LATEST UPDATES » Vol 22, No 05, May 2018 – Dental health - The root to good health       » Drugs to watch 2018       » Dragonfly-inspired nano coating kills bacteria upon contact       » New compound improves stroke recovery in monkeys and mice       » Cancer stem cell therapy breakthrough       » Draft genome of tea plant sequenced      
Putting Numbers into Biology: The Combination of Light Sheet Fluorescence Microscopy and Fluorescence Spectroscopy
by Xue Wen Ng1,2, Thorsten Wohland1,2,3,*

1Department of Chemistry, National University of Singapore, Singapore
2Center for BioImaging Sciences, National University of Singapore, Singapore
3Department of Biological Sciences, National University of Singapore, Singapore
*: Corresponding author

The development of light microscopy is closely linked to requirements in the biological sciences. The invention of the optical microscope made the observation of the intricate organization of biological organisms possible for the first time. Since then, each new development in modern microscopy, including phase contrast, fluorescence and confocal microscopy, brought biologists closer to their goal of observing subcellular structures and/or specific single biomolecules within live specimens in 3D. The latest addition in this list of microscopy techniques is light sheet fluorescence microscopy (LSFM).

Named Method of the Year 2014 by Nature Methods, LSFM is a superior imaging technique that offers high image quality with good signal-to-noise ratios. [1] The first implementation of LSFM, called selective plane illumination microscopy (SPIM), has the illumination and detection pathways aligned orthogonal to each other. The illumination is accomplished by a light sheet that can illuminate a whole cross-section of a specimen, while the detection objective captures the emitted fluorescence and images that whole cross-section on a camera. This has multiple advantages. The fact that optical sectioning can be achieved directly from the illumination path allows the entire plane illuminated by the light sheet to be registered while other planes are not excited. This greatly reduces photobleaching of fluorophores, photodamage to the sample and enables better time resolution. The light sheet intersects the focal plane of the detection objective. Fluorescence collected by the detection objective is then spectrally filtered by emission filters and imaged onto sensitive array detectors. The combination of light sheet illumination along with array detection allows for the simultaneous imaging of an entire plane, as opposed to sequential scanning in confocal microscopy, thus providing much higher time resolution that is solely determined by the detector speed. Furthermore, the excellent penetration depth of SPIM, ~500 μm in a Medaka embryo with a 6 μm axial resolution [2], and its minimum invasive approach makes it the ideal technique to study 3D processes in live organisms.

The next step in LSFM is the quantification of molecular events by combining LSFM techniques with fluorescence spectroscopy approaches. One such example is the combination of LSFM with fluorescence recovery after photobleaching (FRAP) that quantifies the mobility of biomolecules in live samples by monitoring the recovery rate of fluorescence upon photobleaching a pre-defined region of interest with a pulse of high laser power. In a novel application, LSFM-FRAP was used to quantify the rate of synthesis of two fluorescently-expressing proteins involved in the regulation of mRNA translation in live transgenic Caenorhabditis elegans. [3] To study 3D biological processes in the molecular level, single molecule tracking (SMT), another advanced spectroscopy method, was adapted in LSFM. SMT tracks molecular movement by monitoring the mean squared displacement (MSD) of individual molecules with time. The combination of light sheet illumination and high-speed imaging with an electron multiplying charge coupled device (EMCCD) camera enabled the tracking of fast-moving single molecules in 3D biological samples with high sensitivity, contrast, signal-to-noise ratio, and spatiotemporal resolution at large sample depths. The lab of Prof Kubitscheck successfully applied LSFM-SMT 200 μm deep into the living salivary glands tissue of Chiromonus tentans larvae to track the mobility of Balbiani Rings mRNA particles (BR mRNPs) in the cell nucleus. [4] In a recent study, SMT was adapted in reflected light-sheet microscopy, also known as single-objective SPIM [5], which utilizes a microprism-attached standard coverslip that reflects the light sheet onto the sample for ease of sample preparation. [6] The authors quantified three diffusing components of 10 kDa Dextran-Alexa647 molecules in live Drosophila embryos. The fast component likely corresponds to freely-diffusing molecules while the slower components were shown to have directional motion possibly due to active transport of the Dextran molecules in endosomes.

