Leica Sp8 DIVE Multiphoton

The Leica Sp8-DIVE (Deep In Vivo Explorer) Multiphoton Microscope was installed in March of 2019.  70% funding for the DIVE was provided by a National Science Foundation Major Research Instrumentation grant; the remaining 30% funds were provided from the School of Arts and Sciences.

The DIVE instrument is one of the first of its kind in the country; providing a fully tunable non-descanner that can take full advantage of all outputs from the Spectraphysics 800 to 1300 nm pulsed infrared (IR) laser, allowing 2 photon imaging of any dye between 400 and 800 nm.  A secondary fixed output 1040 pulsed IR laser provides excellent imaging from red dyes like RFP and mCherry.  Together, multiple colors can be imaged at the same time from the same sample in very deep tissue samples.  The DIVE 4Tune nondescanner incorporates two ultra-sensitive HyD detectors for the best signal to noise light collection in the industry.    In photon-counting mode, the HyDs allow for Fluorescence Lifetime Imaging (FLIM), which is fully incorporated into the LAS X software as a simple module.  The pulsed lasers can also be easily tuned for Second Harmonic Generation (SHG) imaging of many unprocessed macromolecules like collagen and tubulin. Objectives include a wide range of water and multi immersion dipping lenses as well as a 25X water dipping lens with a software-driven motorized correction collar that continuously adjusts as the image plane moves deeper in the Z axis. In addition to the Infrared lasers, there are an additional 4 visible lasers running from 405 to 638 nm to provide greater flexibility in imaging parameters.  The DIVE scanhead incorporates a x2y Tandem Scanner, providing great Field of View (FOV) and speed via the selection of conventional or resonant galvanometers.  Additional speed can be achieved using the piezo-driven z attachment.  For long time lapse imaging of living samples, an Okolabs temperature and humidity controlled stage mounted environmental chamber is available.  In addition, there is an integrated Trigger module for simple time-stamp and secondary equipment time control.  All image acquisition and processing is run via the powerful and simple to use LAS X software package.  Modules for the DIVE include NAVIGATOR (tilescan and point scan gui), FALCON (FLIM), FLIM-FRET, FRAP, 3D viewer and analysis, Electrophysiology, and LIGHTNING (adaptive deconvolution).

Microscope and Illumination Sources

Leica CFX Flex upright fixed stage microscope.  Large manual working distance Z adjustment to accommodate small animals, slides, petris and multiwell plates.  Fully motorized X Y axes.  Motorized standard galvo and Piezo driven objective turret for fast Z acquisition.  Broadband EL6000 mercury halide lightsource with multicolor (RGB) filter cube.  White transmitted light source for brightfield and DIC optics.  Okolabs purpose-built environmental chamber for precise temperature and humidity control. 

Objective Magnification Numerical Aperture


10X dry



16X multi immersion (water/oil/glycerol/BABB/SCALE) manual correction VISIR



25X water immersion VISIR



25X water immersion Motorized Correction



40X water immersion manual correction


Laser Type Power

Spectraphysics Insight X3 Dual tunable wavelength

Pulsed Femtosecond continuous tuned 680 nm to 1300 nm

Max of 2.0 W at 900 nm

Spectraphysics Insight X3 Dual single wavelength

Pulsed femtosecond single 1045 nm

3.5 W

405 nm DMOD compact < 60 mW, < 5 mW at objective
488 nm DPSS < 12 mW, < 3 mW at objective
552 nm DPSS < 12 mW, < 3 mW at objective
638 nm DPSS < 12 mW, < 3 mW at objective

SP8 LIAchroics Compact RGB x2y Tandem scanner:   X axis 8000 Hz resonant scanner + 10-1800 Hz galvonometric scanner; dual Y axis scanners for increased Field of View.  IR lasers routed to 4TUNE detector on non-descanned side.


Visible lasers routed to single PMT on descanned path.  Two HyD GaAsP Detectors for gated imaging on 4TUNE non-descanned path.  One PMT for brightfield transmitted light detection.


LAS X software with the following modules:

Module Uses


GUI for point scanning and Tiled images

Dye Finder

Dye setup wizard for multicolor imaging

3D visualization

3D viewer, processor and analysis wizard for Z stacks


Wizards for FRAP, FLIP, FRET and timelapse

FRAP Zoomer

FRAP beam control for enhanced photobleaching


Wizard for FLIM and FLIM-FRET setup


Adaptive Deconvolution software


Wizard for Triggered Electrophysiology experiment setup

Applications and Output


FLIM, FLIM-FRET, SHG, THG, Electrophysiology, deep imaging, live imaging, timelapse, Deconvolution, colocalization, point scanning, Tiled scanning, Z stacks, 3D rendering and analysis.  All standard post-acquisition processing.


.lei, .tiff, .avi, .mov, .mpeg4