- Email: [email protected]
Simple and inexpensive hardware and software method to measure volume changes in Xenopus oocytes expressing aquaporins
Journal of Neuroscience Methods 161 (2007) 301–305
Short communication
Simple and inexpensive hardware and software method to measure volume changes in Xenopus oocytes expressing aquaporins Ricardo Dorr a,∗ , Marcelo Ozu a,b , Mario Parisi b a
Laboratorio de Biomembranas, Departamento de Fisiolog´ıa, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155 Piso 7, C1121ABG, Buenos Aires, Argentina b Unidad de Biomembranas, Universidad Favaloro, Argentina Received 26 September 2006; received in revised form 3 November 2006; accepted 10 November 2006
Abstract Water channels (aquaporins) family members have been identified in central nervous system cells. A classic method to measure membrane water permeability and its regulation is to capture and analyse images of Xenopus laevis oocytes expressing them. Laboratories dedicated to the analysis of motion images usually have powerful equipment valued in thousands of dollars. However, some scientists consider that new approaches are needed to reduce costs in scientific labs, especially in developing countries. The objective of this work is to share a very low-cost hardware and software setup based on a well-selected webcam, a hand-made adapter to a microscope and the use of free software to measure membrane water permeability in Xenopus oocytes. One of the main purposes of this setup is to maintain a high level of quality in images obtained at brief intervals (shorter than 70 ms). The presented setup helps to economize without sacrificing image analysis requirements. © 2006 Elsevier B.V. All rights reserved. Keywords: Image analysis; Webcam; Freeware; Xenopus oocyte; Water permeability; Aquaporin
1. Introduction Cellular water channels are known as aquaporins (AQPs), and some AQPs family members have been identified in central nervous system cells. For instance, the AQP1 was detected in the apical domain of the choroid plexus epithelial cells; AQP4 is abundantly expressed in astrocyte foot processes and ependymocytes facing capillaries and brain–cerebrospinal fluid interfaces, whereas AQP9 is localized in tanycytes and astrocytes processes (Lehmann et al., 2004). The use of hardware and software for image acquisition and analysis is a frequent activity in research laboratories studying membrane water permeability. Hundreds of papers have been published using the technique described by Zhang et al. (1990) based on volume changes in intact Xenopus laevis oocytes expressing AQPs. This methodology is con-
∗
Corresponding author. Tel.: +54 11 4964 0503. E-mail address: [email protected] (R. Dorr).
0165-0270/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2006.11.005
sidered a very valuable approach to identify AQPs from all sorts of living beings and to measure their biophysical properties. For these purposes, researchers rely on powerful equipment valued in thousands of dollars, generally provided by companies specialized in the development of hardware and software for image acquisition and analysis. However some scientists such as Tort et al. (2006) consider that new approaches are needed to reduce costs in scientific laboratories, especially in developing countries. Several scientific papers have contributed during the last few years with low cost solutions to different experimental necessities. In recent years, significant progress has been made in the development of digital systems for processing and handling large amounts of data at increasing speeds. Ordinary image-capturing devices have become more sophisticated and inexpensive (Fern´andez-Miyakawa et al., 2007). This is the case with webcams, a very popular device which has arisen in association with the increasing use of the Internet, the presence of high-speed connections and the advanced software for domestic or business videoconferences.