However, the requirement for low labeling density and bright fluorophores for SMT measurements makes it challenging to measure functional proteins in organisms and severely limits the application range in vivo. Therefore, in another pairing of light sheet microscopy and fluorescence spectroscopy, our lab combined fluorescence correlation spectroscopy (FCS) with SPIM (SPIM-FCS) which is viable with more commonly used fluorophores such as fluorescent proteins at physiological expression levels. SPIM-FCS is a camera-based FCS modality that simultaneously registers multiplexed outcomes of FCS measurements, namely diffusion and number of particles, by combining the plane illumination of SPIM and a fast array detector such as an EMCCD camera. [7,8] A SPIM-FCS measurement provides quantitative maps of diffusion coefficient and number of particles of a single sample plane. The deep penetration depth of light sheet illumination enables us to measure protein diffusion in live organisms. In a novel study, our group had recently quantified the membrane diffusion of functional Wnt3-EGFP signaling proteins in the cerebellar cells of live transgenic Wnt3-EGFP zebrafish embryos with SPIM-FCS. [9] Furthermore, we adopted the FCS diffusion law [10] to SPIM-FCS measurements [11] to study the sub-resolution membrane organization in terms of transient trapping of Wnt3 in plasma membrane nanodomains of live zebrafish embryos. The FCS diffusion law monitors the spatial dependence of the diffusion of membrane probes to elucidate the type of membrane organization involved. The theory of the FCS diffusion law, its different variations and applications had been reviewed here [11]. Our results revealed that the palmitoylation of Wnt3 by Porcupine, a membrane-bound O-acyltransferase, is necessary for its confined diffusion in membrane domains in the cerebellar cell membranes of live transgenic zebrafish embryos. The association of Wnt3 with the membrane domains was shown to decrease with increasing dosage of Porcupine inhibitor (C59). We postulated these observations to originate from a possible reduction of membrane-bound Wnt3 in the cerebellar cells and a possible change in the palmitoylation states of Wnt3 from bi-palmitoylated to mono-palmitoylated Wnt3 in the membrane (Figure 1). Wnt3 was also found to be associated to cholesterol-dependent membrane domains in live zebrafish embryos since its domain confinement decreased with the reduction of cholesterol levels by cholesterol-depleting drugs. The association of Wnt3 to cholesterol-dependent membrane domains and the importance of palmitoylation in its membrane domain confinement could very likely be involved in the proper signaling of Wnt3 in vivo. Therefore, this showcases the competence of SPIM-FCS to measure dynamics at different spatial scales in vivo to determine sub-resolution membrane organization of functional proteins through its diffusion mode as characterized by the FCS diffusion law.

The basis of quantitative bioimaging is the ability to decipher intrinsic properties of biological processes at the molecular level. This can be achieved by combining microscopy (to precisely locate a specific region of interest on the sample) and fluorescence spectroscopy (to quantitate molecular properties) without much technical difficulty. The optical sectioning and depth penetration ability of LSFM makes it an ideal microscopy technique for high resolution imaging in live organisms. Therefore, the union of LSFM with fluorescence spectroscopy techniques such as SMT and FCS provides an unprecedented capacity for the quantification of biology at the molecular level in vivo. With the advancement of LSFM technology, it is possible to simultaneously obtain macroscopic information along with quantitative microscopic information in the same organism to accurately compare the time dependent outputs at each spatial scale. Considering that LSFM is a relatively new technique it will be exciting to see what the next years will bring. Already many new LSFM modalities are proposed and implemented in labs worldwide, each with different capabilities and new perspectives for combinations with spectroscopy techniques. [12] These will offer new possibilities of putting numbers into biology by providing detailed quantitative information on molecular processes even in live organisms.


