Name: real-time imaging of plant cell surface dynamics with variable-angle epifluorescence microscopy
Uploaded: 2015-12-12T14:00:00.000+00:00
Duration: 6 min 31 s
Description: Watch this Scientific Journal Video about Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy at JoVE.com

I am grateful to Dr. Masaru Fujimoto for his technical suggestions for VAEM. I am also grateful to Prof. Koh Iba and Dr. Mimi Hashimoto-Sugimoto for providing GFP-PATROL1 transgenic plants, and discussions about PATROL1. I thank Prof. Seiichiro Hasezawa for his continuing support of my work. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 25711017.

Fidan 1. Hazırlık <ol><li> Tohum sterilize edin. <ol><li> Ve 1 ul% 10 Triton X-100 500 ul steril su: 500 ul NaClO (% 5.0 mevcut klorin) eklenerek sterilizasyon solüsyonu hazırlayın. </li><li> Yeri yaklaşık 10 transgenik A. Bir 1.5 ml tüp içine GFP PATROL1 8 taşıyan thaliana tohumları. </li><li> 1 ml% 70 etanol solüsyonu ekleyin ve beş kez ters yüz edilerek iyice karıştırın. 1 dakika boyunca bekletin. </li><li> Tohumlar tüpün dibine çöker gözlemleyin. Temiz bir laminer akış kabininde, yavaşça bir mikropipet kullanılarak% 70 etanolü çıkarmak ve 1 ml sterilizasyon çözeltisi ilave edilir. Beş kez tersini İyice karıştırın ve 5 dakika bekletin. </li><li> Tohum yıkayın. Hala temiz bir bankta aseptik koşullarda çalışan, yavaşça bir mikropipet kullanarak çözüm kaldırmak ve 1 ml steril su ekleyin. Bu beş kez tekrarlayın. Sterilize edilmiş tohumlara, 2 gün boyunca 4 ° C&#39;de steril su içinde depolanabilir. </li></ol></li><li> Yani% 0.5 gellan zamkı katılaşmış 1/2 Murashige ve Skoog levha (pH 5.8) 9 sterilize edilmiş tohumlar, w. Cerrahi bant iki kat kullanarak plaka üzerine kapağı bantlayın. </li><li> Soğuk depolama odası O / N 4 ° C&#39;de karanlıkta plaka inkübe edin. </li><li> 100 umol m-2 sec -1 beyaz ışık kullanarak 12 saat / 12 saatlik bir ışık-karanlık çevrimi ile 23.5 ° C &#39;ye ayarlanmış bir büyütme odasında içine plaka aktarın ve 7 gün boyunca inkübe edilir. Uzun yaklaşık 1 mm kotiledonlarında fideler daha sonra hasat edilebilir. </li></ol> 2. Sky Drop Kotiledon Örnekler montajı NOT: VAEM gözlem numune hazırlama önemli bir faktör numune ve kapak cam arasındaki hava kabarcıkları dahil kaçınmaktır. Kabarcıklar ölçüde kırılma indeksi farklılıkları yol açarak VAEM görüntü kalitesini azaltır. Biz montaj &#39;gökyüzü bırak&#39; çağrısında bulundu basit bir yöntem, kabarcıkları önlemek için de kullanılabilirA arasında thaliana Kotiledonlar ve kapak cam. Bu gözlem hemen önce yapılmalıdır. <ol><li> 76: Bazal tampon [5 mM 2- (N-morfolino) -etansülfonik pH 6.5&#39;te asit tris (MES-Tris), 50 mM KCI, 100 uM CaCl2] Bir cam slayt ortasına (boyut 30 ul koyun × 26 mm, kalınlığı: 1.0-1.2 mm). </li><li> Diseksiyon makas kullanarak 7 günlük bir fide bir kotiledon çıkarın. Gözlem tarafı bazal tampon damla (Şekil 1, aşama 1) üzerinde yukarı bakacak şekilde kotiledon kaydırın. </li><li> Bir kapak cam ortasına bazal tampon 30 ul yerleştirin (boyut: 18 × 18 mm, kalınlığı: 0,12-0,17 mm) (Şekil 1, adım 2). Başaşağı hafifçe cam kapak çevirin. Yüzey gerilimi düşmesini tampon damla önleyecektir. Bir kenarında cımbızla cam kapak tutun. Tampon damla yaklaşık kotiledon üstünde olacak şekilde cam slayt karşı kenarını yerleştirin. </li><li>Bu kotiledon