Our multiphoton imaging system is purpose-built for deep imaging in biological tissue, aimed at revealing both detail and dynamics. Molecules can be visualized deep inside the specimen enabeling 3D imaging of tissue slices, organoids, whole organs, embryos, or even whole animals.
Efficient delivery and detection of photons in scattering media enable high signal-to-noise ratio acquisition, high sensitivity, and high-speed imaging to capture fast in vivo responses.
Objective: water immersion objective with isolated tip
XLPLN25xWMP2: NA 1.05 - WD 2.00 mm
high performance objective for multiphoton excitation (high IR-transmission from 400 to 1600 nm)
The objective maximize resolution and contrast for 3D imaging deep within thick specimens. The objective is equipped with a motorized correction collar (adapting to refractive index mismatch) that can automatically and dynamically compensate for spherical aberration while maintaining focus position.
Find your specimen with transmitted light or wide-field fluorescence:
Multiphoton excitation and non-confocal detection,
incl. 4 non-confocal/non-descanned detectors + one standard external transmitted light detector, IR laser introduction with ultra-fast AOM intensity control.
This section includes all the FluoView specific adaptations to the microscope frame to setup a complete FluoView multiphoton excitation laser scanning microscope system according to the provided system requirements:
Hybrid scan unit for multiphoton excitation imaging:
· The hybrid scan unit is designed for highly effective and highest speed IR laser scanning. High IR transmission is achieved with anti-corrosive silver coated galvanometric scanning mirrors.
· Fibre coupled conventional fluorescence illumination via the scan unit and software controlled motorized switch on/off.
· Scan unit is equipped with a set of high-performance galvanometer point scanners and a set of high speed resonant galvonometer scanners. Switching between standard point and resonant scanning can be done conveniently via software and takes appox. 3 seconds.
Non-descanned detector unit with 4 detection channels in reflection:
· Non-descanned reflection detector module with 4 channels implemented close to the specimen position to observe fluorescence in reflection mode.
2 high-performance multi-alkali PMT detectors
2 GaAsP PMT detectors
Ion deposition filter sets included to efficiently detect fluorescence light, e.g. CFP/YFP, GFP, Calcium indicators, etc. Filter cubes can be exchanged for detection of different dye combinations (see next section " Filter configuration").
Fully automated beam introduction system for pulsed IR laser including AOM laser control
IR laser - for multiphoton laser imaging and manipulation / stimulation
MaiTai HP DS-OL
Z-Axis control with the BX63L:
Stepper motor for control of Z-axis position is integrated in BX63L microscope frame, minimum step size 10 nm (the nosepiece movement is controlled in z-direction whereas microscope stage is fixed).
FluoView software allows specific selection of z-focus working range, maximum working range 20 mm.
Note: Resonant scan is performed at a field number of 18 allowing a big field of view even at highest speed
Standard image pixel formats:
ranging from 64x64 to 4096x4096 pixels. 12 bit per channel acquisition, multi-dimensional TIF image file format.
Flexible subarea Clip Scanning mode with all available image pixel formats to efficiently reduce the amount of acquired data and speed up frame rate.
512x512 pixels, 12 bit per channel acquisition.
Rectangle clip scanning mode to efficiently reduce the amount of acquired data and speed up frame rate.
Multi-area Time Lapse (MATL) functionality:
TruResolution Automatic Correction Collar software control:
FluoView Application Software – post-acquisition display and analysis:
Implemented display and analysis functions:
Additional analysis functions supplied by CellSens Dimension Desktop (also possible using Imaging PC1, which also has IMARIS software):
Along with threshold based phase analysis and offline kinetic functions, cellSens Dimension adds a range of image processing and analysis functions, for example arithmetic and logical operations, edge detection, projections calculations, image smoothing. Constrained Iterative Deconvolution
Ion deposition filter sets included to efficiently detect fluorescence light, e.g. CFP/YFP, GFP, Calcium indicators, etc.
Filter cubes can be exchanged for detection of different dye combinations. Please refer to this FILE to configure your setup. If you need additional filter cubes we will help configuring a cube for you to purchase.
The MPE system is equipped with a femtosecond laser that is tunable from 690nm - 1040nm.
When you need to excite multiple fluorophores, my best guide is to start out with 800nm and detect the signal in all channels. If you are missing signals, please go to this site and search for the fluorophore (with the extension “2P”) and tune the laser towards the specified wavelength.
Laser intensity may drop at extreme wavelengths and it is usually better to use e.g. 1020 than 1040nm
Deep imaging in tissue is challenging because of refractive index mismatch from immersion media and especially within the sample itself. In order to circumvent that issue in deep imaging tissue can be cleared. Tissue clearing can be quite complex, we are not experts yet, but below you will find information we find helpful:
2-p imaging, and access to the animal facility, can be time consuming. We do what we can to make it convenient for our users. After mounting a sample and starting up a long-time experiment, users can access the system via Remote Desktop. All imaging parameters and movements (x,y,z) can be controlled from your office PC (you need LAN cable, not Wifi). See image below for logon information and use the password that you will get from us personally. For detailed guide to remote desktop please follow this link. Shutting off the system requires your presence.
The Skou building, The Animal Facility. Room: 1116-42AA