Nikon Instruments Inc.
1300 Walt Whitman Rd.
Melville, NY, 11747
Website: http://www.nikoninstruments.com
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New Instrumentation For Live Cell Imaging
Jennifer L. Peters, PhD
Figure 1. Components of a
typical live cell imaging system. Image courtesy of Michael Davidson, National High Magnetic Field Laboratory, Florida State University.Click
to enlarge. |
The cell is the basic unit that forms all living matter. Both simple and complex,
the cell orchestrates a stunning array of diverse biochemical processes, including
those that govern its own growth, division and survival.(1) Understanding
these cellular processes is paramount not only to understanding the cell, but
to the living organism as a whole, and is perhaps most important when such cellular
processes break down, causing disease and ultimately death of the organism.
While pioneering techniques in biochemistry and molecular biology have been responsible for many advances including sequencing the genomes of a variety of organisms, many questions concerning the structure and function of cells can only be answered by observing living cells. Extraordinary technical and scientific advances have been made in this arena as well. Beginning with the discovery of the first fluorescent protein in the jellyfish Aequorea Victoria in the early 1960's,(2) scientists have developed fluorescent proteins in an astounding array of colors that can be fused to virtually any protein in the cell, allowing its location and function to be studied.(3) The development of these cellular imaging tools has allowed scientists to attack problems of a complexity never before thought possible. However, as scientists ask biological questions of increasing complexity, the imaging tools used to answer these questions must also advance.
The observation and study of living cells present many challenges, not the least of which is keeping the cells alive. Not only are cells sensitive to temperature, pH and CO2 levels, they can be easily damaged by the very light needed for their observation, making live cell imaging a constant balance between sensitivity of the emitted fluorescence signal and phototoxicity. Add to that the challenge of keeping cells in focus, the need for multi-channel and multi-point automation sequences during long duration experiments, plus the requirements of spatial and temporal resolution, and meeting the instrumentation needs for live cell imaging becomes undeniably challenging. Consequently, the instrumentation needed for live cell imaging experiments can be very complex and typically consists of many components. A typical live cell imaging setup is shown in Figure 1.
Figure 2A. Image of fluorescent microspheres taken at the
indicated times with and without the use of the Perfect Focus System. Arrow indicates the addition of media to the sample.
B. Differential interference contrast (DIC) images of live cells taken at multiple stage positions, demonstrating the ability of the TE2000-PFS to maintain focus during experiments requiring XY movement of the stage. Courtesy of Dr. Alexey Khodjakov, Wadsworth Center, Albany, NY.Click
to enlarge. |
Researchers have been vexed by the tendency of cells to drift out of focus by
thermal and mechanical effects during long term experiments, requiring the researcher
to constantly readjust the focus. The TE2000 Perfect Focus system (Nikon Instruments,
Inc.) works by reflecting a semicircle of red light from a 770 nm LED off of the
coverslip and projecting that reflection onto a linear CCD sensor in order to
track the position of the coverslip by interference methods. By combining this
highly sensitive feedback system with accurate z axis control, focusing precision
of less than 1/3 the focal depth of the objective is easily achieved. The system
contains optical offset technology, which allows the researcher to focus at the
desired height above the coverslip while simultaneously tracking the focus of
the coverslip interface. This technology allows focus correction to be achieved
at a 5 ms sampling rate. Consequently, the instrument is insensitive to thermal
and mechanical drift, allowing live cell time-lapse imaging experiments of 72
hours or longer to be performed. Focus is maintained even during perfusion or
addition of media, (Figure 2A), and it is possible to change the XY position of
the microscope stage without losing focus (Figure 2B), making true 6D multi-point
imaging possible.
Long term live cell time-lapse imaging experiments are also facilitated by BioStation
product family (Nikon Instruments, Inc.), shown in Figure 3, which combine cell
incubation with imaging. The first of these products, BioStation IM, enables users
with minimal microscopy experience to conduct live cell imaging without a steep
learning curve. The unit allows consistent control of environmental factors including
temperature, humidity and gas concentration, while also providing phase contrast
and fluorescence images. Magnifications are from 20× to 80× and imaging
is via a 2 megapixel monochrome cooled camera incorporated into the system. Additional
features include X, Y and Z control, and a no focus drift design, which together
facilitate long duration, in focus, 6D imaging experiments.