302
R. Dorr et al. / Journal of Neuroscience Methods 161 (2007) 301–305
Table 1 Web pages with interesting information on webcam adaptations Site
Author
Article
http://www.astrosurf.com/planetels/planetcam.htm
Jes´us R. S´anchez
http://www.pmdo.com/wintro.htm http://www.microscopy-uk.org.uk/mag/artmar02/dbscope.html http://microscopy-uk.net/mag/artapr02/dbscope2.html
Steve Chambers David Bull David Bull
http://www.microscopies.com/DOSSIERS/Magazine/Articles/ FIXATION%20EN%20EQUERRE1/ FIXATION%20EN%20EQUERRE.htm) http://home.no.net/jonbent/webcam1.html http://www.barrie-tao.com/microscope photo.html http://www.microscopy-uk.org.uk/mag/artsep02/jwvideo1.html
Jean Dexheimer
Observaci´on planetaria con webcam: m´etodo avanzado (in Spanish) Webcams Microscopy on a shoestring Microscopy on a shoestring. II. A home-made microscope adaptor for the Olympus C-960 digicam Une installation de macro- et de microphotographie num´erique peu coˆuteuse (in French)
http://www.modernmicroscopy.com/main.asp?article=52 http://www.nexusresearchgroup.com/research papers/nzms1999b.htm
Jan Hinsch C.D. Fenton and M. Fenton
Jon Bent Kristoffersen Digital Photography Hi-Mag John Walsh
Some webcam adaptations were made for scientific purposes, such as building a cost-effective gel documentation system (Goldmann et al., 2001), achieving automated quantitative measurement of movement in primates (Togasaki et al., 2005) and performing laparoscopic studies (Chung et al., 2005). The aim of this work is to share the necessary information to adapt a low-cost webcam (but with a proper sensor quality) to a zoom stereo microscope system, in order to calculate water permeability in X. laevis oocytes expressing AQPs by means of null-cost software. Information and ideas for this setup were collected from different sources. Adaptations were based on pioneer free distribution projects on the Internet, mainly for astronomical purposes, in which a webcam attached to a telescope is used for sky observation. Websites listed in Table 1 may be consulted for detailed examples of these assemblies and additional interesting webcam adaptations to microscopes. 2. Materials and methods 2.1. Hardware The project required a webcam with a colour CCD sensor capable of obtaining images with a real horizontal resolution not lower than 640 pixels and a vertical resolution of 480 pixels, and with a speed not lower than 15 frames per second (fps). An O’Rite Mc-350 webcam (O’Rite Technology Co. Ltd., Taiwan) priced at 70 US dollars at the moment of purchase was chosen, although other commercial webcams with similar characteristics were also available. The camera (with a colour CCD 1/4-in. image sensor size and 350,000 pixels by Sony, Japan) connects through a USB 1.0 interface to a personal computer (PC) working under Microsoft Windows operating system. It is possible to acquire images at a greater speed (up to 30 pictures per second thanks to hardware video codec) reducing the size of the image to a resolution of 352 × 288 or 176 × 144 pixels. The minimum illumination capable of solving with the default lenses is 5 Lux at f1.4 at 3000 K.
Web camera photography Digital microphotography Field videomicrography. Super-cheap movies through the microscope A webcam looking through the microscope Light photomicroscopy using an internet webcam digital camera
The selected zoom stereoscopic microscope was an Olympus model SZ4045 TR (Olympus Co., Japan), featuring a phototube adapter (PT) for video equipment positioning. 2.2. Mounting the webcam on the microscope The upper portion of the PT was removed, since it interfered with the image formation. A polypropylene tube (outer tube, 3/4-in. in diameter and 1.5 cm long) was affixed to the PT by means of a Teflon platform with half pass screws (ISO metric fitting thread M3 × 14 mm), using the nuts of the PT. The webcam lens was also removed in order to expose the CCD. Another polypropylene tube (inner tube, 1/2-in. in diameter and 2.5 cm long) was affixed to the space left by the lens. The inner tube fitted perfectly to the outer tube preventing the entrance of light. The inner tube could be longitudinally moved along the outer tube to set the proper working distance of the webcam mounted on the microscope. The Teflon platform ensures stability and total darkness along the optic way from the PT to the webcam CCD. Once the optimal working distance was determined both tubes were fixed together using three ordinary screws equidistantly placed on the outer tube. Fig. 1 shows the final setup. The entire assembly was hand-made and all the materials were inexpensive and available in ordinary shops. Although the webcam mounted on the PT is more comfortable for the worker, the adapter can also be fitted to the tube of any eyepiece. 2.3. Software Original webcam drivers were installed according to the manufacturer’s instructions. All the software used to control the webcam, acquire images and analyze data is freeware and downloadable from public Internet sites. AMCap, written by N¨oel Danjou (http://www.noeld.com/ programs.asp?cat=video#AMCap), allows capturing video in a simple form, picking up the image signal from any video device connected to the PC. Both video (with the possibility of including sound) and static images captures can be obtained with
R. Dorr et al. / Journal of Neuroscience Methods 161 (2007) 301–305
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Fig. 1. Detailed setup assembly. Left: the webcam attached to the zoom stereo microscope system. Right: the outer polypropylene tube mounted on the Teflon platform. Four screws affix the Teflon piece to the phototube. The webcam with the attached inner tube slides along the outer tube and the three equidistant screws fix it in the working position.
