Luxendo Light-Sheet Microscopes

InVi SPIM Lattice Pro

The inverted view light-sheet microscope with advanced illumination unit

InVi SPIM Lattice Pro

The InVi SPIM Lattice Pro provides state-of-the-art light sheet technology for high-resolution imaging of live and fixed samples. It uses spatial light modulator (SLM) technology to generate a wide variety of illumination beam patterns , allowing for optimal illumination for each sample and application. An inverted setup of two high-NA objectives (one illumination and one detection lens) and a fully environmentally controlled imaging chamber makes long-term acquisitions using a variety of illumination patterns possible. 

 

Advanced imaging applications, such as structured illumination microscopy (SIM), calcium imaging, fluorescence lifetime imaging microscopy (FLIM), fluorescence correlation spectroscopy (FCS), or photomanipulation (PM) experiments using the add-on PM module are available with the InVi SPIM Lattice Pro.

High-Res
Diffraction-limited imaging
High-NA objective lenses for light sheet microscopy provide the best solution for sample-preserving, long-term imaging at diffraction-limited resolution. 
Multimodal
Specialized imaging made available
Hardware design and software integration allow specialized applications such as SIM, FLIM, or FCS imaging and photomanipulation experiments. 
Flexibility
Instant beam pattern switching
Fully integrated spatial light modulator technology allows sample illumination with a broad range of illumination patterns. 
Features

Inverted Light-Sheet Geometry

The InVi SPIM Lattice Pro is the right tool for high-resolution imaging applications while preserving sample health. Two water dippings and high-numerical aperture objectives ensure the best acquisition quality at diffraction-limited resolution. Different magnification steps allow users to adapt the field of view (FOV) to their requirements. As the z-stack orientation is linear, not skewed, no de-skewing of the stack is required for analysis or projections, making the image processing less computationally demanding.

Unique hardware features of the InVi SPIM Lattice Pro include:

  • Lasers: Up to six laser lines, chromatic correction, up to 20 emission filters, and dual-color acquisition ensure a broad application spectrum.
  • Detection objective: Nikon CFI Apo 25x W @ 1.1 NA, water immersion, with correction collar
  • Illumination objective: Special Optics 28.6x 0.7 NA water immersion objective
  • Magnification changer: 31.3x and 62.5x final magnification optimize the FOV and pixel size
  • Dual-channel detection: Two sCMOS cameras each equipped with up to 10 filters split by two selectable dichroic mirrors.
Gaussian, Bessel, and Lattice Beams

Light-Sheet Illumination Patterns

Every sample and application require diligently optimized experiment settings. The InVi SPIM Lattice Pro provides flexibility for fast adaptation of both illumination and detection to achieve the best results. A simple software command allows the system to switch between beam patterns such as Gaussian, Bessel, or Lattice Beams for multi-pattern illumination imaging in one experiment. Additionally, the Advanced Illumination Module (AIM) employs a fully integrated SLM to generate a broad range of illumination patterns featuring a characteristic pattern scanned vertically across the FOV to generate the light sheet.

The Gaussian beam has a waist at the center and diverges towards the periphery of the FOV. Scanning to generate a light sheet creates a small central area of the highest resolution covered by a thin light sheet. To overcome this limitation, the beam can be tuned for a more extended yet thicker waist, i.e., larger usable FOV at the price of overall resolution and contrast. Overall, a Gaussian beam is robust against scattering and well-suited for fast imaging. The classical static 2D Gaussian beam illumination provides all means for the highest speed continuous imaging. It is suitable for quantitative imaging modalities such as fluorescence lifetime imaging microscopy (FLIM) or fluorescence correlation spectroscopy (imaging FCS).

Self-reconstructing Bessel beams maintain a main lobe as thin as a Gaussian beam waist over a much more axial section than a Gaussian beam. This makes the Bessel beam the optimal choice for a large FOV at optimal resolution.

