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The method consists of imaging developing pollen grains as they form within intact, immature Arabidopsis thaliana anthers. Using two-photon excitation in the infrared wavelength range, the intrinsic fluorescence (autofluorescence) of developing pollen grains and surrounding sporophytic tissues of the anther wall, including the tapetum, middle layer, endothecium and epidermis, can be visualized in the three-dimensional space of an intact anther. In contrast to conventional confocal microscopy, the application of red-shifted light by two-photon microscopy improves depth penetration into specimens, while the scattering of light and subsequent phototoxicity is minimized, making this a superior method for imaging the developing pollen grains and tapetal cells enclosed within anthers. The technique described was optimized for the detection of autofluorescent components of the pollen wall, including sporopollenin and the pollen coat, and provided spatial and developmental data on the autofluorescent metabolites in anthers of wild-type and pollen wall mutant plants (Quilichini et al., 2014). The use of two-photon imaging of live, intact anthers holds potential for future studies aimed at understanding the spatial relationship between gametophytic and sporophytic tissues during pollen development and the distribution of metabolites or fluorescently-tagged proteins within developing anthers.

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Analysis of Developing Pollen Grains within Intact Arabidopsis thaliana Anthers by Olympus Two-Photon Laser Scanning Microscopy
使用Olympus双光子激光扫描显微镜分析完整的拟南芥花药中正在发育的花粉粒

植物科学 > 植物发育生物学 > 形态建成
作者: Teagen D. Quilichini
Teagen D. QuilichiniAffiliation: Department of Botany, University of British Columbia, Vancouver, Canada
Bio-protocol author page: a2772
A. Lacey Samuels
A. Lacey SamuelsAffiliation: Department of Botany, University of British Columbia, Vancouver, Canada
Bio-protocol author page: a2773
 and Carl J. Douglas
Carl J. DouglasAffiliation: Department of Botany, University of British Columbia, Vancouver, Canada
For correspondence: carl.douglas@botany.ubc.ca
Bio-protocol author page: a2774
Vol 5, Iss 23, 12/5/2015, 2068 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1677

[Abstract] The method consists of imaging developing pollen grains as they form within intact, immature Arabidopsis thaliana anthers. Using two-photon excitation in the infrared wavelength range, the intrinsic fluorescence (autofluorescence) of developing pollen grains and surrounding sporophytic tissues of the anther wall, including the tapetum, middle layer, endothecium and epidermis, can be visualized in the three-dimensional space of an intact anther. In contrast to conventional confocal microscopy, the application of red-shifted light by two-photon microscopy improves depth penetration into specimens, while the scattering of light and subsequent phototoxicity is minimized, making this a superior method for imaging the developing pollen grains and tapetal cells enclosed within anthers. The technique described was optimized for the detection of autofluorescent components of the pollen wall, including sporopollenin and the pollen coat, and provided spatial and developmental data on the autofluorescent metabolites in anthers of wild-type and pollen wall mutant plants (Quilichini et al., 2014). The use of two-photon imaging of live, intact anthers holds potential for future studies aimed at understanding the spatial relationship between gametophytic and sporophytic tissues during pollen development and the distribution of metabolites or fluorescently-tagged proteins within developing anthers.

[Abstract] 该方法包括当它们在完整的未成熟拟南芥花药内形成时使发育的花粉粒成像。使用在红外波长范围内的双光子激发,发育花粉颗粒和花药壁的周围孢子体组织(包括绒毡层,中间层,内皮和表皮)的固有荧光(自发荧光)可以在三维空间的完整的花药。与传统的共聚焦显微镜相反,通过双光子显微镜应用红移光改善深度穿透到标本,同时光的散射和随后的光毒性最小化,使这是成像发展中的花粉粒和绒毡层细胞的优良方法封闭在花药内。所描述的技术被优化用于检测花粉壁的自体荧光组分,包括花粉球蛋白和花粉外壳,并且提供关于野生型和花粉壁突变植物的花药中的自发荧光代谢物的空间和发育数据(Quilichini等al。,2014)。活的,完整的花药的双光子成像的使用具有潜在的未来研究,目的在于了解花粉发育期间配子体和孢子体组织之间的空间关系以及代谢物或荧光标记的蛋白质在发育的花药内的分布。

Materials and Reagents

  1. Ruler with millimetre divisions
  2. Glass bottom petri dishes (MatTek Corporation, catalog number: P35G-1.5-14C )
  3. Coverslips (No.1.5, 0.17 mm thick, 22 x 22) (Thermo Fisher Scientific, catalog number 12-541B )
  4. Transfer pipettes 1.5 ml
  5. Arabidopsis thaliana plants in the flowering stage
  6. Non-toxic food-grade paraffin wax maintained in a liquid state at 40 ºC in a paraffin bath (Thera-Band, catalog number: 24050 )
  7. Distilled water