this software. AMCap allows pre-visualization of images and supports the use of several monitors at the same time. It is a compatible application with Microsoft DirectShow (denominated ActiveMovie formally; hence the name of the program). It has support for compression of video with codecs used in Microsoft Windows Media 9 and it requires the installation of Microsoft DirectX 9.0 for full operation. Codecs and DirectX are free to download from the Microsoft web site (www.microsoft.com). For the method presented here, the video had a resolution of 640 × 480 pixels, which allows to obtain 15 consecutive frames per second. Video images were captured in RGB24 bit (True colour) format without compression, which represents the greatest quality offered by the device. VirtualDub software (http://www.virtualdub.org) was used to extract individual images from the video file in BMP format; it is a video capture and processing utility optimized for fast linear operations. Other valuable software applications for image analysis were Scion Image (Scion Corporation; http://www.scioncorp.com/) and ImageJ (developed by Wayne Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, USA; http://rsb.info.nih.gov/ij/). They support standard image processing functions such as contrast manipulation, sharpening, smoothing, edge detection and median filtering. Both allow to apply arithmetic functions to images, to calculate areas from a selected region, to measure distances and angles and to create density histograms. Simple but interesting software for edge detection is Canny Edge Detector, developed by Nikos Papamarkos (Democritus University of Thrace of Greece; http://ipml.ee. duth.gr/∼papamark/). This software applies the Canny edge detection algorithm (known as an optimal image edge detector) in a user-friendly interface allowing the selection of sigma and high and low threshold values. If necessary, uncompressed video files in AVI format could be compressed using the software AVI2MPG2 version 1.24 beta, developed by Brent Beyeler (http://members.cox. net/beyeler/bbmpeg.html). These files can be opened with MPEG-2 Upgrade for VirtualDub (http://fcchandler.home. comcast.net/stable/index.html).
2.4. Oocytes isolation Adult female X. laevis were kept in tanks containing filtered water and were fed twice a week. For surgery, specimens were anesthetized by hypothermia induced by placing the animal in ice (20–25 min). A 1-cm incision was made in the abdominal wall, and a lobe of ovary containing oocytes was excised. The piece was rinsed several times with OR-2 solution (for solutions compositions see below) until the solution was clear. The oocytes were separated in small groups (10–20 oocytes per group) with fine forceps, washed several times with OR-2 solution and incubated with 20 ml of OR-2 solution plus 2 mg/ml of Colagenase A for 1 h at 18 ◦ C with gentle shaking. Cells were then washed five times with OR-2 solution plus Bovine Serum Albumin (BSA) (0.1 g/100 ml) and incubated with 50 ml of a high potassium solution allowing gentle shaking for 1 h. Oocytes were passed through a plastic pipette every 15 min to facilitate their separation. They were then washed five times with Barth’s solution plus BSA (0.1g/100 ml) and finally placed overnight at 18 ◦ C in Barth’s containing gentamicine (1 g/ml). Stage VI cells were selected according to the classification established by Dumont (1972). 2.5. Plasmid construction, in vitro synthesis and translation The complete coding regions of the full length AQP1 clone were inserted into the EcoRI and XhoI sites on both ends of a pSP64T derived Bluescript vector carrying 5 and 3 untranslated sequences of a -globin gene from X. laevis (Abrami et al., 1994). Capped complementary RNAs (Daniels et al., 1994) were synthesized in vitro using T3 RNA polymerase kit and purified as described by Preston et al. (1992). Synthesized products were suspended in RNAse-free water at a final concentration of 1 g/l and stored at −20 ◦ C until microinjection. 2.6. cRNA microinjection Oocytes were microinjected with 50 nL AQP1-cRNA 24 h after isolation, using an automatic injector (Drummond Sci-
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entific Co, Broomall, PA, USA). Injected oocytes were incubated at 18 ◦ C in Barth’s solution supplemented with 1 g/ml gentamicine 24 h before water permeability measurements. 2.7. Permeability measurements To calculate the osmotic permeability coefficient of water (Pf ), AQP1-cRNA or water injected oocytes were maintained in isotonic Barth’s solution before experiments. The hypotonic challenge was done replacing the total volume of the isotonic Barth’s solution (200 mOsm) by a hypotonic one (50 mOsm) in the chamber where the oocyte lays. Pf (cm/s) was estimated from the function Pf = JW / VW A Osm, where JW is the volume of water (in l) transferred
in the unit of time (s) under an initial osmotic gradient Osm (mol/cm3 ), VW is the partial molar volume of water (18 ml/mol) and A is the observed area of the oocyte (mm2 ) at the beginning of the media change. JW values were obtained from the slope of the volume versus time function. Two orthogonal radios were measured from the images and averaged to estimate the volume and the area of the cell. Pf was calculated for each experiment with the data of volume and area obtained from the corresponding video. 2.8. Solutions Barth’s solution (in mM): NaCl 88, KCl 1, NaHCO3 2.4, HEPES 10, Ca(NO3 )2 –4H2 O 0.33, CaCl2 –2H2 O 0.41, MgSO4 –7H2 O 0.82, pH 7.4; high K+ solution: 100 mM