  1. (2015) Methods of the year 2014. Nat Methods 12, 1.
  2. Huisken, J., Swoger, J., Bene, F. Del, Wittbrodt, J., and Stelzer, E. H. K. (2004) Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy. Science 305, 1007–1009.
  3. Rieckher, M., Kyparissidis-Kokkinidis, I., Zacharopoulos, A., Kourmoulakis, G., Tavernarakis, N., Ripoll, J., and Zacharakis, G. (2015) A customized light sheet microscope to measure spatio-temporal protein dynamics in small model organisms. PLoS One 10, 1–15.
  4. Ritter, J. G., Veith, R., Veenendaal, A., Siebrasse, J. P., and Kubitscheck, U. (2010) Light sheet microscopy for single molecule tracking in living tissue. PLoS One 5, e11639.
  5. Galland, R., Grenci, G., Aravind, A., Viasnoff, V., Studer, V., and Sibarita, J.-B. (2015) 3D high- and super-resolution imaging using single-objective SPIM. Nat. Methods 12, 641–644.
  6. Greiss, F., Deligiannaki, M., Jung, C., Gaul, U., and Braun, D. (2016) Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy. Biophys. J. 110, 939–946.
  7. Wohland, T., Shi, X., Sankaran, J., and Stelzer, E. H. K. (2010) Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments. Opt. Express 18, 10627–10641.
  8. Singh, A. P., Krieger, J. W., Buchholz, J., Charbon, E., Langowski, J., and Wohland, T. (2013) The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy. Opt. Express 21, 8652–8668.
  9. Ng, X. W., Teh, C., Korzh, V., and Wohland, T. (2016) The Secreted Signaling Protein Wnt3 Is Associated with Membrane Domains In Vivo: A SPIM-FCS Study. Biophys. J. 111, 418–429.
  10. Wawrezinieck, L., Rigneault, H., Marguet, D., and Lenne, P.-F. (2005) Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization. Biophys. J. 89, 4029–4042.
  11. Ng, X. W., Bag, N., and Wohland, T. (2015) Characterization of Lipid and Cell Membrane Organization by the Fluorescence Correlation Spectroscopy Diffusion Law. Chim. Int. J. Chem. 69, 112–119.
  12. Lim, J., Lee, H. K., Yu, W., and Ahmed, S. (2014) Light sheet fluorescence microscopy (LSFM): past, present and future. Analyst 139, 4758–68.
About the Authors

Thorsten Wohland studied Physics at the Technical University of Darmstadt and the University of Heidelberg in Germany. He completed his diploma thesis in physics at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, with Ernst H.K. Stelzer, and his PhD with Horst Vogel at the Swiss Federal Institute of Technology in Lausanne (ETHL/EPFL), Switzerland. Following a postdoc with Richard N. Zare at Stanford in the USA he joined the National University of Singapore (NUS) as Assistant Professor in June 2002. In June 2008 he was promoted to Associate Professor and in January 2016 to Professor. His interests focus on the development of fluorescence microscopy and spectroscopy techniques to measure molecular processes in live cells and organisms.
Email: twohland@nus.edu.sg

Xue Wen Ng obtained her Bachelors of Science (Honours) degree in Chemistry on 2013 at the National University of Singapore (NUS). She is supported by a NUS research scholarship and is currently (2014 to current) undertaking her doctoral degree in the research group of Thorsten Wohland at NUS. The topics of interest in her doctoral studies include the applications of single plane illumination microscopy-fluorescence correlation spectroscopy (SPIM-FCS) to study the molecular dynamics and localization of biomolecules in live cells and organisms, and technical improvements in SPIM-FCS.
Email: a0054184@u.nus.edu

Click here for the complete issue.

news Shire, Microsoft and EURORDIS form Global Commission to accelerate time to diagnosis for children with rare diseases
news EmTech Asia explores future of life, humanity and economy
news Biology of Ageing II - Impactful Interventions
Asia Pacific Biotech News
Subscribe to APBN E-Newsletter
Find us under 'Others' option to receive APBN e-newsletters thrice a month!

APBN Editorial Calendar 2018
Obesity / Outlook for 2018
Searching for the fountain of youth
Women in Science - Making a difference
Digestive health in the 21st century - Trust your guts
Dental health - The root to good health
Oncology / Biotech landscape in APAC
Water management / Vaccination
Regenerative medicine / Biotech start ups
Digital healthcare / 3D printing
Bones / Breast cancer
Liver health / Top science research nations & institutions
AIDS / Breakthrough of the year/Emerging trends
Editorial calendar is subjected to changes.
About Us
Available issues
Editorial Board
Letters to Editor
Instructions to Authors
Advertise with Us
World Scientific Publishing Co. Pte. Ltd.
5 Toh Tuck Link, Singapore 596224
Tel: 65-6466-5775
Fax: 65-6467-7667
» For Editorial Enquiries:
   biotech_edit@wspc.com or Ms Lim Guan Yu
» For Subscriptions, Advertisements &
   Media Partnerships Enquiries:
Copyright© 2018 World Scientific Publishing Co Pte Ltd  •  Privacy Policy