Intravital Microscopy

Overview

Intravital microscopy enables dynamic, 3D, cellular-level imaging of various biological processes in vivo. It enables scientists to directly verify hypothesis derived from ex vivo or in vitro observations in natural physiological in vivo microenvironments.

Using intravital microscopy, in vivo visualizations of gene expression, protein activity, cell trafficking, cell-cell / cell-microenvironment interactions, and various physiological responses to stimuli have been accomplished, providing novel insights, which have been impossible to obtain with conventional static 2D observation of ex vivo or in vitro samples.

However, up until now, individual users have had to improvise the required functions for each of the intravital imaging applications for the various organs with conventional standalone microscopes, which resulted in non-optimal performance for limited applicability.

The IntraVital Microscopy (IVM) systems from IVIM Technology, is a fully integrated system with optimized optical, mechanical, and electrical components to ensure best-in-class imaging performance for a wide range of in vivo intravital imaging applications.

The IVM systems (IVM-C/M/CM/MS) provide an all-in-one in vivo imaging solution providing integrated physiological monitoring and support, anesthesia, along with state-of-the-art living tissue motion compensation for improved image quality. The software has been fully optimized to allow for rapid image acquisition, while image processing is accelerated by GPU-assisted parallel computing.

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Individual user have had to improvise the required functions for each of the intravital imaging applications with conventional standalone microscope.

• High degree of technical difficulty for non-expert individual user
• Non-optimized intravital imaging performance
• Limited applicability to various organs
• Difficult to standardize intravital imaging procedure
• Difficult to establish universal protocol for experiment
• Limited reproducibility and productivity

IVIM Technology’s All-in-One Intravital confocal/two-photon microscopy system (IVM-C/M/CM/CMS/MS2) is extensively optimized and carefully engineered to provide superb performance in the intravital imaging of live animal models in vivo.

All-in-One IVM Platform

• All-in-One single-box packaged Intravital Microscopy (IVM) system
• Fully integrated with key functionalities for imaging of live animal model
• Unified animal stage to accommodate various accessories for wide-range of intravital imaging applications
• User-friendly design for easy and smooth operation

Feature	Benefit
Integrated temperature support, and physiological monitoring	Ensures animal well-being throughout the imaging session, and consistency between animals within a study
Integrated inhaled anesthesia	Ensures ease of animal handling and consistent plane of anesthesia
Animal motion compensation	Provides enhanced image quality on organs which may be affected by respiratory motion
User friendly design – both software and hardware	Allows for ease of use, and reproducible results
4-color simultaneous imaging	Confocal / Two-Photon Modes – Allows users to track the movement of several cells in vivo to better understand the biological processes being examined
Ultrafast rotating polygonal mirror scanner	Allows for ultrafast uniform laser-beam scanning with uniform excitation illumination over the entire field of view (FOV) Enables ultra-high speed in vivo imaging (max. 100fps @ 512×512 pixels FOV)

IVM Models

IVM-C (Confocal)	IVM-C is optimized for in vivo imaging experiments. It is specially equipped with four (4) different wavelength lasers and four (4) high-sensitivity confocal detectors. IVM-C is the optimal system to observe highly diverse, dynamic multi-cellular behaviors in living animals simultaneously with four (4) different fluorescence colors. Easy and highly efficient multi-color simultaneous imaging High flexibility in the selection of laser/detector wavelength Customizable design for modifications according to the customer’s requests and accommodations for future updates Applicability for conventional ex vivo, in vitro, and in vivo imaging
IVM-M (Two-Photon)	IVM-M is optimized for in vivo imaging experiments. Especially because it is equipped with a fully-automated tunable femtosecond (fs) pulse NIR laser system. IVM-M is the optimal system for users who need to conduct deeper-tissue imaging using less-scattering NIR wavelengths. The fs-laser system’s full control functionality is integrated with the two-photon imaging software for user convenience with various automated algorithms. Laser wavelength tuning for optimal two-photon excitation of a wide-range of fluorescence agents Deeper tissue imaging with a longer-wavelength NIR fs-laser system Capable of label-free, non-linear multi-harmonic generation imaging Motion compensation and ROI tracking for long-term in vivo imaging
IVM-CM (Confocal & Two-Photon)	VM-CM is optimized for in vivo imaging experiments. Especially because both the confocal and two-photon microscopes are integrated into a single-box packaged system. IVM-CM provides the ultimate versatile functionality of the IVM-C and IVM-M in one system Dual-mode IVM-C (confocal) / IVM-M (two-photon) imaging capability One-click automated transition between confocal and two-photon imaging mode
IVM-MS (Two-Photon Smart Version)	IVM-MS is optimized for in vivo imaging experiments and is equipped with a new compact, high-efficiency femtosecond (fs) pulse laser module. Especially because it integrates a compact high-stability maintenance-free fs-pulse laser into a single box. The IVM-MS is the ideal solution for customers in need of a two-photon microscope with limited resources of space and budget. Less expensive, fully-automated fs-laser system Space-saving with no additional fs-laser box configuration Simple hand-free turn-key operation of the fs-laser for two-photon excitation Easy maintenance and management without liquid cooling system requirements

