Multimodal label-free ex vivo imaging using a dual-wavelength microscope with axial chromatic aberration compensation

Abstract

Label-free microscopy is a very powerful technique that can be applied to study samples with no need for exogenous fluorescent probes, keeping the main benefits of multiphoton microscopy, such as longer penetration depths and intrinsic optical sectioning while enabling serial multitechniques examinations on the same specimen. Among the many label-free microscopy methods, harmonic generation (HG) is one of the most intriguing methods due to its generally low photo-toxicity and relative ease of implementation. Today, HG and common two-photon microscopy (TPM) are well-established techniques, and are routinely used in several research fields. However, they require a significant amount of fine-tuning to be fully exploited, making them quite difficult to perform in parallel. Here, we present our designed multimodal microscope, capable of performing simultaneously TPM and HG without any kind of compromise thanks to two, separate, individually optimized laser sources with axial chromatic aberration compensation. We also apply our setup to the examination of a plethora of ex vivo samples to prove its capabilities and the significant advantages of a multimodal approach.

Patient-Derived Scaffolds of Colorectal Cancer Metastases as an Organotypic 3D Model of the Liver Metastatic Microenvironment

“…SHG analysis was done using a custom-built multimodal microscope, described in detail elsewhere [45] and in the supplementary methods.…”

Miniaturized multimodal multiphoton microscope for simultaneous two-photon and three-photon imaging with a dual-wavelength Er-doped fiber laser

“…In the current experiment, the sample is manually shifted by 50 µm to match the imaging planes for the 2PM and 3PM, respectively. To compensate focal shift, A. Filippi et al used separate optical paths for adjusting the beam divergence respectively so that the focal planes of 800-nm wavelength and 1200-nm wavelength were overlapped [18]. The drawback of their approach is the increased complexity and dimension of the setup.…”

“…Since most intrinsic fluorophores in tissue have a two-photon absorption peak at shorter than 900 nm wavelength [15], a wavelength shorter than 900 nm is required in order to excite the intrinsic TPEF signal in tissues. The second type of laser system used in multimodal MPM is dual-wavelength laser source [16][17][18][19]. In dual-wavelength laser system, a longer wavelength (>1 µm) excites 3PM signals such as 3PEF and THG, and a shorter wavelength (<1 µm) excites 2PM signals such as TPEF and SHG.…”

“…In dual-wavelength laser system, a longer wavelength (>1 µm) excites 3PM signals such as 3PEF and THG, and a shorter wavelength (<1 µm) excites 2PM signals such as TPEF and SHG. Dual-wavelength laser excitation has been achieved by tuning a Ti:Sapphire laser with OPO to two different wavelengths [16][17][18]. For example, previously reported dual-wavelength sources used tunable wavelengths at 1180-nm for 3PM and 860-nm for 2PM [17]; 1300-nm for 3PM and 920-nm for 2PM [16]; and 1200-nm for 3PM and 800-nm for 2PM [18].…”

“…Dual-wavelength laser excitation has been achieved by tuning a Ti:Sapphire laser with OPO to two different wavelengths [16][17][18]. For example, previously reported dual-wavelength sources used tunable wavelengths at 1180-nm for 3PM and 860-nm for 2PM [17]; 1300-nm for 3PM and 920-nm for 2PM [16]; and 1200-nm for 3PM and 800-nm for 2PM [18]. Alternatively, dual-wavelength laser excitation has also been achieved by sequentially using two lasers with different working wavelengths [19].…”