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Quantitative Imaging for Life Science: Obtaining Stable, Repeatable Results
“Is this simply an overexpression artifact?”
“Why do my images look different from day to day?”
“Why aren’t we seeing what the other lab reported seeing?”
Such questions still vex many biological researchers as they examine their fluorescence images. The ability to obtain repeatable results relies not only on adherence to tightly defined experimental protocols but on proper use of quantitative imaging techniques.
Even relatively subtle experimental variables like fluorescent marker age, antibody binding efficacy, and cell cycle phase can have an effect on dye uptake and protein expression level. A high-performance quantitative CCD camera system can be utilized to check experimental variables as well as to acquire data for analysis and publication. To verify appropriate experimental conditions, all control images must be measured quantitatively (refer to Figure 1) using the same CCD camera. |
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The analog-to-digital unit (ADU) count registered for each control image can be used to calculate the electron acquisition rate for the image, as shown in Figure 1. The system gain, which defines the relationship between the ADUs generated and the number of electrons collected on the CCD, is also required for this calculation. Any appreciable difference among the electron acquisition rates yielded by the control images indicates a change in one or more experimental variables. Researchers can then modify their experimental conditions and quantitatively compare all newly acquired control images.
The simple technique described above allows biological researchers to analyze and present data with far greater confidence. Having validated their control images, researchers can calculate the electron acquisition rate for each image acquired over the course of the experiment, thus producing a set of quantitative values suitable for direct comparison.
Naturally, the effectiveness of this technique depends on the quantitative stability of the imaging system. In addition to utilizing a digital camera that offers good quantum efficiency, all system gain settings should be calibrated and measured by the manufacturer — with the final test results being provided to the camera user. For this reason, every Photometrics CCD camera is shipped with a Certificate of Performance that lists the camera’s factory-measured system gain and several other key specifications, including system linearity (i.e., the degree to which the relative brightness of any two real-life objects is maintained in an image). The system gain, system linearity, and quantum efficiency of Photometrics CCD cameras all exhibit outstanding stability over time.
To learn more about Photometrics cameras, please visit:
www.photomet.com
Live-Cell Total Internal Reflectance (TIRF) Microscopy
One of the goals of modern microscopy is to correlate the spatial and temporal data gathering ability of fluorescence microscopy to the functional activity of biomechanical events. When imaging molecular interactions and signaling processes in space and time, camera sensitivity and the ability to acquire images at a high rate of speed can have an appreciable impact on the quality of results. Advanced CCD solutions from Photometrics have been demonstrated to produce significant and impressive results in this context.
Endocytosis is a phenomenon by which a cell takes up molecular matter via invaginations on the cell surface that pinch from the plasma membrane and then move inside the cell. Understanding the intricacies of endocytosis more completely is thus of crucial importance to cell biologists; related applications range from cell invasion of pathogens to uptake of hormones and growth factors into cells.
This application article describes the use of simultaneous, multiple-wavelength acquisition coupled with total internal reflection fluorescence microscopy (TIRFM) to successfully image actin and dynamin recruitment during the final steps of clathrin-mediated endocytosis, thereby revealing the sequence in which dynamin and actin proteins are recruited to clathrin-coated pits during the endocytic event.1
Since most of the key information about endocytosis occurs at the membrane surface of cells, an optical technique that can visualize these areas without fluorescence interference from the underlying cellular structure is favorable. In TIRFM, the illumination source imparts on the coverslip at a large angle (critical angle), resulting in the formation of an evanescent wave that illuminates the sample. The strength of the evanescent wave drops off exponentially with increasing distance from the coverslip interface, only exciting fluorochromes within 200 nm of the sample surface. This makes TIRFM a particularly powerful technique for studying endocytic events.2
Dynamic Multicolor Imaging of Clathrin-Mediated Endocytosis
Working with several colleagues in 2002, Dr. Wolfhard Almers, at Oregon Health and Sciences University’s Vollum Institute, used TIRFM and simultaneous multicolor imaging to visualize clathrin-mediated endocytosis and determine the order in which various proteins play a role in this process1.
The study temporally resolved the involvement of certain proteins in clathrin-mediated endocytosis. The timeline of the appearance of dynamin (a protein believed to be involved in severing the clathrin-coated pit from the plasma membrane) and actin (a common structural protein) during the internalization of a clathrin vesicle from the plasma membrane was measured. Since the events associated with endocytosis occur within microseconds, high quantum efficiency (QE), low noise, and fast readout times were critical considerations when choosing a CCD camera for this study.