örnek (Şekil 1, aşama 3) üzerinde yer almaktadır, böylece sürgü hala, kapak camın kenarına ve hala cımbız ile karşı kenarını tutarken, kapak camı altında tamponu damla konumunu ayarlamak. Kapak cam gidelim. Hava kabarcığı olmadan bir hazırlık sonucunda numune üzerinde bir damla monte edin. </li><li> Havsız dokuları kullanılarak aşırı tampon siliniz. Hemen hazırlık gözlemleyin (adım 3). NOT: örnekleri mühür gerek yoktur. </li></ol> 3. VAEM Gözlem ve Toplama Film Bir ters mikroskop, bir TIRF ünitesi ve 1.49 sayısal diyafram ile bir TIRF objektif lens ile donatılmıştır: NOT: aşağıdaki gibi bu çalışmada kullanılan TIRF mikroskop sistemi 9 açıklanmıştır. Lazer giriş açısının bilgisayarlı kontrol için, bir kontrol kutusu kullanılır. Yeşil floresan proteini (GFP) 488 nm optik pompalanan yarıiletken lazer ve t ile heyecanlıO floresans kloroplast gelen otoflüoresanı önlemek için 510-550 nm bant-geçiş filtresi içinden tespit edilir. Fiber çıkış gücü ölçülen maksimum değeri 13,0-13,5 mW olduğunu. Tespiti için, bir elektron kullanılır (EM-CCD) kamera kafası sistemi ve bir C-mount kamera büyütme değişim ünitesi şarj çiftli cihaz çarparak. <ol><li> Lazer üreticinin talimatlarına göre merkezleme ve odaklama kalibrasyonu. Not: Bu adım hassas VAEM gözlemleri için çok önemlidir. Kuvvetle lazer yolu ayar prosedürlerinin periyodik kontroller, mikroskop uygun, yürütülmektedir tavsiye edilir. <ol><li> Mikroskopi odasının tavanında bir objektif lens içermeyen ışık yolu ile aydınlatılmış merkezi konumunu belirleyin. Merkezi konumunu işaretlemek için, tavanda renkli daire mühür koymak (Şekil 2, adım 1, 2). </li><li> Objektif lensi (Şekil 2, aşama 3, 4) sahip tavan yakma. I Taşıorta konumda (Şekil 2, adım 5) &#39;bölgesini lluminated. Lazer (Şekil 2, Adım 6) odaklanın. Ince ayar yapma merkezi (Şekil 2, basamak 7) odaklanmış lazer konumu. </li></ol></li><li> Mikroskop sahnede bir örnek olarak ayarlayın ve parlak alan aydınlatma kullanılarak gözlem hücreleri seçin. </li><li> Floresan protein hücrelerinde görülebilir kontrol edin ve Epifloresans aydınlatma (Şekil 3A) kullanılarak hücre yüzeyinde z -Axis konumunu ayarlamak. </li><li> VAEM gözlemleri gerçekleştirin. <ol><li> Denetleyici kutusu ile yavaş yavaş lazer ışınının giriş açısı eğim. Aynı zamanda, özenle canlı görüntüyü izlemek. Başlangıçta, görüntü (Şekil 3B) bulanık olacaktır. </li><li> Lazer açısı arttıkça, VAEM görüntü sonunda net bir görüntü (Şekil 3C ve D) üreten, daha az bulanık hale gelecektir. Bu noktada, increa durdurmaLazer açısını şarkı. Floresan sinyali kaybolursa, sığ açı azalır. </li><li> İnce ayar lazer giriş açısı daha iyi bir görüntü elde etmek için. İnce ayar z -Axis pozisyon da imajını artırabilir. Gerekirse, optik parametreleri (lazer gücü, dalga boyu ve filtre seti) ve görüntü sensörü parametrelerini [görüntü boyutu, pozlama süresi, kazanç, sayısallaştırıcı ve elektron-çarparak (EM) kazanç] ayarlayın. NOT: TIRF mikroskop ekipman durumda yukarıda açıklanan aşağıdaki gibi, temsilci parametrelerdir. Sadece kamera önünde lens Büyütme: 2X; Lazer çıkışı: 1.0 mW; Resim boyutu: 512 × 512 piksel; Pozlama süresi: 100 ms; Kazanç: 5 ×; Dijitalleştirici: 11 MHz; ve EM kazanç: 100. </li></ol></li><li> Ticari mikroskop yazılımını kullanarak çok sayfalı TIFF görüntü dosyası olarak film edinin. Burada, film stoma