Optical lattice light sheets even out the Bessel beam side lobes through destructive interference, reducing light sheet thickness and non-specific fluorescence. The available lattice patterns have varying beam densities and characteristics along the different spatial dimensions, which affect resolution and FOV. Optical lattices can be used for resolution-enhancing structured illumination microscopy. Additionally, deconvolution can be performed during image processing to improve data quality further.

Beam Pattern Static Light Sheet Scanned Gaussian Scanned Bessel Optical Lattices
Measured Resolution Z [1] 800 nm 750 nm 680 nm 530 nm
Measured Resolution XY [1] 310 x 310 nm 310 x 310 nm 310 x 310 nm 310 x 310 nm
Minimum LS thickness [2] < 800 nm < 800 nm < 690 nm < 550 nm
Max. FOV 426 x 426 µm (31.25x) 426 x 426 µm (31.25x) 426 x 426 µm (31.25x) 26.6 x 57 µm (62.5x) [3]
Acquisition Speed 80 fps [4] 80 fps [4] 80 fps [4] 80 fps [4]
Multi-Channel Imaging simultaneous dual-color simultaneous dual-color simultaneous dual-color sequential dual-color
Number of Preset Beam Patterns 4 13 10 42
Key Advantage Highest-speed imaging and techniques such as FCS, FLIM Beam robustness, fast imaging Homogeneous illumination across a large FOV Optimized and enhanced resolution in 3D

Table depicting the specifications of different illumination patterns.
1 FWHM measured with 170 nm sized fluorescent beads / no deconvolution / @ 515 nm detection
2 Measured in water with 488 nm laser
3 Dithered light sheet
4 Full-frame imaging / max. 500 fps at reduced FOV

Arabidopsis leaf images obtained using a Gaussian Beam (left), Bessel Beam (center), and Optical Lattice (right). Images used mCherry, 150 µm max projection, 0.5 µm step size. Sample courtesy of Dr. Amrit Bhal, Dept. of Biological Sciences, NUS, Singapore.

Advanced Imaging Methods

Illumination
  • SLM-based pattern generation, including wavefront correction for illumination with minimized optical aberrations
Beam Patterns
  • In-experiment switching of beam patterns: Gaussian, Bessel, Static LS, Lattice, and Droplet
  • Customizable beams are possible
Beam Scanning Modes
  • Static: Static Gaussian Beam for applications such as FCS or FLIM
  • Scanned: Scanned Gaussian Beam with extensive beam modulation possibilities (axial positioning; waist length, beam thickness)
  • Dithered: Lattice pattern oscillates for best Z-resolution
  • SIM: Subdiffraction-limited resolution by localized illumination pattern and computational reconstruction from image series
New Advanced Features    

Next-Generation Structured Illumination Module (SIM)

Structured illumination microscopy (SIM) uses spatially structured patterns of excitation light to generate patterns of light interference, called the Moiré effect. When excitation pattern frequencies are correctly applied, SIM can result in a 50% lateral resolution enhancement.

Importantly, light is only maximally structured inside the focal plane. In contrast, out-of-focus light is homogenous enough to allow for its removal in image processing. 

While no specific sample preparation is required, SIM requires longer exposure times and higher signal-to-noise ratios. While increased sub-diffraction resolution applies to the lateral resolution, the axial resolution is still diffraction-limited as the illumination pattern is not structured along the axial direction (Vangindertael et al. 2018 Methods Appl. Fluoresc. 6 022003).

Features

Advantageous Photomanipulation

The ability to spatiotemporally control, manipulate, and alter biological processes is critical to revealing relevant mechanisms. A photomanipulation  module (PM) can be added to InVi SPIM Lattice Pro, allowing for experiments such as photoablation, cauterization, optogenetics, and fluorescence recovery after photobleaching (FRAP) with:

  • Beam coupled into detection objective
  • Diffraction-limited illumination spot
  • Flexible region-of-interest (ROI) generation (point, straight line, freeform, and square)
  • Laser from continuous wave ultraviolet/visible (CW UV/VIS) to pulsed infrared (IR)
Features    

Full Environmental Control for Live Samples

While imaging living specimens, sample health and integrity are critical. The InVi SPIM Lattice Pro is equipped with an environmental control that supports long-term imaging (hours to days) of a wide variety of living specimens. This system is equipped with a generous sample chamber for immersion medium that controls temperature (20–45° C), gas concentration (CO2, O2, N2), and humidity. 

