TriM Scope™ II Multiphoton Microscope

The upright setup is the most common configuration and presents a perfect entry into the world of multiphoton microscopy. Focusing on the essential components required for highly sensitive deep-tissue imaging, it presents a versatile instrument that can be used in a large variety of applications, including in vivo imaging in brain, bone marrow, and lymph node, as well as calcium imaging and combinations with electrophysiology protocols.

Due to the modular design, the TriM Scope II Multiphoton Microscope can be easily modified or upgraded at any time as experimental needs evolve.

The inverted configuration combines a standard scientific-grade inverted microscope with Miltenyi Biotec’s TriM Scope II Scan Head. In various applications, the inverted configuration is superior to an upright configuration:

• Imaging of cultured cells, which adhere to the bottom of a dish and spread across one focal plane while having access to large amounts of medium.
• Applications needing access to the sample from the top (liquid exchange, micro-pipettes).
• Sterile applications as there is no contact between the objective and the sample.
• Any in vivo application dealing with specimens that cannot be rigidly mounted or that change their shape because of breathing or blood flow. 

Selected imaging applications suitable for this configuration include studies of brain slices, cell cultures, fixed tissues, organs ex vivo, and organs in vivo (liver, kidney, and intestine).

This configuration applies the Nikon Eclipse Ti2-U stand. Other stands are possible on request.

The Double-Header configuration combines an upright and an inverted microscope stand with a single scan head. This unique feature makes it a perfect, cost-effective instrument for multi-user environments, or whenever a versatile 2-photon platform for various applications is required.

This configuration uses the same excitation source and scan optics as the upright configuration. Switching between both imaging modalities can be done within seconds without realignment. Each stand offers up to six detection ports in epi-direction plus two ports in transmission-direction.

In epi-direction, the Spectral Detector allows maximum flexibility in experimental design via the simultaneous use of up to six GaAsP, GaAs, and/or Bialkaline PMT detectors. Due to its compact design, all PMTs are equidistant and very close to the objective’s back aperture in order to facilitate the highest collection efficiency. Advanced imaging optics ensure that no fluorescence light gets blocked by apertures.

Key features are:

• Equidistant mounts close to the objective's back aperture.
• Efficient spectral separation by dichroic mirrors and dielectric filters.
• Fast mechanical shutter (optional) to block scattered laser light, e.g., during optical treatment with vis-lasers.

The Transmission Detector module is designed for efficient SHG/THG detection in forward direction for both upright and inverted configurations. It can carry up to two PMTs and is located close to the back aperture of the condenser lens.

Key features are:

• Customized dichroic mirror.
• Customized narrow bandwidth notch filter in front of each PMT for splitting SHG and THG signals.
• IR-blocking filter to protect PMTs from the excitation wavelength.

The Treatment Scanner, an additional independent galvo scanner, stimulates any number of locations like cells, spines, or axons within the sample by VIS- or IR-laser light in real time. Due to the separated beam paths, photo-stimulation, such as uncaging, photo-activation, FRAP, opto-genetics, etc., can be performed independently of the imaging (point, line, ROI). Individual dwell-times for each treatment location can be set by the user within the software, including path optimization options between the set locations.

Many intravital 2-photon microscopy applications dealing with the observation of living samples and dynamic processes like calcium imaging need exceptional performance in terms of frame rates. Setups based on single-beam scanning are limited by sensitivity and speed because higher performance requires higher laser power, leading to sample degradation by bleaching and thermal destruction. The Cloud Scanner parallelizes the excitation process by distributing the laser power to four individual foci that are all arranged within one pixel. This increases the number of fluorescence photons per voxel and per time without raising photodamage, leading to quicker acquisition and much gentler imaging.

The Trim Scope ImSpector software package allows users to choose between single-beam and cloud-scanning modes; no further alignment procedures are required.

In intravital imaging, various tissue components feature significantly different refractive indices. This results in reduced optical resolution and limits imaging depth. The Adaptive Optics module consists of a thin reflective membrane that can be deformed to pre-compensate for aberrations induced by the sample and by the optical system itself.

For optimized imaging results, the user can individually choose between various compensation modes, such as for astigmatism, spherical aberration, coma, and trefoil. All settings for an individual experiment can be saved as a template. While acquiring a z-stack, the correction will be adapted to the penetration depth automatically.

The Adaptive Optics module provides: 

• Aberration correction.
• Increased optical resolution and imaging depth.
• Software interface to Python programming language for custom-made algorithms.

The In Vivo Stabilization module is a software- and hardware-based motion stabilization tool for imaging conscious rodents. It includes a piezo-driven tripod to move the objective in all dimensions, and additional software correction algorithms (x and y) that prevent motion artefacts due to heartbeat and breathing, assuring sharp in vivo images without artefacts. 

In addition, the In Vivo Stabilization module can be used for fast acquisition of z-stacks.

In vivo imaging with subcellular resolution is usually limited to a depth of 1 mm due to the tissue’s optical density, as well as scattering and absorption of the emitted fluorescence light. 2-photon endomicroscopy represents a minimally invasive approach that enlarges the imaging volume by at least one order of magnitude by going deeper into the tissue and recording 360° panorama images.

The Intravital Stage is a motorized xy-table with long z-travel for any upright microscope setup – the perfect choice for in vivo imaging. It provides precise hydraulic height adjustment, ample space for equipment like micromanipulators, and a large working distance below the objective lens (up to 200 mm with the extended version). The Intravital Stage is fully integrated into the ImSpector imaging software package, allowing precise mosaic stitching and multiregional measurements.

The upright TriM Scope II Multiphoton Microscope can be upgraded with a horizontal excitation module to allow sample rotation by 360°, opening up the following opportunities:

• Imaging living samples from different angles.
• Imaging free-standing samples under set conditions.

The Horizontal Imaging module includes a rotatable sample mount inside a chamber filled with liquid medium, supporting the sample during observation.

The Horizontal Imaging module utilizes the same laser sources and scan optics as the upright system. Switching between both imaging modalities can be done within seconds without re-alignment of the laser system.