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.
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.
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.
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.
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.
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.
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.
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.
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
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
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.
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.
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.
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.
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.
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.
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.
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.