Next Generation Imaging    

Next-Level Resolution

The InVi SPIM Lattice Pro is the best solution for sample-preserving, long-term imaging at diffraction-limited resolution. Using two water-dippings and high NA objectives guarantees high acquisition quality while the adaptable magnification feature enables users to tailor the FOV to their needs. Additionally, the system's versatility is enhanced by its capacity for up to six solid-state laser lines, chromatic correction, a choice of 20 selectable filters, and dual-color acquisition, making it suitable for various applications.

Stimulation and measurement created from Gaussian Beam, Bessel Beam, Lattice Light-Sheet, and Static Light-Sheet

Sample Mounting

Organoid imaging with TruLive3D dishes by ibidi GmbH (left). Cells seeded on cover glass, imaged with a glass slide adapter (right).

The InVi SPIM Lattice Pro has an inverted geometric design, easy sample, access, and accurate environmental control. Depending on beam patterns and experiments, sample mounting can be adapted to optimize resolution and experimental outcomes.

Broad Sample Variety

The InVi SPIM Lattice Pro is suitable for a variety of samples in cell biology from organoids and cell cultures to organs. Easy sample access allows straightforward sample placement and manipulation while switching beam patterns via a simple mouse click allows for experiment optimization.

Applications made possible with the InVi SPIM Lattice Pro include:

  • Live samples' dynamic processes and interactions
  • Cell cycle imaging
  • Subcellular structure visualization
  • Tracking of cells or protein movements in 3D
  • Long, time-lapse imaging
  • 2D cells, spheroid, organoid, tissue, or organism development

Subcellular Components

The InVi SPIM Lattice Pro is ideal for imaging subcellular components including cytoskeletal structures like actin.

Left-to-right: Merged image of multi-stained HELA Cells. Green – nuclei, blue- nuclei with deconvolution, red – actin, yellow – actin with deconvolution, white – actin with deep learning-based image reconstruction. Image courtesy of Ulrike Engel Nikon Imaging Center, Heidelberg, Germany.
Applications

The InVi SPIM Lattice Pro in Research

Primary mouse mammary epithelial acini to study oncogenes

Publication: Alladin et al. (2020), Tracking cells in epithelial acini by light-sheet microscopy reveals proximity effects in breast cancer initiation, eLife 9:e54066, DOI:10.7554/eLife.54066

The InVi SPIM Lattice Pro is suitable for 3D Matrigel cultures of mammary acini. The imaging chamber allows for environmental control and has a reservoir for the immersion medium.

In their research work, Alladin et al. (2020) developed primary mouse mammary epithelial acini to study oncogenes.

To follow up their observations over time, they combined a nuclear reporter H2B-mCherry into the T and B mice to mark all the cells. The T/H2B-mCherry acini transduced with reporter virus proliferated upon doxycycline addition, showing an expansion of both the marked and unmarked cells. Time-lapse imaging showed tumor phenotype manifestation as seen by multi-cell-layered rims and pronounced proliferation-associated apoptosis in all acini.

(a) Light-sheet imaging of stochastic tumorigenesis in mammary acini. These 3D images were taken of selected time points during live-cell time-lapse microscopy of induced T acini transduced with Reporter virus. (b) All cells in the acini express H2B-mCherry (magenta) and only cells transduced with lentiviral particles express H2B-GFP (green). (c) Schematic representation of the big-image data analysis pipeline developed to analyze the light-sheet microscopy images. Figure recreated under Creative Commons Attribution License from Alladin et al. (2020), Tracking cells in epithelial acini by light-sheet microscopy reveals proximity effects in breast cancer initiation, eLife 9:e54066, DOI:10.7554/eLife.54066

Stem Cells

Pluripotent cells can self-renew and differentiate, with hundreds of genes expressed stably during self-renewal and directed expression upon signaling for differentiation. Characterizing the pluripotent state requires markers, such as miRNAs, which can also aid in differentiation. Research has shown that miRNA expression in human cells reveals surprising diversity and can be quantitatively mapped to understand stem cell processes. Colonies of mouse embryonic stem cells can be studied to realize pluripotency, involved genes, and regulatory networks.

