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Basic building units and properties of a fluorescence single plane illumination microscope
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16.See EPAPS Document No. for movies of 1)comparison of the bleaching in SPIM and DeltaVision, 2) the drosophila muscle development and 3) rotating maximum projections of a microtubule aster. These documents can be reached via a direct link in the online article’s HTML reference section or via the EPAPS homepage (http://www.aip.org/pubservs/epaps.html).[Supplementary Material]


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FIG. 1.

The SPIM principle. (A) The central element of a SPIM is an essentially regular fluorescence microscope. It consists of an objective lens, a filter, a tube lens, and a wide-field detector. In contrast to the epifluorescence arrangement of conventional wide-field and confocal microscopes, no dichroic beam splitter is required to separate the illumination and detection light. Instead, the specimen is illuminated from the side by focusing a collimated laser beam to a light sheet with a cylindrical lens. Hence, only a thin volume around the geometric focal plane of the objective lens is illuminated. (B) In the specimen, only those fluorophores that are actually observed are also illuminated. This leads to optical sectioning and does not generate photodamage outside the focal plane. (C) A single image out of a stack of 500 planes, which was recorded about inside a fixed PAX6 in situ medaka embryo (Ref. 17), demonstrates the optical sectioning capability, the excellent signal to noise ratio, and the extremely low background. (D) Sagittal projection of the stack of 500 images recorded at different depths. The dashed line indicates the plane in which the image shown in C was recorded. Recording conditions: , detection filter Chroma HQ , detection lens Carl Zeiss Fluar , scale bar , recording time /image, image size , . Sample prepared by Mirana Ramialison.

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FIG. 2.

The building units of a SPIM. The detection unit is a simplified fluorescence wide-field microscope. An objective lens, a filter, and a tube lens form the fluorescence image on the wide-field detector (CCD). The sample can be immersed in a medium-filled chamber for optimal experimental conditions. The illumination unit generates the light sheet for the illumination of the focal plane in the sample. A beam expander (BE) adjusts the diameter of a collimated beam and feeds it into a cylindrical lens. The laser unit delivers a collimated beam to the illumination unit. An acousto-optic tunable filter (AOTF) picks at least one of the several laser wavelengths and adjusts its intensity. The movement unit holds the sample and moves it relative to the optical setup. Three translational and one rotational stages allow positioning and scanning of the sample. Finally, the control unit, a standard computer equipped with data acquisition boards, controls the hardware and acquires the data. Additional optical elements such as beam couplers and splitters for auxiliary units can be introduced in the infinity corrected space (ICS) between the objective lens and the tube lens and will require further lasers and other optical components not shown here.

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FIG. 3.

Optical layout of illumination unit. (A) The most basic setup to create a light sheet uses a single cylindrical lens, which is used to focus a collimated beam into the sample. An adjustable slit adapts the numerical aperture (NA) of the illumination lens and thus the thickness of the light sheet. A second, perpendicular slit (not shown) adapts the field of view, thus making sure that no unobserved parts of the sample are illuminated. (B) The intensity distribution of a simulated light sheet is calculated for a wavelength of and an illumination NA of 0.05 (units along both directions are in millimeters, please note the different scales). The geometry of the light sheet is adjusted to the size of the field of view, which we suggest to be twice the Rayleigh range (indicated by the two lines marked with ). The light sheet is a factor of thicker and a factor of dimmer at the edge of the field of view than in its center. This optimizes the trade-off between the thickness of the light sheet and its uniformity over the area of interest. (C) An improved setup is used to create a diffraction limited light sheet in the SPIM. The optical components and ray traces are shown in planes perpendicular (top) and parallel (bottom) to the light sheet. The adjustable gimbal-mounted mirror allows the light sheet to be aligned to the focal plane of the detection unit (not shown). Lenses L1 and L2 form a relay telescope, and the cylindrical lens and objective (obj) generate the light sheet. BFP: back focal plane.

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FIG. 4.

Sample handling. The sample is embedded in a cylinder of a agarose gel. A glass capillary (e.g., with an inner diameter of ) or a syringe (inner diameter of ) holds the agarose gel. The sample is inserted from above into an aqueous-medium-filled chamber in the SPIM. The refractive index of the agarose and the surrounding aqueous medium differs by only about 0.04%, which avoids image degradation. The capillary or syringe can be translated along three axes and rotated around its center. The rotation axis is parallel to gravity to avoid sample deformation during rotation. The latter feature allows the sample to be accessed along all sides. This is a prerequisite for multiview imaging and the reconstruction of hidden or blurred features. The insert shows the projections through stacks of images of the same sample shown in Fig. 1, but acquired along five additional directions. Each of the six views shows different details in the embryo.