Fig. 2. Image analysis. First row: two 640 × 480 pixels images extracted as singles frames with VirtualDub in BMP format from the AVI files captured with AMCap software. The images of a Xenopus laevis oocyte expressing AQP1 were taken before and after a hypotonic challenge during a typical osmotic experiment. Second row: the edges of the images were calculated using the Canny Edge Detector program. Third row, left: the obtained edges were superimposed using the Scion Image or ImageJ software. This procedure clearly shows how the oocyte volume increases during the experiment. Edges were coloured to emphasize differences. Third row, right: the oocyte diameter (assuming spherical geometry) was indistinctly measured with Scion Image or ImageJ software to calculate volume. A typical “volume vs. time” plot is shown. JW value is represented by the slope of the curve and it is used to calculate the water permeability value. Time 0 represents the beginning of the hypotonic shock.
R. Dorr et al. / Journal of Neuroscience Methods 161 (2007) 301–305
K2 HPO4 –3H2 O, BSA (0.1 g/100 ml), pH 6.5; OR-2 solution (in mM): NaCl 82.5, KCl 2, HEPES 5, MgCl2 –6H2 O 1, pH 7.5. 2.9. Statistical analysis Data obtained from independent experiments with different oocytes were presented as mean ± S.E.M.
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authors wish to thank scientists from the Laboratorio de Biomembranas for critically reading an earlier version of this manuscript and for their valuable suggestions. Mention of trade names or commercial products in the present article is exclusively for providing specific information and does not imply any recommendation. References
3. Results and discussion Fig. 2 shows the results obtained with the webcam and the free software setup. Pf values obtained with this equipment ((33.4 ± 2.6) × 10−3 cm/s, n = 7) are equivalent to data calculated with traditional devices (Agre et al., 1993). The image capture rate provided good time resolution, which could be adjusted if necessary. Data could be collected for long periods of time, the only limitation being the computer storage space. Technically, webcams are devices of low static yield but with high dynamic effectiveness, since isolated photograms sacrifice high quality in order to maintain a high transference speed to the computer. Nonetheless, although webcams do not reach the highest resolution of other professional video devices, with a frame rate of 15 fps, a resolution of 640 × 480 pixels and a sensitivity of 5 lux at f1.4 (3000 K), specifications of the selected camera were satisfactory to obtain motion images of an appropriate quality. Their luminous sensitivity and colour precision can also be considered acceptable. When selecting an adequate model, it is advisable to prioritise the characteristics of the sensor rather than the quality of lenses, which are not used. The webcam option proved to be not only cheap but also efficient and easy to install and maintain. It must be emphasized that the continuous development of personal computers and their peripherals will result in cheaper devices with improved quality in the near future. Acknowledgements This work was supported by grants from Universidad Nacional de Buenos Aires and Fundaci´on Antorchas. The
Abrami L, Simon M, Rousselet G, Berthonaud V, Buhler JM, Ripoche P. Sequence and functional expression of an amphibian water channel, FA-CHIP: a new member of the MIP family. Biochim Biophys Acta 1994;1192(1):147–51. Agre P, Preston GM, Smith BL, Jung JS, Raina S, Moon C, Guggino WB, Nielsen S. Aquaporin CHIP: the archetypal molecular water channel. Am J Physiol 1993;265:F463–76. Chung SY, Landsittel D, Chon CH, Ng CS, Fuchs GJ. Laparoscopic skills training using a webcam trainer. J Urol 2005;173:180–3. Daniels MJ, Mirkov TE, Chrispeels MJ. The plasma membrane of Arabidopsis thaliana contains a mercury-insensitive aquaporin that is a homolog of the tonoplast water channel protein TIP. Plant Physiol 1994;106(4):1325– 33. Dumont JN. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J Morphol 1972;136:153–80. Fern´andez-Miyakawa ME, Dorr R, Fern´andez LE, Uzal FA, Ibarra C. Simple multichannel system for the measurement of the net water flux across biological tissues. Comput Methods Programs Biomed 2007;85(2): 95–100. Goldmann T, Zyzik A, Loeschke S, Lindsay W, Vollmer E. Cost-effective gel documentation using a web-cam. J Biochem Biophys Methods 2001;50:91–5. Lehmann GL, Gradilone SA, Marinelli RA. Aquaporin water channels in central nervous system. Curr Neurovasc Res 2004;1(4):293–303. Preston GM, Carroll TP, Guggino WB, Agre P. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 1992;256:385–7. Togasaki DM, Hsu A, Samant M, Farzan B, DeLanney LE, Langston JW, Di Monte DA, Quik M. The webcam system: a simple, automated, computerbased video system for quantitative measurement of movement in nonhuman primates. J Neurosci Methods 2005;145:159–66. Tort AB, Neto WP, Amaral OB, Kazlauckas V, Souza DO, Lara DR. A simple webcam-based approach for the measurement of rodent locomotion and other behavioural parameters. J Neurosci Methods 2006;157(1):91–7. Zhang R, Logee K, Verkman AS. Expression of mRNA coding for kidney and red cell water channels in Xenopus oocytes. J Biol Chem 1990;265:15375–8.