Equipment

  1. Spectra-Physics MaiTai HP Titanium:Sapphire mode-locked pulsed laser, with tuneable wavelengths spanning 690 to 1,040 nm.
  2. Olympus Fluoview 1000 scan head (Olympus America Inc., model: FV1000MPE ) modified by Olympus for two-photon imaging by the inclusion of a light path for the pulsed laser input and the housing for the non-descanned, multialkali side window photomultiplier tubes (PMTs).
  3. Olympus BX61WI upright microscope
  4. Olympus multiphoton microscope filter cube (440/40 bandpass filter) (OLYMPUS, model: FV10-MRVGR/XR 4CH NDD FILTER )
  5. Olympus multiphoton microscope dedicated objective lens optimized for transmitting IR light (25x, 1.05 numerical aperture, 2.0 mm working distance) (OLYMPUS, model: FV10-SNPXLU )
  6. Light tight, custom made multiphoton microscope enclosure. To build an enclosure, aluminum extruded components (Item North America) and flat-black painted fibre board can be used
  7. Forceps for fine dissections (Electron Microscopy Sciences, catalog number: 72700-D )
  8. Olympus stereomicroscope (magnification range of 6.3-63x) (OLYMPUS, model: SZX10 )

Software

  1. Olympus FluoView FV10-ASW 3.01
  2. Spectra-Physics MaiTai Control laser software
  3. Volocity version 6.1.1 software package
    Note: ImageJ 1.47v can also be used to process images.

Procedure

New users learning to operate an Olympus two-photon microscope or similar system should be supervised by an experienced user or technician. The protocol described herein is specific to the Olympus system and should only be used as a supplementary resource when operating other two-photon imaging platforms.

  1. Microscope set-up
    1. Operating the MaiTai laser.
      Turn the laser power on, tune the laser to the desired wavelength and open the shutter using the Spectra-Physics MaiTai Control (see Figure 1). Although the laser can be operated through FluoView or separately through the Spectra-Physics MaiTai Control application, it is recommended to turn the laser on through the Spectra-Physics application, independently from the FluoView software, to prevent laser shut down in the case of software malfunctions.
      1. Turning the laser on.
        The laser should be set to 800 nm when not in operation (Figure 1, left panel) and should warm up at this wavelength (Figure 1, middle panel). Select and hold down ‘ON’ to turn the laser on (Figure 1, left panel). Wait for the laser to begin pulsing (indicated by the green box under ‘Pulsing’) and the IR Power level (indicated by a red bar) to stabilize (Figure 1, middle panel). Ensure that laser pulsing (mode-lock) status is reached.
      2. Tuning the laser to the imaging wavelength.
        After a 30 min laser warm-up, the infrared (IR) power level should be stabilized. Tune the laser set wavelength from 800 nm to 720 nm by entering the imaging wavelength ‘720’, ‘enter’ to tune the laser to this wavelength (Figure 1, right panel). Ensure the IR Power reaches 2.49 watts when using 720 nm and 3% laser power. Also ensure the correct 720 nm wavelength is entered in the Fluoview software Acquisition settings under Laser.
        Note: As you change laser wavelength, IR Power also changes. To obtain consistent emission power at different wavelengths, the laser power must be adjusted each time the laser wavelength is changed. Enter the correct laser wavelength in FluoView, in the Acquisition Settings Panel under Laser.
      3. Open the reflected light shutter.
        Click on the closed (grey) shutter button to switch the shutter to the open (yellow) position (Figure 1). Manually open the shutter on the microscope unit, located beside the filter wheel.


        Figure 1. MaiTai laser operation through the Spectra-Physics Laser Control interface. From left to right: Adjusting laser parameters as shown turns the laser power on and tunes the laser to the desired wavelength. Starting with the left panel, the laser power is off and set to 800 nm by the previous user. Select and hold down ‘ON’ to turn on the laser on. Wait for the laser to begin pulsing (indicated by the green box under ‘Pulsing’) and the IR Power level (indicated by a red bar) to stabilize. After sufficient laser warm-up, tune the laser to the imaging wavelength of 720 nm. Ensure the IR Power level stabilizes. Click on the closed (grey) shutter button to switch the reflected light shutter to the open (yellow) position.

    2. Install the objective.
      Select a clean objective and screw into position on the microscope. For the Olympus FV1000MPE multiphoton microscope, the XLPLN 25x water immersion objective is recommended, as it is optimized for multiphoton imaging (Figure 2A). Select the correct objective in the FluoView Acquisition Settings Panel, under Microscope.
    3. Filter cube selection.
      Select the Olympus BFP/GFP/RFP/Ds Red filter cube, with 440/40 bandpass filter (420-460 nm; Figure 2B). Always handle the filter cube with gloves. Screw the filter cube into place within the filter cube chamber located behind the filter wheel and sliders.
    4. Adjusting objective lens and stage position.
      The microscope stage can be adjusted in the x- and y-axes with the joystick control (Figure 2C). To move the objective lens in the z-axis, towards or away from the specimen, use the focus remote controller (Figure 2D).


