HoloMonitor M4 base unit incl. Hstudio software

Key Features

Quantifying over time is crucial for a full understanding of cell systems. HoloMonitor M4 allow cultured cells to be individually monitored and quantified with unrivaled temporal resolution.

  • Quantitative measurements
  • High temporal resolution
  • Label-free samples
  • Long-term evaluation
  • Motorized stage
  • Digital auto-focus
  • Reliable and cost-effective operation


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Quantitative measurements

Unlike conventional microscopy, holographic microscopy has the ability to measure cytometric parameters
such as optical cell volume and thickness. This enables Holo­Monitor M4 to automatically identify cells for
quantitative single-cell and population analysis.

High temporal resolution

Conventional cell analysis methods have poor temporal resolution.
Important cellular events are therefore easily overlooked.
Holo­Monitor M4 achieves high temporal resolution by recording and analyzing time-lapse image sequences.

Label-free samples

No staining or contrast agents are required to capture holographic phase images.
Cell cultures can be continuously monitored and analyzed in their natural environment,
before and after treatment, minute by minute and day after day, without any phototoxic effects.

Long-term evaluation

The incubator tolerant design makes HoloMonitor M4 especially well suited for long-term kinetic cellular analysis.
The low intensity, single wavelength, laser generates no heat and reduces the risk of photo-damage to an absolute minimum.

Motorized stage

HoloMonitor M4 may optionally be equipped with a motorized stage.
The high precision stage allows HoloMonitor M4 to record time-lapse movies at multiple locations, in parallel.
Sample locations may be within the same culture or in different cultures.
The stage control software is fully integrated in the proprietary Holo­Monitor software – HoloStudio.
After sample locations have been graphically programed, time-lapse movies will be automatically recorded at each location.

Digital auto-focus

HoloMonitor M4 records a sequence of holograms from which individual images are created,
digitally auto-focused and assembled into a time-lapse movie. Auto-focusing is achieved
by digitally creating images at various focal planes from the same hologram.
The focused image is selected from this image stack as the unfocused images are discarded.
This process makes HoloMonitor M4 insensitive to focus drift and allow images to refocused after they have been recorded.

Reliable and cost-effective operation

A new and innovative mechanical design together with an intuitive software interface makes
HoloMonitor M4 operation simple and reliable, unsurpassed by alternative systems.


Just like water waves, light waves have two principal characteristics; amplitude and phase. The amplitude corresponds to light intensity and is the height of the wave, measured from crest to trough. The phase measures, at specific location, whether a wave is currently at its crest, in its trough or anywhere in between.

Invisible cells

For a cell to be visible by the naked eye or in a normal microscope, the light arriving from the cell must have a different amplitude than the back­ground. Unfortunately, living cells are transparent and only change the amplitude of the illuminating light slightly, if at all.

The problem

To be observed in a normal light microscope, cells must therefore be invasively stained to absorb or emit light and through this have a different amplitude than the back­ground. An unstained living cell do, however, distort the light passing through the cell by phase shifting the light.

By using a special kind of microscope, the phase contrast microscope, phase shifts may be observed, making unstained cells visible. However, phase contrast micro­scopy does not have the ability to quantify phase shifts, only visualize them. This has limited the use of the phase contrast microscope to a visual tool only.

A 3D phase shift image of cells. The height of the cell and its color tone correspond to the phase shift, in a specific image point. The phase shift in turn is pro­por­tional to how much the light has slowed down when passing through the cell.

The solution

Modern computer technology has made it possible to both quantify phase shifts and visualize them in so called phase shift images. 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, the new quantitative counterpart has the ability to give both quantitative data and beautiful images, transforming phase contrast microscopy into a quantitative tool.

Quantitative phase microscopy can be achieved using several different techniques. The most commonly used technique is holographic micro­scopy, which is used in the HoloMonitor time-lapse cytometers.

Besides being able to create phase shift images, images created by holo­graphic microscopy are focused when viewed, not when re­cord­ed. This makes holo­graphic microscopy ideal for long-term observation of living cells, using time-lapse microscopy techniques. Unfocused images, caused by focus drift, are simply refocused at will by letting the computer software recreate the image.

External reading

An ordinary light microscope can only detect variations in light intensity. Translucent objects, like unstained living cells, therefore have very poor contrast when observed with an ordinary microscope. Living cells are more optically dense than the fluid cell culture media they are kept in. This makes it pos­sible to observe living cells with good contrast by using a special kind of light microscope that visualizes variations in optical density – the phase contrast microscope. However, to achieve contrast the phase contrast microscope creates visual artifacts. These artifacts make it difficult to computer process phase contrast images to extract quantitative information. Instead of creating artifacts, a quantitative phase microscope visualizes variations in optical density by displaying the optical thickness of an object in each image point.

Phase shift images

An illustration of the phase shift created by an adherent cell.
As living cells are more optically dense than their sur­round­ings, they reduce the speed of the light wave that passes through them. The speed difference creates a dent in the uniform wave front that illuminates the cells. A quantitative phase microscope quantifies the depth of the dent by measuring how much the phase of the light wave has been shifted when passing through the cells. The measured phase shift is displayed in a so called phase shift or phase image, where color or intensity variations represent the phase shift.

