Femtosecond Stimulated Raman microscopy: home-built realization and a case study of biological imaging

Abstract

The progresses of femtosecond laser physics have had a significant impact on research and development of nonlinear microscopy techniques, such as Stimulated Raman scattering microscopy. The main advantages of this technique, based on vibrational spectroscopy, is its abilities to perform label-free imaging with high sensitivity, high spatial and spectral resolution, 3D sectioning capability and fast time of image acquisition (typically a few seconds). The main aim of this paper is to demonstrates the home-built realization of an imaging system based on Stimulated Raman Scattering (SRS) powered by femtosecond laser sources, using a National Instrument data acquisition (NI-DAQ) card. The SRS microscope is obtained by the integration of an SRS experimental set-up with a commercial laser scanning microscope. The successful implementation of the imaging system is demonstrated reporting label free images of polystyrene beads with diameter of 3 microns and evaluating their signal to noise ratio. Finally, the reliability of our system in a biological environment is tested. Taking advantage of SRS microscopy, subcellular localizations of lipid droplets inside adipocyte cells are demonstrated.

Stimulated Raman Microscopy Implemented by Three Femtosecond Laser Sources

“…This SRS microscope is used to take advantage of two laser combinations. Ti:Sa and OPO laser combination cover the C-H region in SRG modality [17][18][19][20][21][22]. The latter Ti:Sa and SHG laser combination provide the extension of the microscope to the silent region (<1800 cm−1) and fingerprint region (1800-2800 cm−1) in the SRL modality [23].…”

“…We note that typical and widespread commercial laser sources combinations, such as Chameleon compact OPO (Coherent, Inc.) and Ti:Sa, although tailored for multimodal imaging, their minimum Raman shift is 2500 cm -1 . This means that by adopting this laser combination, only the CH-OH region of Raman spectra can be explored, while the and fingerprint regions are out of emission range [17][18][19][20][21][22]. Therefore, they cannot accomplish the demand of biorthogonal platforms based on femtosecond stimulated Raman microscopy.…”

A Spectral Resolution study in Femtosecond Stimulated Raman Scattering Microscopy

“…In our previous paper, we have successfully described the design and execution of femtosecond-based SRS microscopy, which is able to cover the C-H region (>2,500 cm -1 ) [16][17][18][19][20][21][22]. In Figure 1, a femtosecond-SRS spectroscopy setup is integrated with a Nikon C2 confocal microscope, which comprises an inverted Nikon Tieclipse microscope and a scan head.…”

“…Our SRS microscope allows us to probe individual Raman bands at a time. By tuning the wavelength of the pump or Stokes beam with the matching Raman frequency of different samples at the required Raman band, we can obtain the images of the different Raman band matching molecules with high chemical contrast [16][17][18][19][20][21][22][23]. Thus, CH2 (2,845 cm -1 ) and CH3 (2,940 cm -1 ) stretching signals can be collected at one Raman shift at a time, leading in principle to map the distributions of the lipid and protein contents in the same image.…”

Femtosecond Stimulated Raman Microscopy in C▬H Region of Raman Spectra of Biomolecules and Its Extension to Silent and Fingerprint Regions

“…However, although typical and widespread commercial laser sources, such as OPO pumped by Ti:Sa laser, are tailored for multimodal imaging, they present a limitation: Their minimum wavelength difference between pump (Ti:Sa laser) and signal (OPO) is 2500 cm −1 . Therefore, this laser combination allows only the CH-OH region exploration of Raman spectra, while the silent and fingerprint regions are out of the emission range [33][34][35][36][37][38]. As a consequence, this laser combination cannot accomplish the demand of bio-orthogonal platform based on femtosecond stimulated Raman microscopy, compromising its diffusion.…”