Colonies of mouse embryonic stem cells, stably a nuclear and membrane marker. x-y (axial) view (left) and a y-z (lateral) view (right). Samples courtesy of Pierre Neveu EMBL Heidelberg, Germany.
Colony of mouse embryonic stem cells with a stable nuclear (blue) and membrane marker (white). The image shows a single slice of a 3D stack. Samples courtesy of Pierre Neveu EMBL Heidelberg, Germany.

Morphogenesis and Biophysics

The InVi SPIM Lattice Pro is an outstanding tool for visualizing developmental biology and morphogenesis with unprecedented detail. Taking this a step further, the optional photomanipulation module allows for:

  • Control of fluorophores (optogenetics, uncaging, etc.)
  • Study of dynamics (FRAP experiments)
  • Biophysical studies (photoablation and cauterization)
Membrane staining of developing fruit fly (Drosophila). Sample Courtesy of Roland Le Borgne, Université de Rennes, France.
Specifications    

Objective Specifications

Objective Magnification Pixel Size FOV
25x 1.1NA 31.3x 208 nm 426 µm
  62.5x 104 nm 213 µm

System Comparison
* COMPATIBLE MODULE

  MuVi SPIM
Multiview
InVi SPIM Lattice Pro
Inverted View
LCS SPIM
Large Cleared Samples
TruLive3D Imager
Benchtop Design
Configuration
Horizontal Multiple-View
Inverted
Inverted - Dual Illumination
Inverted - Dual Illumination
# Lenses

4 lenses (2 IO, 2 DO)
2 lenses (1 IO, 1 DO) 3 lenses (2 IO, 1 DO)  
Multi-View
     
Live/Fixed Samples
 
Cleared Samples
   
Best Embedding
Capillary/FEP tube w/ agarose 3D stage
FEP foil; glass slides
Quartz-crystal cuvette TruLive3D dishes and FEP foil
Photomanipulation*
 
Environmental Control*  
Destriping/Uniform Illumination* ✓ (w/ Advanced Illumination Module)
Software

All-in-One LuxBundle Software

Intuitive design

Bruker's LuxBundle software saves time and enhances productivity by providing:

  • All-in-one, easy-to-use interface for acquisition, viewing, and post-processing
  • Fully scriptable microscope control and post-processing via open interface (e.g., Python or any other language) ready for custom "smart" microscopy
  • High reproducibility of experiments: all parameters are saved in the metadata and configurations can be saved for future experiments
  • Data formats (.tiff, .hdf5, .ims) compatible with common image processing software: Imaris, Aivia, BigDataViewer, Arivis, Fiji, Python, Matlab, Napari

Impressive Image Post Processor

MuVi SPIM records a sample from different angles/views and generates images composed of multiple tiles. The LuxBundle software ensures high-quality, 360° crisp images of the sample that compensate for absorption and scattering. Features include:

  • Multi-color alignment
  • Tile stitching of hundreds of tiles for large samples
  • Multi-view image fusion and deconvolution

3D Data Viewer

LuxBundle's integrated 3D data viewer allows researchers to inspect the entire dataset directly after acquisition. This gives users control over their data with key capabilities, including:

  • The ability to turn tile stitching on or off
  • Both raw and post-processed images
  • Fast viewing of multi-terabyte data sets
  • Flexible options to draw and annotate regions and landmarks
Cleared mouse embryo labeled with methylene blue (cyan) and showing autofluorescence (magenta). Image composed of 12 tiles and 920 planes, all processed and stitched with LuxBundle (Image courtesy of Montserrat Coll Lladó, European Molecular Biology Laboratory, Barcelona, Spain)

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