      Figure 2. Adjustable microscope hardware. A. The Olympus XLPlan N 25X W MP objective lens, recommended for multiphoton imaging. B. Olympus filter cube (FV10-MRVGR/XR 4CH NDD FILTER, BFP/GFP/RFP/Ds Red, 380-560 nm). C. Stage control joystick. D. Focus remote control.

    5. Directing the light path.
      For transmitted light viewing or two-photon imaging, different light path configurations are required. For transmitted light viewing, the sliders must be in the outward position to allow light to move to the oculars (Figure 3A) and the filter wheel should be in the DICT position (Figure 3C). For four-channel two-photon imaging, ensure both sliders are in (Figure 3B) and move the filter wheel to position 2 (Figure 3D). For the Olympus FV1000 MPE system, the R690 position on the filter wheel is meant for two-photon imaging and the DICT position allows transmitted light viewing.


      Figure 3. Microscope configurations. A. Microscope sliders are in the outward position for transmitted light viewing. B. Microscope sliders are in the inward position for two-photon imaging with emission capture in four channels. C. Filter wheel is in the DICT position for transmitted light viewing. D. Filter wheel is in the R690 position for two-photon imaging.

  2. Specimen preparation
    1. Select anthers in the free microspore stage of pollen development.
      1. With forceps, remove a floral cluster from your Arabidopsis plant of interest (Figure 4A). Discard open flowers.
      2. Under a dissecting microscope, separate each bud from the floral cluster using forceps (Figure 4B).
      3. For stamens in the free microspore stage, select buds measuring 0.7 to 1.2 mm from base to apex (Figure 4B, boxed in the middle row). Buds measuring <0.5 mm, 0.5-0.7 mm typically contain anthers in the microspore mother cell or tetrad stages of pollen development, respectively (Figure 4B, see bracketed floral bud cluster). Buds measuring >1.2 mm typically contain anthers in the late stages of pollen development, characterized by tricellular pollen and tapetum degeneration (Figure 4B, see bracketed bottom row of buds).
        Note: The developmental stage of each stamen can vary between buds of the same length, among the six stamens within one bud and even occasionally between locules of the same anther. For these reasons, numerous anthers for each genotype of interest should be analyzed from multiple buds, to ensure the correct stage(s) of interest are identified.
      4. Following bud size selection, carefully remove sepals and petals from one bud of interest (Figure 4C) and excise stamens into the microwell of a glass bottom Petri dish (Figure 4D). When handling each stamen with forceps, clasp the filament to avoid damage to the anther.
      5. Cover the stamens with distilled water. Gently remove all air bubbles trapped in the microwell. To remove air bubbles along the anther outer surface, hold each stamen underwater with forceps and gently manipulate bubbles away from the anther surface. Removing all air bubbles is critical, as air bubbles interfere with imaging. If air bubbles will not dissipate, the anther is not selected for imaging. Anthers ready for imaging should sink.
      6. Discard anthers with any visible damage due to dissections (such as the shrivelled anther with a detached filament, circled in Figure 4D). Anthers submerged in water (Figure 4E) but associated with air bubbles should be discarded.
      7. Seal your specimens under a coverslip with paraffin along all edges (Figure 4F).


        Figure 4. Stamen selection and preparation for imaging. A. Arabidopsis flower bud cluster with open flowers removed. B. Bud size selection. All buds displayed are from flower bud cluster in (A). Ruler on right edge depicts 1 mm intervals. Buds measuring 0.7-1.2 mm in length (middle row, boxed) are selected, as they contain anthers in the free microspore stages of pollen development before tapetum programmed cell death. Buds exceeding 1.2 mm in length (bracketed bottom row in panel B) or under 0.7 mm in length (in the floral cluster, bracketed in panel B) are discarded. C. Bud showing exposed stamens and carpel, after sepal and petal removal. D. Anthers after removal from flower pedicel but prior to submergence in water. Anthers with damage caused by dissections (such as the circled anther lacking a filament and appearing shrivelled) are discarded. E. Anthers submerged in distilled water, below paraffin sealed coverslip. Anthers associated with air bubbles are not imaged. F. A coverslip covering the microwell and submerged anthers is sealed to the Petri dish on all sides with paraffin.

