ABSTRACT
Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies enlarge the scope of investigations to multi-cellular phenomena. A the highest level of complexity, brain organoids that – in many aspects – recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that belongs to the class of two-photon (2P) light-sheet microsocpes. Its particularity is that – unlike existing two- or three-lens designs – it is using a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petran spinning-disk scheme for achieving wide-field excitation. However, unlike the common Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This design, advantageous for 2P excitation circumvents problems arising with the tandem disk from the large wavelength-difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited in by the light sheet is collected by the same objective and imaged onto a fast sCMOS camera. We demonstrate three-dimensional imaging of TO-PRO3-stained embryonic bodies and of brain organoids, under control conditions and after rapid (partial) transparisation with triethanolamine and /ormamide (RTF) and compare the performance of our instrument to that of a confocal microscope having a similar numerical aperture. 2P-virtual light-sheet microscopy permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.
Footnotes
↵+ co-first authors
↵§ co-last authors
↵* Elements of this work has been published in the Master thesis of I.R. [1]
The authors declare not conflict of interest. Rainer Uhl is the founder and owner of TILL.id, Andreas Deeg and Christian Seebacher are employees of TILL.id.
The funders of this study had no hands on the outcome or interpretation of the obtained results.
List of abbreviations
- 2P
- two-photon
- 3-D
- three-dimensional
- AOI
- angle of incidence
- BSA
- bovine serum albumin
- CLSM
- confocal laser scanning microscope
- DMEM
- Dulbecco’s modified Eagle medium
- EB
- embryonic body
- FWHM
- full-width at half maximumhi
- PSC
- human inducible pluripotent stem cell
- LP
- long-pass (filter)
- MG
- Magnesium Green
- NA
- numerical aperture
- OASIS
- On-axis 2-photon light-sheet generation in-vivoimaging system
- OCT
- opimal cutting temperature
- PBS
- phosphate-buffered solution
- PEG
- polyethylene glycol
- RI
- refractive index
- ROI
- region of interest
- RTF
- Rapid clearing method based on Triethanolamine and Formamide
- sCMOS
- scientific Complementary Metal Oxide Semiconductor
- SD
- standard deviation
- SPIM
- selective-plane illumination
- TDE
- 2,2’-thiodi ethanol
- TO-PRO-3
- a carbocyanine monomer nucleic acid stain with red excitation and far-red fluorescence (642 nm/661 nm) similar to Alexa Fluor 647 or Cy5. It is among the highest-sensitivity probes for nucleic acid detection.