Portable and turn-key multimodal multiphoton microscopy in Life Science

Session

Poster Session

Authors

Stefanie Kiderlen (2), Lukas Krainer (2), Andy Hill (1)

Affiliations

1. Photon Lines
2. Prospective Instruments

Keywords

Multiphoton, Two-Photon, SHG, THG, FLIM, Widefield, CARS, SRS, Three-photon, Fluorescence, microscopes, femtosecond Lasers, deep-tissue, 3D, label-free, in-vivo, intravital, brain, pathology, histology

Abstract text

Summary

The experimental requirements for different imaging scenarios are versatile and varying setups of the microscope are needed. The MPX multiphoton microscope allows a large number of different experiments due to its easy-to-use and flexible design providing a large working space underneath the 360-frontend, which enables microscopy from any angle, positioning and direction (upright or inverted). Multimodal imaging including two-photon (TPEF), higher harmonics (SHG/THG), Coherent anti-Stokes Raman Scattering and Stimulated Raman scattering (CARS/SRS), Fluorescence lifetime (FLIM) and epi-widefield fluorescence gains orthogonal data sets and addresses a broad range of different samples. Non-linear imaging techniques like TPEF or SHG/THG enable deep tissue penetration and the multi-channel approach allows multiplexing to distinguish between different imaging modalities that can be recorded simultaneously. Thus, a broad range of key uses in life science research including 3D imaging, label-free, deep tissue, in-vivo and intravital live-animal, whole organ, and whole slide imaging can be accommodated.

Introduction

Nowadays many labs are focusing on translational and interdisciplinary collaborations, research fields and applications, thus the demand for flexible systems that adapt to these increasingly complex requirements of laboratories is rising. Furthermore, also well-established research areas change their way of performing experiments and obtaining data. For example, in the interest of animal welfare, more and more experiments must be transferred into 3D cell culture experiments. However, until the day of vision zero, different types of experiments will run side by side with different microscopy requirements. In parallel efforts are made to transfer existing 2D cell culture assays also to 3D cell culture for fundamental research or drug testing since it is well known that cells behave different in a 3D environment than in 2D. Thus, high-quality imaging of 2D, 3D and 4D samples as well as label-free and non-destructive imaging is becoming more important. However, biological samples are a highly heterogeneous and complex composition of proteins, cells and other molecules, creating structures on the micro and macro scale. This heterogeneity causes complex optical effects like scattering and absorption that make it challenging to penetrate optically deep into these samples. However, 3D imaging in biological samples is essential to understand cellular mechanisms and cellular interactions with neighbor cells and their surrounding environment. Here standard microscopy techniques are limited in imaging depth because of using wavelength in the visible (VIS) range. In contrast, multiphoton microscopy uses a highly focused laser beam in the near-infrared (NIR) range. Here, nonlinear optical effects results in a very tight volume concentrated in the focal plane, significantly reducing the absorption cross section from surrounding material.

Methods

We have developed an integrated imaging platform which gives easy and economically viable access to technically challenging, but very capable non-linear imaging techniques like 2P, CARS and SRS, so far typically requiring major system installations and maintenance costs, prohibiting a wider spread to a broader user base. Here, we will demonstrate turn-key portable multimodal multiphoton microscopy, combining 3D label-free upright and inverted imaging that can be integrated into a variety of lab and clinical settings to gain orthogonal informational content, enhance imaging depth, and save time in 3D in-vivo and ex -vivo samples as well as large area tissue sections. Combined with an ultrafast resonant scanning option even fast dynamic processes like tracking single erythrocytes in brain capillaries can be captured. Future developments will include improvements for even higher imaging resolutions and depth by implementing adaptive optics techniques and three-photon microscopy. The flexibility of the MPX multiphoton microscope allows different experimental setups and configurations to image any type of sample ranging from singe cells up to living animals.

Figure 1: Concept of the MPX microscope. The microscope consists of the controller unit and the sceanhead that are connected through a flexible umbilical cord. The flexible 5-axis movement of the scanhead unit and the large working distance allows different experimental setups like in-vivo imaging (d), whole organ imaging or imaging of standard objective slides.

Results and Discussion

Especially, in the field of clinical research and pathology, the identification of biomarkers and the need for deep-tissue imaging has rapidly spread in the past few years. Here, changes in diseased or injured tissue can serve as valuable diagnostic tools. The fast and reliable identification of biomarkers using multimodal multiphoton imaging delivers valuable orthogonal information, enhances imaging depth, and saves time in clinical and diagnostical workflows. 

Figure 2: Label-free multimodal imaging showing epi-fluorescence widefield guided multiphoton microscopy of a human label-free skin melanoma FFPE section (a) and CARS microscopy of an adipose cryosection (b). Fast widefield overview scan (a) and respective multiphoton close-ups of healthy tissue (c,d), interface between healthy and diseased tissue (e), and regions in the lesion (f,g), showing the collected autofluorescence in yellow and the SHG signal in blue. Scalebar: 1 mm (a) and 200 µm (c-g). h) CARS microscopy image of an adipose cryosection showing the CARS signal in red and a nuclear stain in blue. 

On the other hand, people working in the scientific field have other needs and demands on their experimental setup and the results. Here the small footprint combined with the high flexibility of the microscope is highly appreciated whereas the time-saving aspect is rather marginal. A large working space provided by the 360-frontend allows for example in-vivo and in-vitro microscopy from any angle and positioning of any required accessories such as stages, heating, and monitoring pads.

Figure 3: In-vitro 4D spheroid time-lapse imaging. Timelapse inside a colorectal adenocarcinoma cell line DLD1 spheroid stained with Spirochrome’s SPY555 tubulin probe. Imaged over 4 hours. Close ups show events like cell divisions. SPY probe was kindly provided by Spirochrome

Here, we present a broad range of different imaging scenarios ranging from clinical research and pathology to R&D applications and fundamental biology using one microscope.

References

S. Kiderlen & L.Krainer: Turnkey Multiphoton Microscopy for 3D samples Label-free and 4D imaging in Life Science and Medicine, Wiley Imaging&Microscopy, 2023

M. Holmes et al.: Next-Generation Laser Scanning Multiphoton Microscopes are Turnkey, Portable, and Industry-Ready, Microscopy Today, 2022

M. Homes et al.: A Multiphoton Microscope Enables Portable 3D Biological Imaging, Biophotonics, 2022