Confocal Microscopy

High-resolution optical sectioning imaging of in vivo tissue
- Rejection of fluorescence signal from out-of-focus volume by confocal pinhole
- High contrast, low background, high quality in vivo imaging

Ultrafast precise Raster scanning of multi-color excitation laser-beam focus
- Video-rate imaging of fast cellular-level dynamics in live tissue in situ

Ideal for high-sensitivity multi-color intravital imaging of in vivotissue

Two-Photon Microscopy

High-resolution optical sectioning imaging of in vivo tissue
- Fluorescence signal is intrinsically generated only at the focus
Deeper tissue imaging with longer-wavelength near-infrared (NIR) fs-pulse laser for two-photon / multi-photon excitation
Capable of label-free, non-linear multi-harmonic generation imaging (SHG, THG)
No photo-bleaching at non-focal plane; reduced phototoxicity
Ideal for long-term 3D intravital imaging of in vivo tissue

Confocal Microscopy	Two-Photon Microscopy
Single-photon excitation	Two-photon excitation
Point scanning + pinhole	Point scanning + no pinhole
Optical sectioning: Fluorescence signal from out-of-focus is blocked	Optical sectioning: Fluorescence signal is intrinsically generated only at the focus
Imaging depth – 100-200µm	Imaging depth – 250-1000µm
Continuous-wave solid-state laser with flexible choice of wavelengths at the range from ultraviolet (UV) and visible (VIS) to near-infrared (NIR)	Femto-second pulsed laser tunable at near-infrared (NIR) wavelength range
Descanned confocal detector	Non-descanned detector (NDD)
Easy and efficient, multi-color 3D intravital imaging	Deeper-tissue 3D intravital imaging

Applications

The IVM series of systems have been designed to provide expandability and flexibility for highly diverse intravital imaging applications of various tissues and organs, including the brain, liver, spleen, kidney, pancreas, lung, heart, gastrointestinal tract, retina, skeletal muscle, bone marrow, peripheral lymph node, prostate, thymus, thyroid gland, adipose tissue, blood, and lymphatic vessels, etc.
In addition, to handle various imaging needs raised by researchers for a wide range of biomedical studies, detailed experimental protocols have been established for high-quality intravital image acquisition. Subsequent quantitative analysis of various cellular-level dynamics and physiological alterations have been established.

In Vivo Live Cell Imaging
- IVM systems allow for dynamic 3D imaging of various cellular-level dynamics such as cell trafficking, cell-cell interaction, cell-microenvironment interactions in vivo; providing insights into the processes of human disease progression and response to therapeutic interventions

Drug Discovery
- The IVM series of systems serve as a highly valuable and versatile tool allowing:
  - Target identification and validation in natural in vivo microenvironments
  - Confirmation of mode of action (MoA) and proof of concept (PoC) of new therapeutics by direct in vivo cellular observation
  - Monitoring of in vivo drug delivery, distribution, retention and in situ efficacy at target tissues and organs
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