Periodic frames from time-lapse acquisition show the presence of clathrin-DsRed molecules (in regions believed to be clathrin-coated pits) at the plasma membrane (see Figure 1). Internalization of the clathrin-coated pit was determined by a decrease in the intensity of the clathrin-DsRed signal (top row). Simultaneously, the appearance of dynamin1-EGFP (bottom row) was measured and found to localize at the clathrin-coated pit just prior to internalization.
The authors then simultaneously observed clathrin-DsRed (first row of panels) over time and found that the appearance of EGFP-actin peaked at the clathrin pit after the clathrin signal started to decrease (see Figure 2).
When both results are plotted on a graph relative to the scission point of the endocytic event, it is revealed that the appearance of dynamin peaks prior to the scission event, while the actin appearance peaks after the event (see Figure 3). This is consistent with dynamin1 playing a role in the pinching of clathrin-coated pits from the plasma membrane. The fact that actin peaks after this event suggests that its role is likely post-internalization. The authors theorize that the actin may actually help provide the force for movement into the cytosol.
This work has since been extended to encompass N-WASP and the Arp2/3 complex3. Recently, it has also demonstrated the role of cortactin in the scission process during endocytosis4. Using this equipment setup and TIRFM, the researchers plan to investigate and further detail other events and protein interactions near the plasma membrane, including exocytosis, the function of caveolae, and signal transduction in lipid rafts.
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Figure 1. Dynamin is recruited to the clathrin-coated pit and leaves the plasma membrane with the vesicle. These time sequences show images of a clathrin-coated structure (top row) and dynamin fluorescence (bottom row) under evanescent illumination. The dimming of the clathrin-coated structure is preceded by transient recruitment of dynamin.1
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Figure 2. Actin is recruited to the clathrin-coated pit and leaves the plasma membrane with the vesicle. The top row is a time sequence of clathrin–DsRed fluorescence imaged under evanescent illumination. The bottom row displays green fluorescence images of EGFP–actin taken under evanescent illumination at the same times as the images in the top row.1
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Figure 3. Due to the high temporal and spatial resolution afforded by this experimental setup, it was found that dynamin recruitment to the site of endocytosis peaks just prior to the endocytic event, whilst actin is recruited just after the endocytic event.1 |
Enabling Technologies
The researchers utilized a high-performance Cascade:512B electron-multiplying CCD (EMCCD) camera from Photometrics and an Optical Insights Dual-View (currently know as the MAG Biosystems™ DV2™) for simultaneous, dual-channel, fluorescence image acquisition. Cascade® cameras employ state-of-the-art detector technology that provides excellent QE across the visible spectrum as well as on-chip EM gain in order to boost signal levels for high-speed imaging.
The Cascade:512B combines the sensitivity of a back-illuminated EMCCD with the high-speed imaging capability of a frame-transfer device. With the detector’s 16-micron square pixels, subcellular structures labeled with GFP can be resolved quite easily. In addition, the camera can collect data continuously, since the photosensitive side of the EMCCD collects light while the stored image is being read out from underneath the permanent mask. When run in standard-mode operation at 10-MHz readout speed, the camera can collect data from 29 frames per second (fps) at full resolution to 300 fps and higher on binned subregions of the EMCCD.
The Cascade:512B also has an additional software-selectable readout speed (5 MHz) for use under conditions where fast frame readout is not as critical. The lower-noise readout performance at this slower speed enables higher signal-to-noise data collection. To minimize the dark noise that can accumulate during longer exposures, the camera is cooled to -30°C.
A newer member of the Cascade family of EMCCD cameras is the Cascade II:512. The Cascade II:512 combines the sensitivity of a back-illuminated, deeply cooled (-80°C) EMCCD with the speed of a frame-transfer device. This camera offers up to 92% QE, wide dynamic range (16-bit digitization), low dark noise, and high-speed readout in a single, versatile instrument. In standard-mode operation at 10 MHz, the Cascade II:512 can collect full-resolution images at 29 fps; adjacent pixels can be binned for even greater sensitivity and speed. For applications that require longer exposures and exceptionally low noise, the Cascade II:512 provides an additional software-selectable readout speed of 5 MHz. While this slower speed reduces the camera’s readout noise, cooling the EMCCD to -80°C minimizes the dark noise that can accumulate during longer exposures.