    2. Specimen loading and alignment.
      Place a large water droplet onto the sample coverslip and lower objective lens until it is partially submerged. Bring the filter wheel to the DICT position for transmitted light. Turn the transmitted light lamp on (in FluoView under Image Acquisition Control) and pull both microscope sliders to the outward position to allow light to move to the oculars. Using the stage control joystick and the focus remote controller (Figure 2C) to centre your region of interest and bring your specimen into focus. Turn the transmitted light off and move the filter wheel to position 2 (R690) for two-photon imaging.
    3. Enter the two-photon imaging mode.
      In the FluoView program Image Acquisition Control panel, select the Dye List menu. Clear all dyes from the list before selecting ‘Two Photon’.
    4. Check the light path.
      For emission capture in the channels of interest, move the SDM570 dichroic mirror into the light path for four-channel imaging by sliding both microscope sliders inward. This configuration allows emission collection from channels 1-4 (RXD1-4, Figure 5). In the Quilichini et al. (2014) study, emissions for hydroxycinnamic acids and related compounds were collected in the low wavelength emission range of 420-460 nm (in RXD1), while chlorophylls and related compound emissions were collected in higher wavelengths, from 495-540 nm (in RXD2).
      Note: The two-photon microscope system described in this protocol consists of a separate non-descanned light path with no pinhole in front of the PMTs. However, opening the pinhole for two-photon imaging on other systems with conventional confocal capabilities may be required.


      Figure 5. Light path for two-photon imaging on the Olympus FV1000MPE multiphoton microscope. For the correct light path for imaging anther autofluorescence, check that the laser path (depicted by a yellow line) in the Light Path and Dyes window of the Image Acquisition Control panel includes Laser Unit 3 at 720 nm, the excitation dichroic mirror, RDM690, and the correct emission capture channels, RXD1 and RXD2.

    5. Image acquisition parameters.
      In Fluoview, enable the two emission channels of interest by selecting RXD1 and RXD2 within the Image Acquisition control window. During scans and image collection, the live view window can be configured to display RXD1 and RXD2 emission, or an overlay of these channels. Different display pseudocolour options are available through the Live View window under LUT. The Hi-Lo display function is recommended, as it provides information on the intensity of emission over the imaging area. Using this display, overexposed pixels will appear red, while underexposed, dim pixels appear blue. Laser power and channel voltage settings, as detailed in this protocol below, were selected to maximize the signal intensity from the mixture of cell types within developing anthers, while avoiding sample rupture and pixel saturation.
    6. Minimize sample damage.
      During alignment, use a fast scan (called focus x 2 or focus x 4) to align your specimen without causing significant sample bleaching. Collect images using the xy scan function to collect a single frame image. To collect a z-stack, set the start and end z-positions for your sample of interest, typically surrounding one anther locule, with a 1 µm step size. Capture the z-stack by selecting depth (under the xy scan function) prior to clicking xy scan.
    7. Minimize light pollution.
      Detectors in the two-photon system are not protected by a pinhole, as in conventional confocal microscopy, to ensure all light in the focal volume is collected. To minimize light collection from external sources, ensure lighting in the room is minimized by sealing off the microscope within an opaque enclosure, performing imaging in a dark room, and operating the Fluoview program in the dark view.
    8. Optimal imaging parameters for Arabidopsis anthers.
      See table below for Acquisition Settings and Image Acquisition Controls. When different magnifications are used, it is recommended that the aspect ratio be adjusted accordingly, to keep pixel size as close to 0.2 µm/pixel as possible.

      Laser power
      3%
      Laser wavelength (IR power)
      720 nm (2.49 W)
      Scan mode
      XY
      Scan direction
      One-way
      Image size (Aspect ratio)
      800 x 800 pixels (1:1)
      Zoom
      3x
      Pixel size
      0.212 µm/pixel
      Pixel dwell time
      4.0 μs/pixel
      Z-stack step size
      1.0 μm
      Integration type
      Kalman Line Averaging
      Integration count
      3
      RXD1 HV, Gain, Offset
      650 V, 1, none
      RXD2 HV, Gain, Offset
      650 V, 1, none

    9. Correcting brightness at depth.
      To improve emission capture from fluorophores located deep below the surface, either laser power or high voltage (HV, which controls the voltage applied on the PMTs, post-acquisition) can be adjusted for different z-positions (depths). This can be a useful tool, particularly for large specimens where valuable information is located at depth. Although this feature can be useful for imaging dim samples or locules positioned at great depths, it is time consuming, can cause specimen rupture if laser power settings are too high, and optimal settings over the z-series are generally sample specific. For consistency between genotypes and because anther tissues of interest are sufficiently bright without these adjustments, this correction was not applied for the imaging in our study (Quilichini et al., 2014).
    10. Image processing.
      Save each image or z-stack as an .oif file for processing in Volocity v 6.1.1, or import the image sequence into ImageJ equipped with the Bio-Formats plug-in (Abramoff et al., 2004; Linkert et al., 2010). Image processing commonly includes applying false-colour to the emission from each channel, overlaying channels into one merged image or stack, adjusting brightness and/or contrast in a consistent manner, adding scale bars, or creating a z-projection from a z-stack.
    11. Instrument shutdown.
      After imaging, return the laser to 800 nm prior to turning off laser power. Close the shutter on the microscope and through the software used to operate the laser. Move the objective to the uppermost position prior to closing the Fluoview program.

