE
DESCRIPTION
PRICE
BUY
PHM4020
1
HoloMonitor M4 Image Analysis System w/ Morphological Tracking and Analysis Software
Inquire
PHM4030
1
HoloMonitor M4Image Analysis System + XYZ Mortorized Stage w/ Morphological Tracking and Analysis Software
Inquire
PHM6060
1
Optional Upgrade to XYZ Mororized Stage (Upgrade Perfrormed by Manufacturer)
Inquire