A second product, BioStation CT, allows imaging experiments to be conducted without ever removing the cells from the incubator. Consisting of a standard sized tissue culture incubator with an inverted design microscope inside, BioStation CT can hold up to 30 flasks or well plates, which are moved between the microscope stage and the vessel rack via a robotic device. BioStation CT provides images from 2× to 40×: phase images with apodized phase contrast (APC) optics and fluorescence images with three color LED illumination. A "bird's eye" macro view allows the entire vessel to be viewed from above. Complete security is provided: users access only the samples for which they have clearance. Experimental results are reliably traced.
Figure 3A. BioStation CT (left) and
BioStation IM (right).
B. Fluorescence and phase contrast live cell timelapse imaging using BioStation
IM. Peroxisomes are labeled with GFP (green) and mitochondria are labeled
with DsRed (red). Courtesy of Michael Davidson, National High Magnetic Field
Laboratory, Florida State University. |
Perhaps the biggest challenge that researchers utilizing live cell imaging techniques
face is limiting phototoxicity and photodamage to cells while maximizing signal.
Since cells can be easily damaged by illuminating light, especially during fluorescence
observation, cell biologists and instrument designers alike live by the adage
"every photon counts," taking care to collect every resultant photon as efficiently
as possible, and thereby reducing the amount of illumination needed to generate
a detectable signal. These efforts have resulted in brighter fluorescence filters,
optics with remarkable transmission, and highly efficiently and sensitive quantitative
CCD cameras.
One approach for limiting light exposure during live cell confocal imaging is
utilized in the C1 CLEM system (Nikon Instruments, Inc.). CLEM, or Controlled
Light Exposure Microscopy, was developed by Erik Manders and his team at the University
of Amsterdam(5) and is licensed exclusively to Nikon. CLEM reduces
laser illumination by looking at the resulting fluorescence and using sub-microsecond
feedback to adjust the laser light incident on the sample accordingly. For example,
when there is no fluorescence signal at a particular pixel, it is not illuminated
for the remainder of the pixel period. Similarly, when the fluorescence emission
will saturate at a particular pixel, the illumination at that pixel is attenuated.
This results in significantly reduced photobleaching, and allows meaningful data
to be collected for a longer period. Additionally, phototoxicity and photo-damage
are also greatly reduced, increasing the length of time for which healthy cells
can be imaged.
Figure 4. Nikon’s LiveScan Swept
Field Confocal (SFC) microscope. |
Nikon's LiveScan Swept Field Confocal (SFC) works both as a high speed slit scanner
and a multi point field scanner. Various confocal apertures, including 3 pinhole
diameters and 4 slit sizes, allow the user to balance resolution and low phototoxicity
against sensitivity and speed. The LiveScan SFC works by illuminating the selected
apertures and scanning the image of those apertures over the specimen using galvo
and piezo driven mirrors. Emission photons are then de-scanned and focused through
a complementary array of apertures on the imaging side, and from there are re-scanned
onto the face of a highly sensitive CCD camera. This design allows incredibly
fast imaging speeds for dynamic measurements such as intracellular Ca2+:
frame rates upwards of 1000 fps can be achieved in slit modes with the appropriate
camera. Alternatively, pinhole settings allow imaging light sensitive samples
with axial resolution, high signal-to-noise levels and cell viability.
About the author
Dr. Jennifer L. Peters is the Advanced BioSystems Application Scientist at Nikon Instruments Inc., where she is actively involved in the continued development of many of Nikon's live cell imaging products. More information about live cell imaging is available from:
References
1. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P. Molecular biology of the cell. New York, New York: Garland Press, 2002.
2. Shimomura, O., Johnson, F.H. and Saiga, Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol. 59, 223-39 (1962).
3. Lippincott-Schwartz, J. and Patterson, G. Development and use of fluorescent protein markers in living cells. 300, 87-90 (2003).
4. http://www.microscopyu.com/articles/livecellimaging/automaticmicroscope.html
5. Hoebe, R., van Oven, C. and Manders, E. (2006) Focus on microscopy; http://www.focusonmicroscopy.org/2006/PDF/061_Hoebe.pdf
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