hücrelerinin yüzeyi üzerinde yanıp sönen GFP-etiketli noktalar taşımaktadır. NOT: Temsili görüntü alma koşulları fol gibidiralçak. Toplama Modu: Akış RAM&#39;e; Kare sayısı: 600; ve çoklu sayfa TIFF dosya boyutu: ~ 302 MB. </li></ol> 4. kymograph Analizi Niceleme için GFP etiketli Dot Residence Time Fiji Yazılım Kullanma <ol><li> Fiji Install yazarların yönergeleri kullanarak yazılım 10 (&#39;Fiji Just ImageJ olduğu&#39;) (http://fiji.sc/Fiji). </li><li> Fiji çalıştırın ve Fiji menüsü &quot;Dosya-Aç&quot; kullanılarak elde çoklu sayfa TIFF dosyasını açın. </li><li> Fiji araç çubuğu menüsü &quot;Düz Çizgi Seçim Aracı&quot; seçeneğini kullanarak, ilgi sitede bir çizgi yerleştirin. İsteğe bağlı olarak, &quot;Segment Hattı Seçim Aracı&quot; veya &quot;Serbest Hattı Seçim Aracı&quot; kullanın. Burada sunulan sonuçlar şekilde, (8.0 um karşı gelen) 100 piksellik bir düz çizgi bir yan hücre (Şekil 4A) üzerine yerleştirilmiştir. </li><li> Fiji menüsü &quot;Resim-Yığınları-Dinamik Reslice kullanarak bir kymograph görüntü olun </em&gt; &quot;(http://fiji.sc/Dynamic_Reslice) (Şekil 4B). İsteğe bağlı olarak, bir onay kutusu penceresinde ve &quot;Döndür 90 Degrees&quot; &quot;Dikey Döndürme&quot; de (100 piksel tekabül (Burada sunulan sonuçlar kullanılmaz) kymograph görüntüde. X ve y ekseni hattı konumunu belirtmek mevcuttur 8.0 mikron) ve gözlem süresi (600 piksel sırasıyla) 60 saniyeye tekabül etmektedir. kymograph görüntü tatmin edici değilse, pozisyon ve tip (düz, parçalı ve çok sayfalı görüntü hattın serbest) gibi. değiştirilebilir çizgi değişikliği, kymograph görüntü dinamik olarak değişir. Fiji menüsü &quot;Dosya-Farklı Kaydet-Tiff&quot; kullanarak bir TIFF dosyası olarak bir kymograph görüntüsünü kaydedin. </li><li> Zaman ekseni yönde blob uzunluğunu ölçün. <ol><li> Gürültü azaltma gerçekleştirmek için, Fiji menüsü &quot;Süreç Filtreler-Gaussian Blur&quot; seçeneğini kullanarak kymograph görüntüleri Gauss filtre uygulamak. &quot;Sigma (Radius) &quot;parametresi ayarlanabilir (1 piksel yarıçapı sunulan sonuçlar için kullanılan) &#39;dir. </li><li> Segment Fiji menüsünü kullanarak eşikleme sinyal bölgeleri, &quot;Resim-ayarlayın-Eşik&quot;. NOT: Aralarından seçim yapabileceğiniz çeşitli eşikleme algoritması vardır. Sunulan sonuçlarda, &quot;Yen&quot; algoritması (Şekil 4C) seçildi. </li><li> Menüsünü kullanarak blob&#39;un -Axis uzunluğu süresi (y) ölçmek için hazırlanın &quot;Analiz-Set Ölçümleri&quot;. &quot;Set Ölçümleri&quot; penceresindeki &quot;Sınırlayıcı dikdörtgen&quot; seçeneğini işaretleyin. İdeal Alan seti gürültü çıkarmak, mümkün olduğunca küçük olmalıdır. Burada asgari alan 50 piksel olarak belirlendi. Zaman (y) menüsünü kullanarak blob&#39;un -Axis uzunluğunu ölçün &quot;Analiz-Analiz Parçacıklar&quot;. Yöneticisi Ekle &quot;sonuç değerleri elde ve ölçüm bölgelerinin güvenilirliğini kanıtlamak,&quot; sonuçlarını görüntüle &quot;kontrol etmek ve &lt;/ em&gt; &quot;kutuları. </li><li> &quot;Yükseklik&quot; sütununda değerler (y) ölçülen damla (Şekil 4D) için uzunlukları -Axis zaman vardır. Sonuçlar tablo menüsü &quot;Dosya-Farklı Kaydet&quot; seçeneğini kullanarak değerleri kaydetmek ve hesaplanması ve istatistiksel paket ve / veya elektronik tablo yazılımı (Şekil 4E) kullanarak blob görüntü süreleri veri kümesi işlemek. </li></ol></li></ol>

