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.
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.
En av de viktigaste frågorna i förståelse växtens utveckling är hur enskilda celler beter under organdifferentiering och tillväxt. Helst cellulära händelser, såsom gen-uttrycksmönster och intracellulär proteinlokalisering, kan ses i ljuset av ett större sammanhang av vävnaden. Detta mål innebär tekniska utmaningar och kräver hela organ observation med en hög rumslig liksom temporal upplösning över längre tidsperioder, vilket kan orsaka fototoxisk effekt. Eftersom växter snabbt anpassa sig till förändringar i miljön, måste de växande villkor hårt kontrollerad. För att göra en långsiktig avbildning utan att störa den fysiologiska tillstånd av anläggningen, har tre saker att säkerställas, 1) odlingsförhållanden i provkammaren, 2) stabil prov montering under långa tidsperioder, och 3) bild med låga ljusintensiteter för att undvika bild-skador och icke-fysiologiska betingelser.
Fysiologisk växande conditions i mikroskop provkammaren är avgörande för långtidsförsök. Det finns ett antal protokoll som finns tillgängliga som beskriver avbildande odlingskammare för konfokala mikroskop 1 – 3. Emellertid introducerar konfokalmikroskopi hög ljusintensitet till anläggningen, vilket kan orsaka stressreaktioner och vanligtvis hämmar tillväxten 4. Dessutom har de flesta konventionella mikroskop tillåter endast horisontell positionering av provet, vilket inte är optimalt för växter eftersom de försöker att omorientera sig och växa till vektorn av tyngdkraften. Under de senaste tio åren har ljus ark mikroskop framträtt som ett kraftfullt verktyg för att fånga utvecklingen av stora prover på cell upplösning för tidsperioder av upp till flera dagar 5 – 9. Ljus ark mikroskopi tillåter placering av provet vertikalt och används alltmer inom växtforskning studerar rotutveckling 10-21, som nyligen granskats av Berthet och Maizel 22. Många av de nämnda studierna 10,13 – 18,21 var optimerade och genomförs i laboratoriet Ernst HK Stelzer utnyttjar ett speciellt sätt att provet montering präglas av växande roten på ytan av en gel 17. I dessa studier var en skräddarsydd mikroskop används, där växten hålls från botten. Däremot var majoriteten av i stort sett tillgängliga mikroskop ljus ark hålla provet från toppen. Således, denna speciella framställningsmetod kan inte lätt anbringas. Den metod som presenteras här ger ett protokoll för den väletablerade på ytmontering metod som är tillämplig för OpenSPIM 23, en öppen tillgång plattform för att tillämpa och förbättra Selektiv Plane Belysning Microscopy (SPIM).
Det övergripande målet med detta protokoll är att möjliggöra en långsiktig avbildning av Arabidopsis rötter i OpenSPIM ljus ark mikroskop. Detta åstadkoms genom att odla en växt upprätt på de surface av en gel med rötterna i ett vätskemedium medan bladen förblir i luften. För att säkerställa en fotosyntetisk aktivitet av anläggningen, belyser en skräddarsydd belysningssystem bladen men inte rötterna (figur 1).
Light Sheet Fluorescence Microscopy has the great advantage to combine low phototoxicity and ultrafast acquisition speed, which can be used to capture a large volume with a high spatio-temporal resolution while keeping the sample in a physiological state. The resolution of a light sheet microscope can be compared to that of a confocal microscope9. However, light scattering and absorption occurs along the excitation and emission path individually and the overall image quality can be significantly lower inside opaque tissues compared to the surface. To circumvent this complication one can use the possibility to rotate the sample along the vertical axis and observe the same volume from different directions. But this is not always advantageous, e.g. lateral roots emerge on one side of the root and imaging from behind results in a low image quality without gaining more information. However, the rotation can be principally used to position the sample in the best way. The classic horizontal arrangement of the objective lenses allows for new ways of sample mounting. Plants benefit from a vertical position. Presented here, the "on the surface of the gel" mounting method has several advantages compared to other mounting methods such as embedding the root inside of a gel24,25. 1) The root system is in direct contact with the liquid medium. The sample chamber is connected to a perfusion system which provides continuously fresh medium. It can also be used to rapidly exchange the entire volume of the sample chamber to apply different media or drugs. 2) Prior to sample preparation plants grow as they are used to grow in laboratories. Plants can be selected under a fluorescence microscope and only the desired plants need to be prepared. 3) The plant is transferred from the Petri dish to the sample holder without being touched. Thereby the plant can further develop on the same gel it was growing on in the growth incubator and mechanical stress is reduced to a minimum. 4) The view on the specimen is unobstructed and optical aberrations are minimized because the space between the sample and the detection objective is solely filled with medium and no other materials with differing refractive indices.
