Raman spectroscopy is extremely powerful for charactering materials through their intrinsic molecular vibrational contrasts. This capability, when extended to microscopy, becomes especially useful for investigating spatially inhomogeneous specimens. With the availability of commercial confocal Raman microscopy systems in recent years, the method has been increasingly used to study biological samples 1–3
. With few exceptions, spontaneous Raman scattering is severely limited by low signal intensity, and acquiring a two dimensional Raman map of a biological sample could take a fraction of an hour to complete, hindering its utility for the study of dynamic processes, especially those happening in living biological systems. Coherent anti-Stokes Raman scattering (CARS) microscopy has been developed over the past decade to address this problem 4,5
. CARS makes use of two pulsed laser beams with an energy difference tuned to the vibrational frequency of the molecule of interests to obtain much stronger vibrational signals at the anti-Stokes frequency. When applied to the high wavenumber region (CH2
stretching modes) of biological specimens, video-rate imaging can be achieved with good signal to noise ratio (SNR) 6
. In order to obtain spectroscopic information, multiplex CARS can be employed, where both a picosecond narrowband laser and a femtosecond broadband laser are used. The narrowband laser determines spectral resolution and the broadband laser determines the spectral bandwidth 7–10
. The imaging speed is generally limited by the readout speed of the CCD detector to 10–100ms per pixel. It is also possible to use narrowband CARS and sweep the resonance frequency which can achieve 1 sec/frame imaging speed11,12
. This method typically requires mechanical tuning of an optical parametric oscillator (OPO) and only a limited range can be scanned without time-consuming adjustments. The third approach uses two synchronized broadband lasers and the Raman spectrum is acquired by scanning the time delay between the two lasers when they are appropriated chirped. This is known as the spectral focusing approach13
. It has the advantage of tunable bandwidth and simple scanning mechanism. However, all approaches based on CARS have a well-known non-resonant background problem that distorts vibrational spectra and causes image artifacts5
. The nonlinear dependence of CARS signal on chemical concentration also significantly hinders the quantitative analysis of chemical composition. Although phase retrieval using the maximum entropy method or the Kramers-Kronig method allows reconstruction of real Raman spectra 14
, the analysis process is complicated and it has not been shown that individual spatially and spectrally overlapping chemical components in a complex sample can be quantified using this approach.
In recent years, stimulated Raman scattering (SRS) microscopy has emerged as an alternative to CARS microscopy that is free from the aforementioned issues 15–18
. SRS eliminates the non-resonant background problem because the generated third order SRS nonlinear polarization is directly heterodyne mixed and amplified by the input beam with the exact same phase, therefore always resulting in a zero non-resonant contribution. Consequently, the spectral shape of SRS is identical to that of spontaneous Raman, allowing straightforward comparison. Moreover, the SRS signal has a linear dependence on the chemical concentration, allowing for simple quantitative analysis. These capabilities have facilitated applications in many different research areas such as lipidomics, pharmacokinetics, biofuel production, and cancer detection 16,19,20
. Typical SRS microscopy uses synchronized picosecond lasers with narrow bandwidth (<10cm−1
) to excite the sample, thereby acquiring Raman information on a specific predetermined Raman band. To obtain information on a different Raman band, one of the lasers has to be tuned to a different wavelength, which typically involves either cavity length change or crystal temperature change or both 21
. This spectral scanning process is usually slow and susceptible to the optical power drift and wavelength drift. It is also possible to employ a picosecond and femtosecond laser source to acquire spectroscopic information in a manner similar to multiplex CARS microscopy 22,23
. However, SRS microscopy uses a high frequency lock-in amplifier and a multiplex detection approach requires a lock-in amplifier array, which is impractical with current technology. Recent developments in our group used spectrally-tailored or multiplex excitation to achieve molecular selectivity 24,25
. Those approaches work well for simple systems in which all the component spectra are well characterized, but the instrumentation is complicated and has limited sensitivity. Most recently, SRS spectroscopy and imaging using the spectral focusing approach has also been demonstrated by using a femtosecond oscillator and a fiber-generated secondary source26,27
. It allows acquisition of reliable Raman spectrum over a large spectral range. However, the low power output and large bandwidth mismatch between the pump and the Stokes significantly limits the imaging speed to ms/pixel.
In this manuscript, we present SRS hyperspectral imaging using the spectral focusing approach, which improves the imaging speed of the previous works by a thousand fold. We present SRS spectra imaging of bead mixture as well as biological cells that is orders of magnitude faster than confocal Raman and multiplex CARS while obtaining reliable Raman spectra in the CH stretching region. We demonstrate that the chemical concentration of spectrally overlapping species can be determined with sensitivity and accuracy down to a few millimolar. We further demonstrate that with a nonnegative least square algorithm, SRS hyperspectral imaging provides detailed chemical mapping of several species in fixed mammalian cells. We believe the flexibility of choice among fast spectroscopy, single band imaging and hyperspectral imaging provides an extremely powerful tool for studying material and biological systems that is unparalleled by either spontaneous Raman or previous CARS/SRS imaging approaches.