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FIG. 5.

Flowchart of the hardware control and image acquisition program. The kernel of SPIM’s control program performs the image acquisition. It is optimized to minimize the exposure of the specimen to light. The status of the peripheral components (stages, filter wheel, and lasers) is checked prior to every sample illumination, thus making sure that everything is ready for the next image acquisition. The time needed for the image transfer from the camera to the computer and for saving the data to disk is used to set the hardware (camera settings, stage and filter positions, and the AOTF) for the next exposure. To ensure minimal exposure, the laser is switched on just prior to the image acquisition and switched off immediately after the sample has been imaged, via the AOTF. The portion of the program responsible for the acquisition of time lapses is placed external to the central loop and restarts the core routine according to time interval and duration chosen by the user.

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FIG. 6.

Examples of images recorded with high numerical aperture. [(A) and (B)] The maximum-intensity projections of a three-dimensional stack of a tubulin-Alexa-488-labeled aster after it formed in BRB80 buffer. (A) The projection along the detection axis illustrates the diffraction limited resolution of the objective lens in the lateral direction. No background is visible, because out-of-focus fluorophores are not excited. Scale bar . (B) The projection along the illumination axis demonstrates the axial resolution of the system. The detection axis is horizontal in this image. Scale as in A. (C) Maximum-value projection of a part of a stack showing Ady2-GFP-labeled yeast cells. This is one frame out of a time lapse (movie included as online supplement S1) with 191 time points. This long movie is possible because of the reduced photobleaching in the SPIM, when compared to other sectioning microscopic techniques. , detection filter Chroma HQ 500 LP, detection lens Carl Zeiss Achroplan , scale bar . Microtubule aster prepared by Francesco Pampaloni and yeast sample prepared by Christof Taxis.


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Table I.

The basic building units of the SPIM setup.

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Table II.

Calculated point spread function (PSF) extents for different lenses and imaging systems. Extents are provided as a pair of numbers. The upper value is the lateral extent, and the lower the axial extent. They are determined by the full width at half maximum (FWHM) of the PSF. For mvSPIM the extent is isotropic, so only one value is given. We consider conventional (cvFM), confocal (cfFM), and two-photon fluorescence microscopes, and single- and multiview SPIMs (mv). Objectives are designed for use in either air (A) or water (W). Illumination occurs at or (all others), detection above . The size of the field of view of the SPIM is provided in the final column, assuming a pixel pitch of and .


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The critical issue of all fluorescencemicroscopes is the efficient use of the fluorophores, i.e., to detect as many photons from the excited fluorophores as possible, as well as to excite only the fluorophores that are in focus. This issue is addressed in EMBL’s implementation of a light sheet based microscope [single plane illuminationmicroscope (SPIM)], which illuminates only the fluorophores in the focal plane of the detection objective lens. The light sheet is a beam that is collimated in one and focused in the other direction. Since no fluorophores are excited outside the detectors’ focal plane, the method also provides intrinsic optical sectioning. The total number of observable time points can be improved by several orders of magnitude when compared to a confocal fluorescencemicroscope. The actual improvement factor depends on the number of planes acquired and required to achieve a certain signal to noise ratio. A SPIM consists of five basic units, which address (1) light detection, (2) illumination of the specimen, (3) generation of an appropriate beam of light, (4) translation and rotation of the specimen, and finally (5) control of different mechanical and electronic parts, data collection, and postprocessing of the data. We first describe the basic building units of EMBL’s SPIM and its most relevant properties. We then cover the basic principles underlying this instrument and its unique properties such as the efficient usage of the fluorophores, the reduced photo toxic effects, the true optical sectioning capability, and the excellent axial resolution. We also discuss how an isotropic resolution can be achieved. The optical setup, the control hardware, and the control scheme are explained in detail. We also describe some less obvious refinements of the basic setup that result in an improved performance. The properties of the instrument are demonstrated by images of biological samples that were imaged with one of EMBL’s SPIMs.


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Scitation: Basic building units and properties of a fluorescence single plane illumination microscope