Short communication
Simple and inexpensive hardware and software method to measure volume changes in Xenopus oocytes expressing aquaporins Ricardo Dorr a,∗ , Marcelo Ozu a,b , Mario Parisi b a
Laboratorio de Biomembranas, Departamento de Fisiolog´ıa, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155 Piso 7, C1121ABG, Buenos Aires, Argentina b Unidad de Biomembranas, Universidad Favaloro, Argentina Received 26 September 2006; received in revised form 3 November 2006; accepted 10 November 2006
Abstract Water channels (aquaporins) family members have been identified in central nervous system cells. A classic method to measure membrane water permeability and its regulation is to capture and analyse images of Xenopus laevis oocytes expressing them. Laboratories dedicated to the analysis of motion images usually have powerful equipment valued in thousands of dollars. However, some scientists consider that new approaches are needed to reduce costs in scientific labs, especially in developing countries. The objective of this work is to share a very low-cost hardware and software setup based on a well-selected webcam, a hand-made adapter to a microscope and the use of free software to measure membrane water permeability in Xenopus oocytes. One of the main purposes of this setup is to maintain a high level of quality in images obtained at brief intervals (shorter than 70 ms). The presented setup helps to economize without sacrificing image analysis requirements. © 2006 Elsevier B.V. All rights reserved. Keywords: Image analysis; Webcam; Freeware; Xenopus oocyte; Water permeability; Aquaporin
1. Introduction Cellular water channels are known as aquaporins (AQPs), and some AQPs family members have been identified in central nervous system cells. For instance, the AQP1 was detected in the apical domain of the choroid plexus epithelial cells; AQP4 is abundantly expressed in astrocyte foot processes and ependymocytes facing capillaries and brain–cerebrospinal fluid interfaces, whereas AQP9 is localized in tanycytes and astrocytes processes (Lehmann et al., 2004). The use of hardware and software for image acquisition and analysis is a frequent activity in research laboratories studying membrane water permeability. Hundreds of papers have been published using the technique described by Zhang et al. (1990) based on volume changes in intact Xenopus laevis oocytes expressing AQPs. This methodology is con-
∗
Corresponding author. Tel.: +54 11 4964 0503. E-mail address: [email protected] (R. Dorr).
0165-0270/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2006.11.005
sidered a very valuable approach to identify AQPs from all sorts of living beings and to measure their biophysical properties. For these purposes, researchers rely on powerful equipment valued in thousands of dollars, generally provided by companies specialized in the development of hardware and software for image acquisition and analysis. However some scientists such as Tort et al. (2006) consider that new approaches are needed to reduce costs in scientific laboratories, especially in developing countries. Several scientific papers have contributed during the last few years with low cost solutions to different experimental necessities. In recent years, significant progress has been made in the development of digital systems for processing and handling large amounts of data at increasing speeds. Ordinary image-capturing devices have become more sophisticated and inexpensive (Fern´andez-Miyakawa et al., 2007). This is the case with webcams, a very popular device which has arisen in association with the increasing use of the Internet, the presence of high-speed connections and the advanced software for domestic or business videoconferences.
