by Tim Richardson
Figure 1. Detail picture of Richardson's Live Cell Imaging System (LCIS™) comprising a disposable microscope chamber slide, perfusion system and temperature-control module. |
Imaging cells in real timeImaging living samples of cells or other organisms in real time places new demands on the microscope, the chamber needed to support the sample and the microscopist. This article details recent efforts to answer the first two of these areas.
The value of a microscope's image depends on the integrity of the specimen, the fineness of the detail resolved, the ability of the system to detect and image features smaller than the resolution limit, and the presentation of the image information with adequate contrast, and in a readily-perceived form.
The Real Time Microscope (RTM) system, designed and developed by Richardson Technologies Inc. (RTI), provides easy access to images of living cells and organisms with maximum resolution, contrast and color fidelity. It causes minimum impact on the sample, and avoids the need for the normal techniques of fixation, staining or dehydration.
The basic principle behind the RTM is inverted dark-field contrast in an ultra-stable, ultra-clean microscope system with full color imaging capability. The RTM system provides an all-analog, all-optical set of techniques; it is not digitally enhanced microscopy. The digital portion of the system is used for image acquisition and for analysis, and in some cases for repositioning image data.
Light and energy management enhance resolutionThe RTM differs from standard upright microscopes in the following ways:microscope vibration is significantly reduced; infrared energy is removed from the illuminating light; ultraviolet energy is removed from the illuminating light; a condenser color filter wheel selects the most efficient wavelengths of light that will reach the sample; the sample is illuminated through a transmitted-light extreme-dark-field condenser, and stray light is reduced in the illuminating light. All of these modifications ensure that the illuminating beam contains as little damaging energy as possible. The illuminating light interacts with the sample producing image information, which is then conveyed to the camera. RTI has made the following additional changes to standard microscopes: stray light is reduced between the sample and the camera; a full-color, real-time camera is used to provide subtle color information; the fluorescence source is a 120-watt, pre-focused, long-life, modified mercury arc; and all optical components are cleaned in an ISO Class 7 clean room to reduce stray light and image artifacts.
RTI has found that this group of design, integration and precision assembly innovations are ideal for imaging living cell systems, and that they work synergistically to provide the resolution, contrast, and color fidelity.
The RTM microscope is combined with microscope hardware, image management software, and data acquisition and storage systems to store, analyze and edit images.
The high contrast achieved in the 'RTM Modes' has several origins. Because the sample is illuminated in extreme-dark-field, the normal RTM image is built only from photons which carry information about the sample. These are photons whose trajectories have been sufficiently altered by their interaction with the sample to enter the objective's cone of acceptance. This produces images of very high signal-to-noise ratio in which the signal is white against a dark black background. In addition, the 100 objective lens used on the RTM incorporates an adjustable diaphragm which offers novel possibilities for contrast enhancement. These high-contrast 'RTM-mode' images may also be combined with fluorescence images to produce valuable image data.
Invert black background to whiteIn addition to its imaging modes, the RTM contains novel display modes for its images, achieved by inverting the luminance, chrominance or both. When the luminance alone is inverted the black background of the image becomes bright white. Very fine features are displayed as fine black, subtly shaded or colored lines against a white background. These inverted images are similar in appearance to conventional bright-field images. In many cases, this inversion enables images to be more readily interpreted and for fine detail to be discerned. Subtle color shading, which is not apparent in the black background image, also becomes clearly visible in this display mode. Inversions are linear transformations. The image is not altered or thresholded, nor is its contrast stretched. The image data remain accurate and can be reverted to their original form by applying the same process in reverse. The luminance inverted image is color-correct, and because of its appearance is often described as 'color living Transmission Electron Microscope imaging'. RTI has just recently been granted a patent on these display modes.
The high image resolution and contrast of the RTM, and the 'true' color rendering of the sensitive three-CCD camera, together enable the RTM to operate as an efficient fluorescence microscope. The resolution in fluorescence is also equal to that of the RTM imaging modes, due to the vibration-dampening design and careful attention to stray light reduction and cleanliness.
The three-CCD camera presents the real colors as generated by fluorescence in the sample, and, as a result, may display much greater chemical detail in subtle color shading than is displayed in conventional systems. This detail, if observed by a monochrome camera, would not be visible, or it would overlie the desired image data.
Autofluorescence of untreated samples which are imaged by the RTM presents color data that may contain coded structural or functional information. RTM image-management software allows this wealth of new color data to be studied fully, particularly through isolation of the desired spectral signatures. This is particularly true for multi-color dye systems and for autofluorescence. Examples of such multi-color dye systems are the following: Acridine Orange- where the color of the emission depends on the state of the RNA and DNA to which the dye is bound; Nile Red- a lipid stain, where the color ranges from green through yellow to red depending on the type of lipid to which the dye is bound; and JC-1 and JC-9-, two mitochondrial probes which change color depending on the level of membrane potential in the dyes.
The RTM is able to present simultaneously the RTM-mode images along with transmitted-light fluorescence or epi-fluorescence images. Special filter cubes specifically designed for the RTM allow the transmitted-light image to be viewed simultaneously in more than one spectral region. This allows the transmitted-light image to appear on both the long- and short-pass sides of the dichroic and emission filters. For example, green light could be used to excite yellow and red fluorophores, while the transmitted-light RTM image could be positioned as 'purple' light- a mixture of red and blue. These images are very helpful in following cell morphology during experiments with fluorescent proteins, fluorescent dyes or autofluorescence.
View internal cell processes over time
The RTM operates with a variety of accessories and supplies specifically designed to enhance live cell imaging. This includes the Live Cell Imaging System (LCIS) which provides the ability to sustain and culture living cells in a closed chamber environment. The LCIS allows specimens to be maintained in a temperature-controlled environment while providing nourishment or challenge substances through an automated micro-perfusion system. LCIS performance is improved with use of ultra-clean live-cell chamber slides permitting provision of nutrients and removal of waste.
RTI has been successful at culturing HeLa - human cervical cancer; L6 - rat skeletal muscle; CIHA - human cervical cancer; AtR1 clone 3-3 from human A549 lung carcinoma; and BXPC-3 - human epithilial pancreatic cancer cells. These cell lines have grown and divided in the LCIS chambers for periods of time ranging from 24 to 90 hours. The following cells have survived longer than two days but so far division has not been observed: Panc-1 - human pancreatic cancer; H9C2 - rat cardiomyocytes; and Fibroblasts - human.
The LCIS provides an unprecedented ability to view the internal processes of cells and their response to their environments over time periods ranging from a few hours to more than three and a half days of continuous time lapse imaging. Challenge substances can be added to the LCIS and the response of the cells can be directly observed. This has the potential to greatly increase the speed with which substances can be tested, and it improves the level of understanding about the effects of the substances.
The RTM system, with the LCIS, combines well-established upright microscope design with materials, optical systems and procedures to provide high contrast, high resolution images of living cells, in surroundings more similar to their natural environments.
About the authorsTim Richardson is the founder, Chairman and Chief Scientific Officer of Richardson Technologies Inc (RTI). An accomplished microscopist, he holds numerous patents in this area.
More information about RTM microscopy and live cell imaging is available from:
Richardson Technologies Inc.,
888-494-4541,
www.richardson-tech.com