Looking Downside Up
- First and the Only System for in toto Imaging of Mammalian Development from Zygote to Blastocyst
- Very High Imaging Speed
- Maximized Photo-eﬃciency
- Close-to-Confocal Resolution
- Long-term 3D Fluorescence Imaging of Live Specimens
In toto imaging of biological model organisms such as worm, fly and fish has revolutionized the understanding of cell division and cell fate specification in their early development. However, because of high light sensitivity and demanding in vitro culture requirements, in toto imaging of mammalian pre-implantation embryos has not been possible, and a number of important questions, including the time of cell fate specification, remain controversial as a result. In addition, because of the high degree of variability in early mammalian development, a statistical analysis of accurately reconstructed cell lineages of several embryos is a prerequisite to drawing solid conclusions. The need to place the specimen in the narrow space between the illumination and detection objectives, however, has led to light-sheet microscope designs in which the specimen is held vertically by embedding into agarose cylinders or small capillaries, which is incompatible with mammalian embryo development.
To overcome this limitation, we developed an inverted light-sheet microscope in which both the illumination and the detection objective face upward in an immersion water reservoir. The specimen is held by gravity in a long, narrow channel of a microscope slide–sized holder and can therefore be lowered between the two objectives, much like a standard glass-bottom dish on an inverted microscope. Because the transparent plastic of the sample channel has the refractive index of water, the specimen is optically fully accessible for high-resolution immersion objectives, yet it is physically isolated from the immersion medium and can be maintained in standard microdrop in vitro embryo culture. Full environmental incubation of the imaging system allows maintenance of stable temperature and atmospheric composition. Furthermore, free movement along the long axis of the specimen channel makes it possible to place many embryos in a row for high-throughput imaging.
Invi-SPIM system is dedicated to live imaging, It is conﬁgured as an inverted microscope that has been optimized for long-term 3D ﬂuorescence imaging of living specimens. Easy access to the sample chamber, maximized photo- eﬃciency, and short illumination times enable long-term imaging without harming live specimens. The optical conﬁguration combined with the fast acquisition speed of InVi- SPIM enables 3D reconstruction, tracking of cellular and subcellular positions, and morphological analysis in real time. InVi-SPIM system use scanned light sheet technology, with adjustable light sheet thickness of 2-8 um and ﬂexible sheet area. It also has 2 detection modes: Area mode for fast and high sensitivity imaging, and Line scanning mode for background suppression.
The InVi-SPIM provides a unique autoclavable, biologically inert sample chamber with accurate temperature control and optional environmental control. Sample can be easily accessed for patch clamp, photoactivation and/or photoablation.
Typical applications of InVi-SPIM are in toto imaging of small animal models such as whole mouse embryos, imaging of dynamic processes in mammalian cell culture applications, and even live imaging of intact and living plant models.
|Laser||Laser combiner with six laser positions|
|445, 488, 515, 532, 561, 594, 642, and 685nm @ 50mW|
|Fast modulation and high extinction|
|Chromatic correction from 440 to 660nm|
|Light-sheet generation by beam scanning|
|Variable light-sheet thickness (optional)|
|Detection Optics||Water-dipping objective lenses|
|Fast filter wheels with 10 positions and 50ms between adjacent positions|
|Illumination objective: 10x @0.3NA, water immersion|
|Detection objective: 25x @1.1NA, water immersion|
|High-speed sCMOS camera Hamamatsu ORCA-Flash 4.0|
|Sample Chamber and Stage||Water-sealed inert PEEK plastic chamber, autoclavable and biocompatible|
|Sample supported from below for improved stability|
|Easy access from above for sample mounting and manipulation|
|Fast and precise temperature control, range 15-40oC|
|Environmental control (optional)|
|3cm long sample holder|
|System Control||Embedded microscope software with open communication interface|
|Open GUI control for interface control and microscope automation|
|High-speed RAID controller for data streaming|
|HIVE high speed centralized data system (optional)|