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Bu video makalede, protokoller izlenmesi ve Arabidopsis thaliana&#39;nın stoma kompleksi üzerine GFP-PATROL1 noktaların dinamik davranışını ölçmek için verilmiştir. Burada gösterildiği gibi, VAEM gözlem bitki hücre yüzeylerinin canlı görüntüleme için güçlü bir araçtır. Son derece hassas EM CCD VAEM optik bir nispeten zayıf uyarım lazer kullanımına izin verir, çünkü GFP PATROL1 izlenmesi için kullanılan deney koşulları altında, bir video yakalama 1 dakika için kullanılan n...

The plant cell surface, including the plasma membrane and its immediately adjacent cytoplasm, is the main region of a plant cell&#8217;s perception and integration of biotic and abiotic cues from the extracellular environment. In response to these cues, cell surface components including plasma membrane proteins and the cortical cytoskeleton undergo dynamic changes, on a time scale of seconds to minutes1-4. Thus, real-time and high-resolution imaging of fluorescent proteins on the cell surface can illuminate a plant&#8217;s responses to environmental cues at the molecular level.
Confocal laser scanning microscopy is a powerful tool for determination of fluorescently-tagged protein localization3, however, it is often difficult to monitor the real-time protein dynamics because of its relatively long capturing times. An emerging technique for real-time monitoring of proteins in the plant cell is variable angle epifluorescence microscopy (VAEM), which is an adaptation of equipment usually used for total internal reflection fluorescence (TIRF) microscopy. In TIRF microscopy, the fluorescence-excitation light source is an evanescent light field that is generated when the entry angle of the laser is shallow enough to totally internally reflect light at the glass&#8211;water interface. The penetration depth of the evanescent light field is around 100 nm. TIRF microscopy is an outstanding tool for single molecule imaging, such as the detection of exocytosis in animal cells5. However, evanescent light cannot reach plasma membranes or the cortical cytoplasm in plant cells, because they have thick cell walls. Recently, TIRF microscopy equipment has been adapted by plant cell biologists, observing that a laser, if angled slightly more deeply than when being used to induce total internal reflection phenomena, could excite the surface of plant cell samples, resulting in high-quality plant cell imaging6,7. The excitation illumination depth is varied by adjusting the entry angle of the laser; therefore, this technique is described as VAEM. This optical system is also called variable angle TIRF microscopy (VA-TIRFM) because there is a possibility that total reflection may take place at the cell wall-periplasm interface7, however, the term VAEM is used in this article, as per the first report in plants6.
The goal of this protocol is to demonstrate practical procedures for using VAEM to visualize fluorescently-tagged protein dynamics on plant cell surfaces. Additionally, an image analysis protocol to quantify the residence time (duration of presence) of molecules is described for VAEM movie analysis. GFP-PATROL1 dot blinking on stomatal complex cells in Arabidopsis thaliana cotyledons is used as an example. PATROL1 was identified by forward genetic approaches as a causal gene of a stomatal response defect mutant in A. thaliana8. PATROL1 is a plant homolog of MUNC-13, which is a priming factor in synapse vesicle exocytosis8. In response to environmental cues, such as light or humidity, it is thought that PATROL1 reversibly regulates the delivery of a proton pump to plasma membranes in the stomatal complex. Stomatal complexes each comprise a pair of guard cells8 and subsidiary cells9, and they require a proton pump for stomatal movement. In these cells, GFP-tagged PATROL1 localizes to dot-like structures that remain on the plasma membrane for less than 1 min9.