Notes

Readers are encouraged to consult the Olympus Microscopy Resource Center (link below) for further information on the theory and applications of multiphoton fluorescence microscopy.
http://www.olympusmicro.com/primer/techniques/fluorescence/multiphoton/multiphotonintro.html.

Acknowledgements

We thank the University of British Columbia BioImaging Facility, particularly Kevin Hodgson, for training, technical assistance and discussions. This work was supported by Canadian Natural Sciences, Engineering Research Council (NSERC) Discovery Grants and the Working on Walls NSERC CREATE program. This protocol was developed for a study on pollen wall formation in Arabidopsis (Quilichini et al., 2014).

References

  1. Abramoff, M. D., Magalhaes, P. J. and Ram, S. J. (2004). Image Processing with ImageJ. Biophotonics International 11(7): 36-42.
  2. Linkert, M., Rueden, C. T., Allan, C., Burel, J. M., Moore, W., Patterson, A., Loranger, B., Moore, J., Neves, C., Macdonald, D., Tarkowska, A., Sticco, C., Hill, E., Rossner, M., Eliceiri, K. W. and Swedlow, J. R. (2010). Metadata matters: access to image data in the real world. J Cell Biol 189(5): 777-782.
  3. Olympus FV1000 MPE Microscope User Guide.
  4. Quilichini, T. D., Samuels, A. L. and Douglas, C. J. (2014). ABCG26-mediated polyketide trafficking and hydroxycinnamoyl spermidines contribute to pollen wall exine formation in Arabidopsis. Plant Cell 26(11): 4483-4498.

材料和试剂

  1. 带毫米分隔的标尺
  2. 玻璃底培养皿(MatTek Corporation,目录号:P35G-1.5-14C)
  3. 盖片(1.5,1.07mm厚,22×22)(Thermo Fisher Scientific,目录号12-541B)
  4. 移液器1.5 ml
  5. 拟南芥植物在开花期
  6. 在石蜡浴(Thera-Band,目录号:24050)中在40℃下保持液体状态的无毒食品级石蜡
  7. 蒸馏水

设备

  1. 光谱物理MaiTai HP钛:蓝宝石锁模脉冲激光,可调波长跨越690至1,040 nm。
  2. 奥林巴斯Fluoview 1000扫描头(奥林巴斯美国公司,型号:FV1000MPE)由奥林巴斯修改为双光子成像,包括脉冲激光输入的光路和非减速,多碱性侧窗光电倍增管PMT)。
  3. Olympus BX61WI直立显微镜
  4. 奥林巴斯多光子显微镜滤光片(440/40带通滤光片)(OLYMPUS,型号:FV10-MRVGR/XR 4CH NDD FILTER)
  5. 奥林巴斯多光子显微镜专用物镜,优化用于传输红外光(25x,1.05数值孔径,2.0毫米工作距离)(OLYMPUS,型号:FV10-SNPXLU)
  6. 光密,定制多光子显微镜外壳。为了构建外壳,可以使用铝挤压部件(北美洲项目)和平黑漆纤维板
  7. 用于精细解剖的钳子(Electron Microscopy Sciences,目录号:72700-D)
  8. Olympus立体显微镜(倍率范围为6.3-63x)(OLYMPUS,型号:SZX10)

软件

  1. Olympus FluoView FV10-ASW 3.01
  2. 光谱物理麦泰控制激光软件
  3. Volocity 6.1.1软件包
    注意:ImageJ 1.47v也可以用来处理图像。

程序

学习操作奥林巴斯双光子显微镜或类似系统的新用户应由有经验的用户或技术人员监督。本文描述的协议是特定于奥林巴斯系统,并且应当仅在操作其它双光子成像平台时用作补充资源。