In order to perform long-term imaging, the plant illumination system is necessary to ensure photosynthetic activity of the plant. In most laboratories plants grow on a transparent gel, i.e. the roots are exposed to light. This may cause different responses to their environment and induces changes in their biochemistry and development26,27. In order to reduce the amount of light on the root system, black plastic foil was used to cover the water surface as well as a lid made of black aluminum foil covered the sample chamber. Light can reach the plant leaves through the central hole in the lid. In this setup, no increase in background light was observed, suggesting that the amount of stray light from the red and blue LEDs was significantly reduced by the GFP filter and the shading approaches. This allowed keeping the light turned on during image acquisition without increasing the camera background noise.
The sample holder is designed for 3D printing. However, the choice of material is crucial as several plastics that were tested were not 100% stable, resulting in a drift of the sample. Therefore, it is recommended to use resins instead or build the sample holder by milling a Polyethylene (PEP) rod. When using a light sheet microscope setup with double-sided illumination system the sample holder might interfere with one of the light sheets depending on the rotation angle. To reduce mechanical stress during scooping the plant from the plate, use a flat angle of the spatula. The plant can quickly dry out and experience air flow for the very first time. Try to avoid any air draft (rapid movements, air-condition flow), work uninterruptedly and slide the sample holder into a 1,000 µL pipet tip whenever possible. Inside the microscope, it is crucial to not dip the whole plant in liquid and keep the leaves dry.
The technique is ideal for imaging early stages of lateral root formation. When performing long term imaging of mature root tips one must keep in mind that Arabidopsis roots grow with 100-300 µm/h rapidly out of the field of view. A very useful future implementation could be an automated tracking algorithm, which would allow following root tip growth over prolonged periods of time. The ability to control environmental conditions such as light and nutrient composition of the medium during the acquisition process allows investigating how plants adapt to changes. The root is in direct contact with the liquid medium, which can be used to apply drugs to chemically activate gene expression, for example using the dexamethasone inducible28 or the β-estradiol inducible system29. However, it takes time to exchange the entire volume of the sample chamber to wash out a drug. The setup could be improved by minimizing the volume of the sample chamber to accelerate medium exchange. Nevertheless, this technique has a great potential. The combination of mounting procedure, standardized growing conditions and the gentle image acquisition using light-sheet microscopy allows long-term studies of plant development with high resolution at a physiological level. This will help researchers to explore fundamental mechanisms of plant development.
The authors have nothing to disclose.
We thank Matyáš Fendrych for critical reading/viewing and Stephan Stadlbauer for the audio equipment. Thanks to the Miba Machine Shop at IST Austria for their contribution to the OpenSPIM. The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement n° [291734] and European Research Council (project ERC-2011-StG-20101109-PSDP).
Agarose, low melting | VWR | AFFY3282125GM | |
Black aluminum foil | Thorlabs | BKF12 | |
Black plastic foil | Carl Roth | HT83.2 | |
LED blue (453 nm) | OSRAM | LD CN5M-1R1S-35-1 | |
LED red (625 nm) | OSRAM | LR T66F-ABBB-1-1 | |
LED board – PCB design software | Cadsoft Eagle | ||
MES monohydrate | Duchefa | M1503.0100 | |
Micropore Surgical Tape | 3M | 1530-1 | |
Murashige & Skoog Medium (MS-Medium) | Duchefa | M0221 | |
Phytagel | Sigma-Aldrich | P8169 | |
Sample holder 3D print | i.materialise | https://i.materialise.de/shop/item/sampleholder-openspim-zeisslightsheetz1 | |
Square petri dishes (245x245x25 mm) | VWR | 734-2179 | |
Sucrose | Sigma-Aldrich | 84097-1KG |