<table><tbody><tr><td>Inverted microscope</td><td>Olympus</td><td>IX-73</td><td></td></tr><tr><td>TIRF unit</td><td>Olympus</td><td>IX3-RFAEVAW</td><td></td></tr><tr><td>TIRF objective lens&amp;nbsp;</td><td>Olympus</td><td>UAPON 100 &amp;times; OTIRF&amp;nbsp;</td><td>NA = 1.49</td></tr><tr><td>Laser angle control box</td><td>Chuo Seiki</td><td>QT-AK</td><td></td></tr><tr><td>Optically pumped semiconductor laser</td><td>Coherent</td><td>Sapphire&lt;sup&gt;TM&lt;/sup&gt; LP USB 488-20 CDRH Laser</td><td></td></tr><tr><td>510&amp;ndash;550 nm band-pass filter</td><td>Olympus</td><td>U-FBNA</td><td></td></tr><tr><td>EM CCD camera</td><td>Hamamatsu Photonics</td><td>ImagEM C9100-13</td><td></td></tr><tr><td>C-mount camera magnification change unit&amp;nbsp;</td><td>Olympus</td><td>U-TVCAC</td><td></td></tr><tr><td>MetaMorph software</td><td>Molecular Devices</td><td>MetaMorph version 7.7.11.0</td><td></td></tr><tr><td>TIRF microscopy manual</td><td>Olympus</td><td>AX7385</td><td>Instructions: Total Internal Reflection Illumination System (Printed in Japan on August 24, 2012)</td></tr><tr><td>Immersion oil</td><td>Olympus</td><td>Immersion Oil Typr-F</td><td>ne = 1.518 (23 degrees)</td></tr></tbody></table>

real-time imaging of plant cell surface dynamics with variable-angle epifluorescence microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

Bu video makalede, A. GFP-PATROL1 ve VAEM gözlem için protokollerde yer thaliana kotiledon stoma karmaşık hücrelerin sağlanmaktadır. Gökyüzü açılan montaj A. VAEM hazırlıkları hava kabarcıklarının oluşumunu azaltmaya yardımcı olabilir basit bir hazırlık yöntemi thaliana kotiledonları (Şekil 1). Giriş lazer ve / veya VAEM için numunelerin z-konumlandırma Overtilting belirsiz bir görüntü sağlayacaktır. Eğer bu gerçekleşirse, bu Epifl...