  1. 显微镜设置
    1. 操作迈泰激光。
      打开激光器电源,调谐激光器 到所需的波长并使用Spectra-Physics打开快门 ?麦泰控制(见图1)。虽然激光器可以操作 通过FluoView或单独通过Spectra-Physics麦泰 控制应用中,建议打开激光器通过 Spectra-Physics应用程序,独立于FluoView软件, 以防止在软件故障的情况下激光关闭
      1. 打开激光。
        不使用时,激光器应设置为800 nm(图1,左边) ?面板),并应在此波长下预热(图1,中间图)。 选择并按住"ON"以打开激光(图1,左面板)。 等待激光开始脉冲(由下面的绿色框指示) '脉冲')和IR功率电平(由红色条表示)来稳定 (图1,中间图)。确保激光脉冲(模式锁定)状态 达到。
      2. 将激光调谐到成像波长。
        30分钟激光预热后,红外(IR)功率电平应为 稳定。调整激光设置波长从800 nm到720 nm 进入成像波长'720','进入'以对激光器进行调谐 ?波长(图1,右图)。确保红外功率达到2.49 当使用720nm和3%激光功率时的瓦特。还要确保正确的720 nm波长输入Fluoview软件采集设置 在激光下。
        注意:当您更改激光波长时,IR功率也 变化。为了在不同波长处获得一致的发射功率, 每次激光波长时必须调整激光功率 改变。在FluoView中输入正确的激光波长 激光下的采集设置面板。
      3. 打开反射光快门。
        单击关闭(灰色)快门按钮将快门切换到 打开(黄色)位置(图1)。手动打开快门 显微镜单元,位于滤光轮旁边。


        图1.麦泰 激光操作通过光谱物理激光控制接口。 从左到右:调整激光器参数如图所示打开激光器 打开电源并将激光器调谐到所需的波长。从...开始 左面板,激光功率关闭并设置为800 nm的前一个 ?用户。选择并按住"ON"以打开激光器。等待 激光开始脉冲(由"脉冲"下的绿色框表示)和 红外功率电平(由红色条表示)稳定。后 充分的激光预热,调谐激光到成像波长 720nm。确保IR电源电平稳定。点击关闭(灰色) ?快门按钮将反射光快门切换到打开状态 (黄色)位置。

    2. 安装目标。
      选择一个 清洁物镜和螺丝固定在显微镜上的位置。为了 奥林巴斯FV1000MPE多光子显微镜,XLPLN 25x水浸 推荐使用物镜,因为它针对多光子成像进行了优化 (图2A)。在FluoView采集中选择正确的目标 设置面板,在显微镜下。
    3. 筛选多维数据集选择。
      选择Olympus BFP/GFP/RFP/Ds红色滤镜立方体,具有440/40带通 过滤器(420-460nm;图2B)。始终处理过滤器立方体 手套。将过滤器立方体拧入过滤器立方体腔室内的适当位置 位于滤光轮和滑块后面
    4. 调整物镜和镜台位置。
      显微镜载物台可以在x轴和y轴上进行调整 操纵杆控制(图2C)。为了沿z轴移动物镜, 朝向或远离标本,使用对焦遥控器 (图2D)。


      图2.可调整的显微镜硬件 Olympus XLPlan N 25X W MP物镜,推荐用于多光子 成像。 B.奥林巴斯滤镜立方体(FV10-MRVGR/XR 4CH NDD FILTER, BFP/GFP/RFP/Ds红,380-560nm)。 C.舞台控制操纵杆。 D.焦点 遥控器
    5. 引导光路。
      对于 透射光观察或双光子成像,不同的光路 配置。对于透射光观察,滑块 必须处于向外位置以允许光移动到目镜 (图3A),滤光轮应处于DICT位置(图3) 3C)。对于四通道双光子成像,确保两个滑块都在 (图3B),并将滤光轮移动到位置2(图3D)。为了 ?奥林巴斯FV1000 MPE系统,滤光轮上的R690位置是 意味着双光子成像和DICT位置允许传输 光观察。


      图3.显微镜配置。A.显微镜 滑块处于用于透射光观察的向外位置。乙。 显微镜滑块位于向内位置,用于双光子成像 在四个通道中进行发射捕获。 C.滤光轮在DICT中 透射光观察位置。 D.滤光轮在R690 双光子成像的位置。