Optical thickness and volume

A spherical cell (top) and its optical thick­ness (bottom). The optical thickness is the height of the cell (h) multiplied by the difference in optical density between the cell (nc) and the surrounding cell culture media (nm).
The measured phase shift is proportional to the optical thickness. By integrating the optical thickness over the cell area, the optical volume of a cell can be calculated. The ability to measure optical cell volume is unique to quantitative phase microscopy and cannot be done with conventional microscopy. When a cell is dying, surrounding cell culture media enters the cell through the punctured cell membrane. This causes a gradual reduction in the cells optical density and optical volume. The health status of a cell culture can thus be assessed without stains by identifying cells with a gradual decrease in optical volume.
Holography is based on that light waves create inter­ference patterns just like water waves. A hologram is created by dividing the illuminating laser light in two beams. One beam illuminates the sample, the sample beam. The other beam bypasses the sample, the reference beam. By either reflection or transmission, the sample will make an imprint on the illuminating sample beam. To be able to record the imprint, the sample beam is rejoined with the reference beam. The resulting interference pattern is the hologram.


An illustration of the phase shift imprint created by an adherent cell.

Traditional holography

Traditionally holograms are recorded on a photographic plate. After develop­ment, the photographic plate is again illuminated with the reference beam. Amazingly the imprinted sample beam reappears. As the recreated sample beam is a perfect copy of the original, the 3-dimen­sional sample will visually appear as if it is physically present.


Digital holography

Modern image sensors allow holograms to be digitally recorded. Instead of physically recreating the imprinted sample beam and the final image, the image creation process is simulated by a computer.
A holographic microscope like the HoloMonitor differs from a tradi­tional microscope in that the illuminating light is split into a sample beam and a reference beam by a beam splitter (above). After the sample beam has illuminated the sample, it is re­joined with the reference beam by a beam combiner to create the hologram.


Digital lens

Another distinction from a traditional micro­scope is that a holographic micro­scope records the information needed to create image, not the image itself. The tradi­tional image creating lens is replaced by a com­puter algorithm – a digital lens.


Digital autofocus

The flexibility of a digital lens allow images to be refocused after they have been recorded. In a holo­graphic microscope, re-focusing to com­pensate for focus drift is entirely done in software. This is achieved by creating images at several focal planes. From this temporary stack of images, the best in focus image is automatically selected to produce the final holographic image. Alternatively, the focal distance may be manually set to focus on a plane of interest.


Quantitative phase imaging

The recorded hologram contains both intensity and phase information. A holo­graphic micro­scope therefore create two separate images, an ordinary bright field image and a phase shift image.
With good reason, most cell biologist view time-lapse video micro­scopy as compli­cated to setup, difficult to keep cells alive and frustratingly time-con­suming just to render some pretty pictures, without any hard data. Never­theless, to understand a living system is to understand how the funda­mental building blocks of life interact over time to form the system at its present state.


A time-lapse image sequence of an un­stain­ed DU145 cell under­going apoptosis induced by etoposid. The images and the video clip were captured using Holo­Monitor.

Semiconductor revolution

Semiconductor technology has over the past decade ad­vanced to a point where it enables affordable time-lapse microscopes for routine use, without the issues commonly associated with conventional time-lapse microscopy.    



  • State of the art digital image sensors are extremely light sensitive, reducing the amount of required light and consequently reducing the risk of photo­toxicity.
  • Semiconductor light sources dissipate very small amounts of heat, allowing time-lapse microscopes to be placed inside standard cell incubators.
  • Phase shift imaging techniques allow unstained cell populations to be imaged, tracked and automatically quantified for extended periods of time.
  • Microfluidic devices, adapted for microscopic observation, are rapidly becoming available to further enhance in vitro cell environment and push the limit of how long living cells can be kept for observation under the microscope.
  • The average computer hard disk has the capacity to store millions of images. This is sufficient storage capacity to record years of cellular dynamics with time-lapse microscopy.


Time-lapse cytometry

The HoloMonitor time-lapse cytometers utilize these technology advancements to allow time-lapse image sequences of cultured cells to be effortlessly recorded over long time periods. From recorded time-lapse sequences, the Holo­Monitor software aids the user to automatically extract individual cell data of a cell population. Individual cell data — cell count, cell morphology, cell velocity and cell division rate — can be used to achieve a range of applications, from a single time-lapse video recording.

At the beginning of the 20th century the image quality of the optical microscope had reached the quality of modern optical microscopes. However, it was still difficult to obtain high contrast images of living cells.

In 1932 Frits Zernike approached Carl Zeiss AG,the leading microscope manufacturer at the time, with a new type of microscope which improved the contrast of living cells. Zernike's own words from his Nobel Lecture (Zernike 1953) — I went in 1932 to the Zeiss Works in Jena to demon­strate. It was not received with such enthusiasm as I had expected. Worst of all was one of the oldest scientific associates, who said: «If this had any practical value, we would ourselves have invented it long ago».

Zeiss did eventually build a microscope based on Zernike's invention in 1936. His inven­tion, the phase contrast microscope, is today an everyday tool for all cell biologists. In 1943, the first time-lapse film imaged with the Zeiss phase contrast microscope was a sensation, showing cell division.

Two years after Zernike received his Nobel price, in 1953, Georges Nomarski published the theory for the second popular phase contrast microscopy method — differential interference contrast (DIC) microscopy. Just like Zernike's phase contrast, Nor­marski's phase contrast enhances the contrast of transparent objects. But, instead of high­lighting edges, the Normarski method achieves contrast by artificially creating shadows.





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