The monolithic light sheet-based fluorescence microscope (mDSLM)

1Patrick Theer, 1Fabian Haerle, 1Alfons Riedinger, and 1,2Ernst H. K. Stelzer1European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany

2Physical Biology (FB15, CEF-MC, BMLS), Goethe Universität, Max-von-Laue-Str. 15, D-60438 Frankfurt am Main, Germanyernst.stelzer@physikalischebiologie.de

Introduction: Our design for a LSFM, which relies on the DSLM principles, is based on a monolithic platform, which provides a high stability and a small footprint. Self-calibrating and -monitoring optics and electronics ensure an optimal performance under real-time conditions in a multi-user environment. The specimen is moved by four motors below the specimen chamber. The specimen is inserted from above but otherwise freely accessible through the top of the specimen chamber. Vertical line-scanning of a diffraction limited Gaussian beam generates the light sheet. Hence, the light sheet is not static as in a SPIM but dynamic. The laser power can be modulated arbitrarily and synchronously with the beam movement providing e.g. structured illumination. Piezo-actuators scan and position the light-sheet as well as the illumination and detection lenses. The system is com-pletely self-regulated. The mDSLM hardware is controlled by a real-time PC and is a device with an IP-address in a net-work. A second PC provides the user interface. Further processors, such as an Arduino, can be used to control e.g. external light sources and pumps. Their operations are triggered by and synchronized with the actual recording process.

six filters in a wheel96A351 (Ludl)

CCD/CMOS-CameraClara or Neos (Andor)

tube lens f = 164.5 mm

425308 (Zeiss)

broadband telecentricf-theta scan lens, f=60.52 mm

S4LFT0061/065 (Sill Optics)

piezo tip-tilt scannerS-334.2SL (Physik Instrumente)

x/y/z/ motorized stages,3 x M-111.2DG and 1x M-116.DG

(Physik Instrumente)

piezo objective scannerP-725 PIFOC

(Physik Instrumente) specimen chamberwith free access from top

broadband monomodeoptical fiber and

collimator (Pointsource)

monolithic platform

user interface

real-time controller electronicsand laser light engine (SOLE, Omikron)

Seminal References:

Strobl, F., & Stelzer, E. H. K. (2014). Non-invasive long-term fluorescence live imaging of Tribolium castaneum embryos. Development (Cambridge, England), dev.108795–. doi:10.1242/dev.108795Vermeer, J. E. M., von Wangenheim, D., Barberon, M., Lee, Y., Stelzer, E. H. K., Maizel, A., & Geldner, N. (2014). A spatial accommodation by neighboring cells is required for organ initiation in Arabidopsis. Science , 343(6167), 178–83.Maizel, A., von Wangenheim, D., Federici, F., Haseloff, J., & Stelzer, E. H. K. (2011). High-resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy. The Plant Journal, 68(2), 377–85.Keller PJ, Schmidt AD, Santella A, Khairy K, Bao Z, Wittbrodt J, Stelzer EHK (2010) Fast high-contrast imaging of animal development with scanned light sheet-based structured illumination microscopy, Nat Methods, 7(8):637-42Keller PJ, Schmidt AD, Wittbrodt J, Stelzer EHK (2008) Reconstruction of zebrafish early embryonic development by Scanned Light Sheet Microscopy, Science, 322(5904):1065-1069 Verveer PJ, Swoger J, Pampaloni F, Greger K, Marcello M, Stelzer EHK (2007) High-resolution three-dimensional imaging of large specimens with light-sheet based microscopy, Nat Methods, 4:311-313Swoger J, Verveer PJ, Greger K, Huisken J, Stelzer EHK (2007) Multi-view image fusion improves resolution in three-dimensional microscopy, Optics Express, 15(13):8029-8042.Greger K, Swoger J, Stelzer EHK (2007) Basic building units and properties of a fluorescence single plane illumination microscope, Rev Sci Instrum., 78(2):023705Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer EHK (2004) Optical sectioning deep inside live embryos by selective plane illumination microscopy, Science, 305:1007-1009Stelzer, E. H. K., & Lindek, S. (1994). Fundamental Reduction of the Observation Volume in Far-Field Light-Microscopy by Detection Orthogonal to the Illumination Axis - Confocal Theta Microscopy. Optics Communications, 111(5-6), 536–547.

Set-up:

single-sided set-up

double-sided set-up

schematic of light sheet generation

Recording of entire process of lateral root formation. (A) Three-dimensional reconstruction of lateral root growing out of primary root. (B) Single slices along x-y (front view) 10 μm inside epidermis cell layer. (C) Single slices along z-y (sideview) 80 μm deep inside primary root. (D) Single slices along y-z (radial view) through center of primordium. Six days old Arabidopsis stably expresses UBQ10::YFP-PIP1;4 (membranes, green), UBQ10::H2B-RFP (nuclei, red) and GATA23::GUS-GFP-NLS (promotor activity, nuclear localization signal, green). Time points are relative to gravity stimulation. Images were collected with 40x/0.75 ob-jective lens in detection path and 5x/0.16 objective lens in illumination path. Scale bar: 20 μm. (Daniel von Wangenheim).

(Keller et al. Science 2008)