  2. 标本制备
    1. 在花粉发育的自由小孢子阶段选择花药。
      1. 使用镊子,从您感兴趣的拟南芥植物中除去花簇(图4A)。丢弃开放的花。
      2. 在解剖显微镜下,使用镊子(图4B)从花簇分离每个芽。
      3. 对于在自由小孢子阶段的雄蕊,选择0.7的芽 到1.2mm(图4B,装在中间一排)。芽 测量<0.5mm,0.5-0.7mm通常在其中包含花药 小孢子母细胞或花粉发育的四联体阶段, (图4B,见括号花芽簇)。芽 测量> 1.2mm通常在晚期阶段包含花药 花粉发育,其特征在于花粉和绒毡层 退化(图4B,参见括号底芽行)。
        注意: 每个雄蕊的发育阶段可以在芽的芽之间变化 ?长度,在一芽内的六个雄蕊,甚至偶尔 在同一花药的室之间。由于这些原因,许多花药 对于每个感兴趣的基因型应该从多个芽分析 确保确定感兴趣的正确阶段。
      4. 选择芽大小后,小心地从中去除萼片和花瓣 一个感兴趣的芽(图4C)和切除雄蕊进入微孔 ?玻璃底培养皿(图4D)。当处理每个雄蕊 钳子,扣住细丝,以避免损伤花药。
      5. 盖 ?雄蕊用蒸馏水。轻轻取出所有被困住的气泡 ?在微孔中。为了沿着花药外表面除去气泡, ?用镊子保持每个雄蕊水下,轻轻操纵泡沫 远离花药表面。除去所有气泡是至关重要的 气泡干扰成像。如果气泡不会消散, 不选择花药用于成像。花药准备好成像应该 ?水槽。
      6. 丢弃花药,造成任何可见的损坏 解剖(例如具有分离的细丝的萎缩的花药, 在图4D中圈出)。花药浸没在水中(图4E) 与气泡相关的应该被丢弃。
      7. 用石蜡沿着所有边缘将样本密封在盖玻片下(图4F)。


        图4.雄蕊的选择和成像的准备。拟南芥花芽簇除去开花花。芽尺寸选择。所有 ?显示的芽来自(A)中的花芽簇。右边的标尺 描绘了1mm间隔。长度为0.7-1.2mm的芽(中间行, ?boxed),因为它们在游离小孢子中含有花药 阶段的花粉发育先于绒毡层程序性细胞死亡。芽 ?长度超过1.2mm(图B中的括号底行)或下面 长度为0.7mm(在花簇中,包围在图B中) 丢弃。 C.芽显示暴露的雄蕊和心皮,后萼片和 花瓣去除。 D.花药从花花中除去之后,之前 ?淹没在水中。花药由解剖造成的损害(如 如圆圈的花药缺乏丝和看起来蜷缩) 丢弃。 E.将花药浸没在蒸馏水中,低于石蜡 密封盖玻片。与气泡相关的花药不成像。 F。 ?将盖住微孔和浸没的花药的盖玻片密封 所有方面的培养皿用石蜡。

    2. 样品装载和校准。
      将大水滴放在样品盖玻片上并放低 物镜直到其被部分浸没。装上滤光轮 到DICT位置用于透射光。转动透射光 灯(在FluoView下的图像采集控制下)并拉动两者 显微镜滑块到向外位置以允许光移动到 ?眼睛。使用舞台控制操纵杆和对焦遥控器 控制器(图2C),以您的感兴趣区域为中心,并带来您的 标本成焦点。关闭透射光并移动过滤器 轮到位置2(R690)用于双光子成像。
    3. 进入双光子成像模式。
      在FluoView程序的图像采集控制面板中,选择染料 ?列表菜单。在选择"Two Photon"之前,从列表中清除所有染料。
    4. 检查光路。
      对于感兴趣的通道中的发射捕获,移动SDM570 二向色镜进入光路进行四通道成像 两个显微镜滑块向内。这种配置允许发射 从通道1-4(RXD1-4,图5)收集。在Quilichini等人 al。(2014)研究,羟基肉桂酸的排放和相关 在420-460的低波长发射范围内收集化合物 ?nm(在RXD1),而叶绿素和相关化合物的排放 收集在更高的波长,从495-540 nm(在RXD2)。
      注意: 该协议中描述的双光子显微镜系统包括a ?在PMT前面没有针孔的单独的非转换光路。 ?然而,在其他系统上打开双光子成像的针孔 可能需要常规的共焦能力。


      图5。 用于在Olympus FV1000MPE多光子上进行双光子成像的光路 显微镜。用于成像花药的正确光路 自发荧光,检查激光路径(由黄线描绘) 在图像采集控制面板的光路和染料窗口中 ?包括在720nm的激光单元3,激发二向色镜, RDM690,以及正确的发射捕获通道RXD1和RXD2
    5. 图像采集参数。
      在Fluoview中,通过选择启用两个感兴趣的发射通道 RXD1和RXD2在图像采集控制窗口内。扫描期间 和图像采集,实时查看窗口可以配置为显示 RXD1和RXD2发射,或这些信道的覆盖。不同 通过实时查看窗口可以显示伪色选项 在LUT下。推荐使用Hi-Lo显示功能,因为它提供 关于成像区域上的发射强度的信息。使用 这种显示,曝光过度的像素会出现红色,而曝光不足, 昏暗的像素显示为蓝色。激光功率和通道电压设置,如 详细在下面的协议中,被选择以最大化信号 来自发育中的花药内的细胞类型的混合物的强度, 同时避免样品破裂和像素饱和。
    6. 最小化样品损坏。
      对准期间,使用快速扫描(称为对焦x 2或对焦x 4) 对齐您的样品,而不会导致显着的样品漂白。 使用xy扫描功能收集图像以收集单个帧 图片。要收集z堆栈,请为您的开始和结束z位置设置 感兴趣的样品,通常围绕一个花药室,具有1μm ?步长。通过选择深度(在xy扫描下)捕获z-stack 函数),然后单击xy扫描。
    7. 最大限度减少光污染。
      双光子系统中的检测器不受针孔保护,如 在常规的共聚焦显微镜,以确保所有的光在焦点 体积。为了最小化从外部光源收集的光, ?通过密封显微镜确保房间照明最小化 在不透明的外壳内,在暗室中执行成像,以及 在黑暗视图中操作Fluoview程序
    8. 拟南芥花药的最佳成像参数。
      有关采集设置和图像采集,请参见下表 控件。当使用不同的放大倍数时,建议使用 ?相应地调整宽高比,以保持像素大小接近 到0.2μm/像素。