302
R. Dorr et al. / Journal of Neuroscience Methods 161 (2007) 301–305
Table 1 Web pages with interesting information on webcam adaptations Site
Author
Article
http://www.astrosurf.com/planetels/planetcam.htm
Jes´us R. S´anchez
http://www.pmdo.com/wintro.htm http://www.microscopy-uk.org.uk/mag/artmar02/dbscope.html http://microscopy-uk.net/mag/artapr02/dbscope2.html
Steve Chambers David Bull David Bull
http://www.microscopies.com/DOSSIERS/Magazine/Articles/ FIXATION%20EN%20EQUERRE1/ FIXATION%20EN%20EQUERRE.htm) http://home.no.net/jonbent/webcam1.html http://www.barrie-tao.com/microscope photo.html http://www.microscopy-uk.org.uk/mag/artsep02/jwvideo1.html
Jean Dexheimer
Observaci´on planetaria con webcam: m´etodo avanzado (in Spanish) Webcams Microscopy on a shoestring Microscopy on a shoestring. II. A home-made microscope adaptor for the Olympus C-960 digicam Une installation de macro- et de microphotographie num´erique peu coˆuteuse (in French)
http://www.modernmicroscopy.com/main.asp?article=52 http://www.nexusresearchgroup.com/research papers/nzms1999b.htm
Jan Hinsch C.D. Fenton and M. Fenton
Jon Bent Kristoffersen Digital Photography Hi-Mag John Walsh
Some webcam adaptations were made for scientific purposes, such as building a cost-effective gel documentation system (Goldmann et al., 2001), achieving automated quantitative measurement of movement in primates (Togasaki et al., 2005) and performing laparoscopic studies (Chung et al., 2005). The aim of this work is to share the necessary information to adapt a low-cost webcam (but with a proper sensor quality) to a zoom stereo microscope system, in order to calculate water permeability in X. laevis oocytes expressing AQPs by means of null-cost software. Information and ideas for this setup were collected from different sources. Adaptations were based on pioneer free distribution projects on the Internet, mainly for astronomical purposes, in which a webcam attached to a telescope is used for sky observation. Websites listed in Table 1 may be consulted for detailed examples of these assemblies and additional interesting webcam adaptations to microscopes. 2. Materials and methods 2.1. Hardware The project required a webcam with a colour CCD sensor capable of obtaining images with a real horizontal resolution not lower than 640 pixels and a vertical resolution of 480 pixels, and with a speed not lower than 15 frames per second (fps). An O’Rite Mc-350 webcam (O’Rite Technology Co. Ltd., Taiwan) priced at 70 US dollars at the moment of purchase was chosen, although other commercial webcams with similar characteristics were also available. The camera (with a colour CCD 1/4-in. image sensor size and 350,000 pixels by Sony, Japan) connects through a USB 1.0 interface to a personal computer (PC) working under Microsoft Windows operating system. It is possible to acquire images at a greater speed (up to 30 pictures per second thanks to hardware video codec) reducing the size of the image to a resolution of 352 × 288 or 176 × 144 pixels. The minimum illumination capable of solving with the default lenses is 5 Lux at f1.4 at 3000 K.
Web camera photography Digital microphotography Field videomicrography. Super-cheap movies through the microscope A webcam looking through the microscope Light photomicroscopy using an internet webcam digital camera
The selected zoom stereoscopic microscope was an Olympus model SZ4045 TR (Olympus Co., Japan), featuring a phototube adapter (PT) for video equipment positioning. 2.2. Mounting the webcam on the microscope The upper portion of the PT was removed, since it interfered with the image formation. A polypropylene tube (outer tube, 3/4-in. in diameter and 1.5 cm long) was affixed to the PT by means of a Teflon platform with half pass screws (ISO metric fitting thread M3 × 14 mm), using the nuts of the PT. The webcam lens was also removed in order to expose the CCD. Another polypropylene tube (inner tube, 1/2-in. in diameter and 2.5 cm long) was affixed to the space left by the lens. The inner tube fitted perfectly to the outer tube preventing the entrance of light. The inner tube could be longitudinally moved along the outer tube to set the proper working distance of the webcam mounted on the microscope. The Teflon platform ensures stability and total darkness along the optic way from the PT to the webcam CCD. Once the optimal working distance was determined both tubes were fixed together using three ordinary screws equidistantly placed on the outer tube. Fig. 1 shows the final setup. The entire assembly was hand-made and all the materials were inexpensive and available in ordinary shops. Although the webcam mounted on the PT is more comfortable for the worker, the adapter can also be fitted to the tube of any eyepiece. 2.3. Software Original webcam drivers were installed according to the manufacturer’s instructions. All the software used to control the webcam, acquire images and analyze data is freeware and downloadable from public Internet sites. AMCap, written by N¨oel Danjou (http://www.noeld.com/ programs.asp?cat=video#AMCap), allows capturing video in a simple form, picking up the image signal from any video device connected to the PC. Both video (with the possibility of including sound) and static images captures can be obtained with
R. Dorr et al. / Journal of Neuroscience Methods 161 (2007) 301–305
303
Fig. 1. Detailed setup assembly. Left: the webcam attached to the zoom stereo microscope system. Right: the outer polypropylene tube mounted on the Teflon platform. Four screws affix the Teflon piece to the phototube. The webcam with the attached inner tube slides along the outer tube and the three equidistant screws fix it in the working position.