Watch this Scientific Journal Video about Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy at JoVE.com

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

Bu protokolün amacı stoma kompleksinin hücre korteks içinde, GFP etiketli PATROL1, bir membran kaçakçılığı protein noktalar yanıp sönen gösteren değişken açılı Epifloresans mikroskobu ile bitki hücre yüzeylerinde floresan etiketli protein dinamikleri izlemek için nasıl göstermek için Arabidopsis thaliana.

Değişken açılı Epifloresans Mikroskopi ile Bitki Hücre Yüzey Dynamics Gerçek zamanlı Görüntüleme

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

real-time-imaging-plant-cell-surface-dynamics-with-variable-angle

Research

JoVE Journal

Biology

9.0K Görüntüleme.  The University of Tokyo.  Bu protokolün amacı stoma kompleksinin hücre korteks içinde, GFP etiketli PATROL1, bir membran kaçakçılığı protein noktalar yanıp sönen gösteren değişken açılı Epifloresans mikroskobu ile bitki hücre yüzeylerinde floresan etiketli protein dinamikleri izlemek için nasıl göstermek için Arabidopsis thaliana. 

Video: Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

Mikroorganizmalarda Cortex Organizasyon ve Dinamiği Görselleştirme, Toplam İç Yansıma Floresan Mikroskobu kullanılarak

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Biyomühendislik

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

Arabidopsis Çiçek Gelişmekte Konfokal Görüntüleme Canlı

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Gelişim Biyolojisi

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

Kök ve Hipokotil Gelişimin Uzun Süreli Konfokal Görüntülemesi için Basit Bir Oda

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Bitkisel Dokular Gen İfade Araştırmalar Robotik ve Dinamik Görüntü Analizi

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Biyoloji

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In general, for observations over a long period, the technique requires transformation of a fluorescent protein; however, for most systems, genetic transformation is either time-consuming or technically unavailable. This manuscript presents a protocol for 3-D time-lapse imaging of cell wall dynamics over a 3 day period using calcofluor dye (which stains cellulose in the plant cell wall), developed in the moss Physcomitrium patens. The calcofluor dye signal from the cell wall is stable and can last for 1 week without obvious decay. Using this method, it has been shown that the detachment of cells in ggb mutants (in which the protein geranylgeranyltransferase-I beta subunit is knocked out) is caused by unregulated cell expansion and cell wall integrity defects. Moreover, the patterns of calcofluor staining change over time; less intensely stained regions correlate with the future cell expansion/branching sites in the wild type. This method can be applied to many other systems that contain cell walls and that can be stained by calcofluor.

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

This manuscript presents a detailed protocol to image the 3-D cell wall dynamics of living moss tissue, allowing the visualization of the detachment of cell walls in ggb mutants and thickening cell wall patterns in the wild type during development over a long period.

Moss Physcomitrium patens'te Calcofluor Kullanarak Hücre Duvarı Dinamiğinin 3-D Hızlandırılmış Görüntülenmesi

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Görüntüleme için Basit Bir Yöntem Arabidopsis Infiltratif Görüntüleme Orta Perfluorodecalin kullanarak bırakır.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Uzun süreli, yüksek çözünürlüklü Konfokal Time Lapse Görüntüleme<em&gt; Arabidopsis Kotiledon</emÇimlenme sırasında&gt; Epidermis

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

Bir Gel Yüzeyinde Büyüyen Bitki kökleri Işık Sac Flüoresan Mikroskopi

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

Arabidopsis montaj için çok yönlü bir yöntem Intravital zaman hata görüntüleme için yaprakları

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

ERL1 Dinamiğinin Konfokal Görüntüleme ile Uzay-Zamansal Olarak İncelenmesi

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Yazar Spotlight: Stoma Soy Hücrelerinde Membran Kaçakçılığı Olaylarını İncelemek için Görüntü Tabanlı Yöntemler  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Stoma Soy Hücrelerinde Membran Kaçakçılığı Olaylarını İncelemek için Görüntü Tabanlı Yöntemler

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...