Additional Information
To learn more about Dr. Wolfhard Almers’ research, please visit:
www.ohsu.edu/vollum/faculty/almers
To learn more about the Cascade II:512, please visit:
www.photomet.com/pm_products/cascade_2_512.php
To learn more about the MAG Biosystems DV2, please visit:
www.magbiosystems.com/products/DV2.php
Citations
1. Merrifield, C.J., Feldman, M.E., Wan, L., and Almers, W. (2002). Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nature Cell Biology 4, 691-698.
2. Lambert, A. (2005). Microscopy is moving on. American Biotechnology Laboratory 23, 8-10.
3. Merrifield, C.J., Qualmann, B., Kessels, M.M., and Almers, W. (2004). Neural-Wiskott Aldrich Syndrome Protein (N-WASP) and the Arp2/3 complex are recruited to sites of clathrin-mediated endocytosis in cultured fibroblasts. European Journal of Cell Biology 83, 13-18.
4. Merrifield, C.J., Perrais, D., and Zenisek, D. (2005). Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell 121, 593-606.
CCD vs. CMOS
Which technology is best suited to your research?
Today's digital cameras, whether they are intended for scientific or nonscientific use, employ one of two fundamental types of solid-state sensor technologies: CCD (charge-coupled device) or CMOS (complementary metal-oxide semiconductor). Both CCD and CMOS image sensors feature an array of photosensitive elements (i.e., pixels). Incident photons are converted into electrons, which are accumulated and counted. The resultant count is directly related to the intensity or brightness of the photons that strike the pixels.
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CMOS Technology |
CCD Technology |
| CMOS sensors typically read the charge level right at the pixel. |
CCD sensors transport the electrons across the array and then read the charge level. |
| CMOS sensors offer greater flexibility. |
CCD sensors are more sensitive. |
| CMOS sensors, traditionally, are more susceptible to noise. |
CCD sensors provide high-quality, low-noise images. |
| Each pixel on a CMOS sensor has several transistors located next to it; some incoming photons hit these transistors instead of the photodiode. |
CCD sensor technology has been in production for a longer period of time than CMOS sensor technology. |
| CMOS cameras are typically are less expensive. |
CCD sensors are generally regarded as a more stable technology suitable for use in low-light-imaging cameras. |
For life science researchers, the choice comes down to the level of sensitivity versus cost. For pathology applications, or to acquire images suitable for publication, CMOS-based cameras offer good resolution at a lesser cost. For image analysis, or low-light-level applications, CCD cameras are the right choice.
Photometrics’ Interline CCD cameras from its CoolSNAP line offer a full range of options optimized to meet moderate to low light level requirements with high speed, high resolution results.
Designed for high resolution color imaging for image archiving, documentation, publication for the life sciences and many industrial applications, QImaging’s “Go” Series utilize CMOS sensor technology and offer USB 2.0 plug-n-play interfaces under Windows supported operating systems.
Partner Spotlight: QImaging
Retiga-EXL Camera with IEEE-1394b Speed with Low-Noise Readout
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QImaging’s Retiga-EXL is the world’s first 30MHz, 14-bit, cooled CCD camera available for the bio-imaging market. This new addition to QImaging’s popular Retiga™ product line offers the remarkable speed of Lightwire™ 800, an optimized version of the IEEE-1394b protocol. The camera utilizes a Sony® ICX285 interline sensor to provide megapixel resolution and excellent sensitivity.
The Retiga-EXL is ideally suited to many life science and industrial imaging applications, including real-time DIC, real-time spinning-disk confocal microscopy, cell-motility studies, biofilm imaging, particle tracking, semiconductor inspection, solar panel inspection, and high-speed, low-light, live-cell fluorescence imaging.
Lightwire 800 delivers 800Mb/s bandwidth capacity and enables the camera to read out 15 full-resolution frames per second when operated at 30MHz with 14-bit digitization. The camera can also provide 14-bit digital output at 20 and 10MHz, as well as 8-bit digitization at all three of these readout frequencies. Flexible binning and ROI capabilities further increase sensitivity and speed. Variable exposure times range from 10 microseconds to 17.9 minutes.
The Retiga-EXL offers low read noise (6.5e- at 10MHz), along with regulated cooling of the CCD down to 0°C. QCapture Suite software is included with every Retiga-EXL and the new camera can be run on Windows® and Mac operating systems. A tightly synchronized RGB filter option enables the Retiga-EXL to be utilized for quantitative, high-resolution color imaging. |
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Lightwire 800 bandwidth capacity and a low-read-noise implementation of Sony’s ICX285 make the Retiga-EXL the high-performance value leader in rapid image acquisition, user convenience, and instrument versatility.
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For more information regarding QImaging’s products, please visit QImaging at www.qimaging.com.
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