this software. AMCap allows pre-visualization of images and supports the use of several monitors at the same time. It is a compatible application with Microsoft DirectShow (denominated ActiveMovie formally; hence the name of the program). It has support for compression of video with codecs used in Microsoft Windows Media 9 and it requires the installation of Microsoft DirectX 9.0 for full operation. Codecs and DirectX are free to download from the Microsoft web site (www.microsoft.com). For the method presented here, the video had a resolution of 640 × 480 pixels, which allows to obtain 15 consecutive frames per second. Video images were captured in RGB24 bit (True colour) format without compression, which represents the greatest quality offered by the device. VirtualDub software (http://www.virtualdub.org) was used to extract individual images from the video file in BMP format; it is a video capture and processing utility optimized for fast linear operations. Other valuable software applications for image analysis were Scion Image (Scion Corporation; http://www.scioncorp.com/) and ImageJ (developed by Wayne Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, USA; http://rsb.info.nih.gov/ij/). They support standard image processing functions such as contrast manipulation, sharpening, smoothing, edge detection and median filtering. Both allow to apply arithmetic functions to images, to calculate areas from a selected region, to measure distances and angles and to create density histograms. Simple but interesting software for edge detection is Canny Edge Detector, developed by Nikos Papamarkos (Democritus University of Thrace of Greece; http://ipml.ee. duth.gr/∼papamark/). This software applies the Canny edge detection algorithm (known as an optimal image edge detector) in a user-friendly interface allowing the selection of sigma and high and low threshold values. If necessary, uncompressed video files in AVI format could be compressed using the software AVI2MPG2 version 1.24 beta, developed by Brent Beyeler (http://members.cox. net/beyeler/bbmpeg.html). These files can be opened with MPEG-2 Upgrade for VirtualDub (http://fcchandler.home. comcast.net/stable/index.html).
2.4. Oocytes isolation Adult female X. laevis were kept in tanks containing filtered water and were fed twice a week. For surgery, specimens were anesthetized by hypothermia induced by placing the animal in ice (20–25 min). A 1-cm incision was made in the abdominal wall, and a lobe of ovary containing oocytes was excised. The piece was rinsed several times with OR-2 solution (for solutions compositions see below) until the solution was clear. The oocytes were separated in small groups (10–20 oocytes per group) with fine forceps, washed several times with OR-2 solution and incubated with 20 ml of OR-2 solution plus 2 mg/ml of Colagenase A for 1 h at 18 ◦ C with gentle shaking. Cells were then washed five times with OR-2 solution plus Bovine Serum Albumin (BSA) (0.1 g/100 ml) and incubated with 50 ml of a high potassium solution allowing gentle shaking for 1 h. Oocytes were passed through a plastic pipette every 15 min to facilitate their separation. They were then washed five times with Barth’s solution plus BSA (0.1g/100 ml) and finally placed overnight at 18 ◦ C in Barth’s containing gentamicine (1 g/ml). Stage VI cells were selected according to the classification established by Dumont (1972). 2.5. Plasmid construction, in vitro synthesis and translation The complete coding regions of the full length AQP1 clone were inserted into the EcoRI and XhoI sites on both ends of a pSP64T derived Bluescript vector carrying 5 and 3 untranslated sequences of a -globin gene from X. laevis (Abrami et al., 1994). Capped complementary RNAs (Daniels et al., 1994) were synthesized in vitro using T3 RNA polymerase kit and purified as described by Preston et al. (1992). Synthesized products were suspended in RNAse-free water at a final concentration of 1 g/l and stored at −20 ◦ C until microinjection. 2.6. cRNA microinjection Oocytes were microinjected with 50 nL AQP1-cRNA 24 h after isolation, using an automatic injector (Drummond Sci-