      激光功率
      3%
      激光波长(红外功率)
      720 nm(2.49 W)
      扫描模式
      XY
      扫描方向
      单程
      图片大小(宽高比)
      800 x 800像素(1:1)
      缩放
      3x
      像素大小
      0.212μm/像素
      像素驻留时间
      4.0μs/像素
      Z堆叠步长
      1.0微米
      集成类型
      卡尔曼线平均
      整合计数
      3
      RXD1 HV,Gain,Offset
      650 V,1,无
      RXD2 HV,Gain,Offset
      650 V,1,无

    9. 校正深度处的亮度。
      改善位于深层下方的荧光团的排放捕获 表面,激光功率或高电压(HV,其控制 施加在PMT上的电压,采集后)可以调节 不同的z位置(深度)。这可以是一个有用的工具,特别 对于有价值的信息位于深处的大样本。 虽然此功能可用于成像模糊样本或腔 位于大深度,耗时,可引起试样 如果激光功率设置太高则破裂,并且最佳设置结束 z系列通常是样品特异性的。为了一致性 并且因为感兴趣的另一组织足够亮 ?没有这些调整,这个校正不适用于 成像在我们的研究(Quilichini等人,,2014)。
    10. 图像处理。
      将每个图像或z-stack保存为.oif文件,以便在Volocity v ?6.1.1,或将图像序列导入装有ImageJ的ImageJ Bio-Formats插件(Abramoff等人,2004; Linkert等人,2010)。图片 ?处理通常包括对来自的发射应用伪色 ?每个通道,将通道叠加到一个合并的图像或堆栈中, 以一致的方式调整亮度和/或对比度,添加 缩放条,或者从z-stack创建z投影。
    11. 仪器关闭。
      成像后,将激光器返回到800 nm,然后关闭激光器 功率。在显微镜和通过使用的软件关闭快门 ?以操作激光器。将物镜移动到最高位置 在关闭Fluoview程序之前。

笔记

鼓励读者参考奥林巴斯显微镜资源中心(链接下面)关于多光子荧光显微镜的理论和应用的进一步信息。
http://www.olympusmicro.com/primer/techniques/fluorescence/multiphoton /multiphotonintro.html

致谢

我们感谢不列颠哥伦比亚大学生物图像设施,特别是Kevin Hodgson,提供培训,技术援助和讨论。这项工作是由加拿大自然科学,工程研究委员会(NSERC)发现补助和墙上工作NSERC创建程序的支持。该方案用于拟南芥中花粉壁形成的研究(Quilichini等人,2014)。

参考文献

  1. Abramoff,M.D.,Magalhaes,P.J.and Ram,S.J。(2004)。 ImageJ的图像处理。 Biophotonics International 11(7):36- 42.
  2. Linker,M.,Rueden,CT,Allan,C.,Burel,JM,Moore,W.,Patterson,A.,Loranger,B.,Moore,J.,Neves,C.,Macdonald,D.,Tarkowska, A.,Sticco,C.,Hill,E.,Rossner,M.,Eliceiri,KW和Swedlow,JR(2010)。 元数据重要:可访问现实世界中的图片数据 Biol 189(5):777-782
  3. Olympus FV1000 MPE显微镜用户指南。
  4. Quilichini,T. D.,Samuels,A.L。和Douglas,C.J。(2014)。 ABCG26介导的聚酮化合物运输和羟基肉桂酰基spermidine促成拟南芥中的花粉壁外壁形成,植物细胞 26(11):4483-4498。
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How to cite this protocol: Quilichini, T. D., Samuels, A. L. and Douglas, C. J. (2015). Analysis of Developing Pollen Grains within Intact Arabidopsis thaliana Anthers by Olympus Two-Photon Laser Scanning Microscopy . Bio-protocol 5(23): e1677. DOI: 10.21769/BioProtoc.1677; Full Text



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