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entific Co, Broomall, PA, USA). Injected oocytes were incubated at 18 ◦ C in Barth’s solution supplemented with 1 g/ml gentamicine 24 h before water permeability measurements. 2.7. Permeability measurements To calculate the osmotic permeability coefficient of water (Pf ), AQP1-cRNA or water injected oocytes were maintained in isotonic Barth’s solution before experiments. The hypotonic challenge was done replacing the total volume of the isotonic Barth’s solution (200 mOsm) by a hypotonic one (50 mOsm) in the chamber where the oocyte lays. Pf (cm/s) was estimated from the function Pf = JW / VW A Osm, where JW is the volume of water (in l) transferred
in the unit of time (s) under an initial osmotic gradient Osm (mol/cm3 ), VW is the partial molar volume of water (18 ml/mol) and A is the observed area of the oocyte (mm2 ) at the beginning of the media change. JW values were obtained from the slope of the volume versus time function. Two orthogonal radios were measured from the images and averaged to estimate the volume and the area of the cell. Pf was calculated for each experiment with the data of volume and area obtained from the corresponding video. 2.8. Solutions Barth’s solution (in mM): NaCl 88, KCl 1, NaHCO3 2.4, HEPES 10, Ca(NO3 )2 –4H2 O 0.33, CaCl2 –2H2 O 0.41, MgSO4 –7H2 O 0.82, pH 7.4; high K+ solution: 100 mM
Fig. 2. Image analysis. First row: two 640 × 480 pixels images extracted as singles frames with VirtualDub in BMP format from the AVI files captured with AMCap software. The images of a Xenopus laevis oocyte expressing AQP1 were taken before and after a hypotonic challenge during a typical osmotic experiment. Second row: the edges of the images were calculated using the Canny Edge Detector program. Third row, left: the obtained edges were superimposed using the Scion Image or ImageJ software. This procedure clearly shows how the oocyte volume increases during the experiment. Edges were coloured to emphasize differences. Third row, right: the oocyte diameter (assuming spherical geometry) was indistinctly measured with Scion Image or ImageJ software to calculate volume. A typical “volume vs. time” plot is shown. JW value is represented by the slope of the curve and it is used to calculate the water permeability value. Time 0 represents the beginning of the hypotonic shock.
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K2 HPO4 –3H2 O, BSA (0.1 g/100 ml), pH 6.5; OR-2 solution (in mM): NaCl 82.5, KCl 2, HEPES 5, MgCl2 –6H2 O 1, pH 7.5. 2.9. Statistical analysis Data obtained from independent experiments with different oocytes were presented as mean ± S.E.M.
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authors wish to thank scientists from the Laboratorio de Biomembranas for critically reading an earlier version of this manuscript and for their valuable suggestions. Mention of trade names or commercial products in the present article is exclusively for providing specific information and does not imply any recommendation. References
3. Results and discussion Fig. 2 shows the results obtained with the webcam and the free software setup. Pf values obtained with this equipment ((33.4 ± 2.6) × 10−3 cm/s, n = 7) are equivalent to data calculated with traditional devices (Agre et al., 1993). The image capture rate provided good time resolution, which could be adjusted if necessary. Data could be collected for long periods of time, the only limitation being the computer storage space. Technically, webcams are devices of low static yield but with high dynamic effectiveness, since isolated photograms sacrifice high quality in order to maintain a high transference speed to the computer. Nonetheless, although webcams do not reach the highest resolution of other professional video devices, with a frame rate of 15 fps, a resolution of 640 × 480 pixels and a sensitivity of 5 lux at f1.4 (3000 K), specifications of the selected camera were satisfactory to obtain motion images of an appropriate quality. Their luminous sensitivity and colour precision can also be considered acceptable. When selecting an adequate model, it is advisable to prioritise the characteristics of the sensor rather than the quality of lenses, which are not used. The webcam option proved to be not only cheap but also efficient and easy to install and maintain. It must be emphasized that the continuous development of personal computers and their peripherals will result in cheaper devices with improved quality in the near future. Acknowledgements This work was supported by grants from Universidad Nacional de Buenos Aires and Fundaci´on Antorchas. The
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